Novel immunologically active interleukin 2 analogue conjugates and methods for their preparation

Abstract

The present invention relates to long-acting conjugates of interleukin-2 analogues that have altered binding avidity to the interleukin-2 receptor.

Description

[Technical field]

[0001] The present invention relates to a novel interleukin-2 analogue conjugate, a method for producing the same, and uses thereof. [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] There are three types of subunit receptors for interleukin 2 receptors. The subunits are formed by the α chain (IL-2Rα, CD25), the β chain (IL-2Rβ or CD122) and the 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. A 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 prominently seen in CD8+ cells and natural killer (NK) cells. Also, a 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 [Patent Document 2] International Publication No. 97 / 034631

Patent Document 3

Non-licensed literature

[0006] [Non-licensed document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

[0007] As part of the development of therapeutic agents for immune-related diseases, there is a demand for the development of drugs that contain interleukin 2 analogues and have excellent long-lasting and superior pharmacological effects. [Means for solving the problem]

[0008] An object of the present invention is to provide a long-acting conjugate comprising an interleukin-2 analogue.

[0009] Another object of the present invention is to provide a method for producing the persistent conjugate.

[0010] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the interleukin-2 analog or a long-acting conjugate thereof.

[0011] Furthermore, the present invention aims to provide a method for preventing or treating cancer, which comprises the step of administering the interleukin 2 analogue, its long-acting conjugate, or a composition containing same to an individual in need thereof.

[0012] A further object of the present invention is to provide use of the interleukin-2 analog, its long-acting conjugate, or a composition containing same for the prevention or treatment of cancer.

[0013] A further object of the present invention is to provide a use of the interleukin-2 analog, its long-acting conjugate, or a composition containing the same for providing a medicament for preventing or treating cancer. Effect of the Invention

[0014] The interleukin 2 analog or the long-acting conjugate containing the same according to the present invention is an analog with increased binding ability to the in vivo interleukin 2β receptor, and has the effect of improving the convenience of administration and side effects for the purpose of anti-cancer therapy. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the results of SDS-PAGE of long-acting interleukin-2 analogue conjugates (analogs 21, 41 and 52). [Diagram 2] FIG. 1 shows the results of a purity analysis of interleukin-2 analogue long-acting conjugates (analogs 21, 41 and 52). [Diagram 3]3 shows the results of analyzing the pharmacokinetics of long-acting interleukin-2 analogue conjugates in ICR and C57BL / 6 mice. A shows the results of analyzing the pharmacokinetics of an interleukin-2 analogue no. 41 conjugate and an interleukin-2 analogue no. 52 conjugate in ICR mice, and B shows the results of analyzing the pharmacokinetics of an interleukin-2 analogue no. 21 conjugate, an interleukin-2 analogue no. 86 conjugate, an interleukin-2 analogue no. 104 conjugate, and an interleukin-2 analogue no. 105 conjugate in C57BL / 6 mice. In FIG. 3, the values ​​of the dosage and serum concentration of the long-acting conjugates are based only on the weight occupied by the interleukin-2 analogue moiety in the entire conjugate. [Figure 4] 4 shows the results of confirming the anticancer efficacy in a mouse animal model of an interleukin-2 analogue no. 21 conjugate, an interleukin-2 analogue no. 41 conjugate, and an interleukin-2 analogue no. 52 conjugate. In Fig. 4, the dose of the long-acting conjugate is based only on the weight of the interleukin-2 analogue moiety in the entire conjugate. [Diagram 5] 5 shows the results of confirming changes in memory T cells in an animal model of an interleukin-2 analogue long-acting conjugate. A shows the results of analyzing the tumor growth inhibitory effect of an interleukin-2 analogue no. 21 conjugate, an interleukin-2 analogue no. 41 conjugate, and an interleukin-2 analogue no. 52 conjugate, and B shows the results of analyzing the number of memory T cells of an interleukin-2 analogue no. 21 conjugate, an interleukin-2 analogue no. 41 conjugate, and an interleukin-2 analogue no. 52 conjugate. In FIG. 5, the dosage of the long-acting conjugate is a value based only on the weight occupied by the interleukin-2 analogue moiety in the entire conjugate. [Figure 6]6 shows the results of evaluating the antitumor efficacy of a long-acting interleukin-2 analogue conjugate in an animal model of malignant melanoma. A shows the results of tumor size determination for a conjugate of aldesleukin and an interleukin-2 analogue no. 86, and B shows the results of survival rate determination for a conjugate of aldesleukin and an interleukin-2 analogue no. 86. In FIG. 6, the dosage of the long-acting conjugate is a value based only on the weight occupied by the interleukin-2 analogue moiety in the entire conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] One aspect of the present invention is a long-acting conjugate containing a novel interleukin 2 analog (IL-2 analog). The interleukin 2 (IL-2) analog may be an interleukin 2 analog having increased interleukin 2β receptor binding ability compared to natural interleukin 2 or an interleukin 2 analog, aldesleukin, and the interleukin 2 analog may include a sequence in which at least one amino acid in natural interleukin 2 is mutated.

[0017] As a specific example, the persistent conjugate is characterized by being represented by chemical formula (1).

[0018] X-La-F···(1)

[0019] wherein X is an interleukin 2 analog that is not part of a long-acting conjugate, and X alone is an interleukin 2 analog that has increased interleukin 2β receptor binding ability compared to aldesleukin, L is a polyethylene glycol linker, a is 0 or a natural number, but if a is 2 or more, each L is independent of the others, F is an immunoglobulin Fc region in a dimeric form, and - indicates a covalent bond between X and L and between L and F, and the interleukin 2 analog is The present invention relates to 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 of native interleukin 2 has been mutated.

[0020] In another embodiment, the long-acting conjugate comprises an interleukin-2 analog in which at least one amino acid of natural interleukin-2 has been mutated in a manner selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0021] The long-acting conjugate according to any of the above-mentioned specific examples is characterized in that it contains as part of the conjugate an interleukin 2 analogue that has altered binding affinity to the interleukin 2α receptor and increased binding affinity to the interleukin 2β receptor compared to native interleukin 2 or aldesleukin.

[0022] The long-acting conjugate according to any of the above-mentioned specific examples is characterized in that it comprises, as a part of the conjugate, an interleukin-2 analog having at least one amino acid added to the amino acid corresponding to position 133.

[0023] The long-acting conjugate according to any of the above-mentioned specific examples is characterized in that it contains, as part of the conjugate, an interleukin-2 analog having a sequence in which the first amino acid of natural interleukin-2 is deleted and the 125th amino acid is replaced with another amino acid.

[0024] The long-acting conjugate according to any of the above-mentioned specific examples is characterized in that it contains, as a part of the conjugate, an interleukin-2 analogue further having 1 to 10 amino acid substitutions.

[0025] The long-acting conjugate according to any of the above-mentioned embodiments is characterized in that it comprises an interleukin-2 analog in which at least one of the amino acids corresponding to the 18th, 19th, 20th, 22nd, 38th, 42nd, 43rd, 45th, 61st, 68th, 69th, 74th, 80th, 81st, 84th, 85th, 86th, 88th, 89th, 91st, 92nd, 94th and 96th positions of the interleukin-2 analog site is further replaced with another amino acid.

[0026] The long-acting conjugate according to any of the above-mentioned embodiments is characterized in that it comprises as part of the conjugate an interleukin-2 analogue in which at least one of the amino acids corresponding to positions 18, 22, 38, 42, 61, 68, 80, 81, 85, 86 and 92 is further replaced with another amino acid.

[0027] The long-acting conjugate according to any of the above-mentioned embodiments is characterized in that it comprises as part of the conjugate an interleukin 2 analogue, which 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, 42nd, 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. (bo) 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, 86th, and 92nd positions are replaced with other amino acids. (bp) 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, and 86th positions are replaced with other amino acids.

[0028] The long-acting conjugate according to any of the above embodiments is characterized in that it comprises an interleukin-2 analog having at least one substitution 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 94 with phenylalanine or valine (ae) Substitution of amino acid 95 with aspartic acid (af) Substitution of amino acid 96 with phenylalanine, valine, or isoleucine (ag) Substitution of amino acid 125 to serine (ah) Substitution of amino acid 126 to threonine

[0029] The long-acting conjugate according to any of the above-mentioned specific examples is characterized in that it comprises an interleukin-2 analog having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-106.

[0030] The long-acting conjugate according to any of the above embodiments is characterized in that it comprises an interleukin 2 analogue, which 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 amino acids at the 125th, 38th, 42nd, 80th, 81st, and 92nd positions 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 amino acids at the 125th, 80th, 81st, 85th, 86th, and 92nd positions 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 amino acids at the 125th, 18th, 22nd, 80th, 81st, 85th, 86th, and 92nd positions 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 amino acids at the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, and 92nd positions are replaced with other amino acids; (e) 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; (f) 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;

[0031] The long-acting conjugate according to any of the above-mentioned embodiments is characterized in that it comprises an interleukin-2 analog having at least one amino acid substitution selected from the group consisting of the following amino acid substitutions: (a) Substitution of amino acid 18 with arginine (b) Substitution of amino acid 22 with glutamic acid (c) Substitution of amino acid 38 with alanine (d) Substitution of amino acid 42 with lysine (e) Substitution of amino acid 61 with aspartic acid (f) Substitution of amino acid 68 with aspartic acid (g) Substitution of amino acid 80 with phenylalanine (h) Substitution of amino acid 81 with glutamic acid (i) Substitution of amino acid 85 with valine (j) Substitution of amino acid 86 with valine (k) Substitution of amino acid 92 with phenylalanine (l) Substitution of amino acid 125 with serine

[0032] The long-acting conjugate according to any of the above embodiments is characterized in that it comprises an immunoglobulin Fc region that is derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof.

[0033] The long-acting conjugate according to any of the above embodiments is characterized in that it comprises an IgG4 Fc region.

[0034] The long-acting conjugate according to any of the above embodiments is characterized in that it comprises an aglycosylated immunoglobulin Fc region.

[0035] In the long-acting conjugate according to any of the above-mentioned embodiments, the immunoglobulin Fc region is characterized in that it is a non-glycosylated Fc region derived from human IgG4.

[0036] In any of the above-mentioned specific examples of the long-acting conjugate, the immunoglobulin Fc region is characterized in that it has a structure in which two polypeptide chains are linked by disulfide bonds, and is linked only via a nitrogen atom of one of the two chains.

[0037] In a long-acting conjugate according to any of the above-mentioned embodiments, the immunoglobulin Fc region is characterized in that it comprises a monomer having the amino acid sequence of SEQ ID NO: 438.

[0038] In any of the above-mentioned embodiments, the immunoglobulin Fc region is characterized in that it is a homodimer consisting of monomers of the amino acid sequence of SEQ ID NO: 438.

[0039] In the long-acting conjugate according to any of the above-mentioned embodiments, the immunoglobulin Fc region is characterized in that it is linked via the nitrogen atom of its N-terminal proline.

[0040] In any of the above specific examples of the long-acting conjugate, L contains an ethylene glycol repeating unit, and the chemical formula weight of the ethylene glycol repeating unit moiety is in the range of 1 to 100 kDa.

[0041] The long-acting conjugate according to any of the above specific examples is characterized in that one molecule of X is covalently linked to any Fc region of the dimeric immunoglobulin Fc region via the polyethylene glycol linker.

[0042] In any of the above-mentioned specific examples of the long-acting conjugate, the ethylene glycol repeating unit is [OCH2CH2]n, where n is a natural number, and is characterized in that the average molecular weight, for example the number average molecular weight, of the [OCH2CH2]n portion in the interleukin 2 analogue conjugate is determined to be 1 to 100 kDa.

[0043] In a long-acting conjugate according to any of the above-mentioned specific examples, the value of n is characterized in that the average molecular weight, for example the number average molecular weight, of the [OCH2CH2]n portion in the interleukin 2 analogue conjugate is determined to be 3.4 kDa.

[0044] In any of the specific examples of the long-acting conjugates described above, a is 1, and one molecule of X is covalently linked to any Fc region chain of the dimeric immunoglobulin Fc region via the linker containing the ethylene glycol repeat unit.

[0045] The long-acting conjugate according to any one of the above-mentioned specific examples is characterized in that one end of the linker is linked only to one of the two Fc domain chains of the dimeric immunoglobulin Fc domain.

[0046] In the persistent conjugate according to any of the above-mentioned embodiments, the conjugate is characterized in that one end of L is linked to F and X by covalent bonds formed by reaction of one end of L with an amino group or a thiol group of F and the other end of L with an amino group or a thiol group of X, respectively.

[0047] Another embodiment of the present invention is a long-acting conjugate of an interleukin-2 analogue represented by chemical formula (2).

[0048] XZ-Fc···(2)

[0049] Here, X is an interleukin 2 analogue comprising any one of the polypeptide sequences selected from the amino acid sequences of SEQ ID NOs: 3 to 106, Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa, Fc is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X and Z and between Z and Fc. The long-acting conjugate is one in which one end of Z is covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X is covalently linked to the opposite end of Z.

[0050] 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.

[0051] In another 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, 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.

[0052] The long-acting conjugate according to any of the above-mentioned embodiments is characterized in that it comprises an interleukin-2 analog having any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105 and 106.

[0053] The interleukin-2 analogue of the long-acting conjugate according to any of the above-mentioned embodiments is characterized in that it further comprises at least one amino acid at the C-terminus.

[0054] Yet another embodiment of the present invention is a long-acting conjugate of an interleukin-2 analogue represented by chemical formula (3).

[0055] X'-Z-Fc...(3)

[0056] Here, X' is an interleukin 2 analogue comprising an amino acid sequence represented by general formula 1, Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa, Fc is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X' and Z and between Z and Fc. The long-acting conjugate is one in which one end of Z is covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X' is covalently linked to the opposite end of Z.

[0057] [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)

[0058] 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).

[0059] As a 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).

[0060] In another embodiment, the interleukin-2 analogue is characterized in that it further comprises at least one amino acid at the C-terminus.

[0061] Yet another embodiment of the present invention is a long-acting conjugate of an interleukin-2 analogue represented by chemical formula (4).

[0062] X''-Z-Fc...(4)

[0063] Here, X″ is an interleukin 2 analogue comprising an amino acid sequence represented by general formula 2, Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa, Fc is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X″ and Z and between Z and Fc. The long-acting conjugate is one in which one end of Z is covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X″ is covalently linked to the opposite end of Z.

[0064] [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)

[0065] 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).

[0066] 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.

[0067] Yet another aspect of the present invention is a method for producing a long-acting conjugate having an interleukin-2 analog, comprising the step of linking the interleukin-2 analog to an immunoglobulin Fc region via a non-peptidyl polymer.

[0068] In one embodiment, the interleukin-2 analogue is characterized in that 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 of naturally occurring interleukin-2 is mutated.

[0069] Yet another embodiment of the present invention is a pharmaceutical composition for preventing or treating cancer, comprising an interleukin-2 analog or a long-acting conjugate thereof.

[0070] In one embodiment, the pharmaceutical composition comprises a pharma- ceutical effective amount of a long-acting conjugate of an interleukin-2 analogue and a pharma- ceutical acceptable excipient.

[0071] In another embodiment, the pharmaceutical composition of the interleukin 2 analogue is characterized by comprising 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 natural interleukin 2 is mutated.

[0072] The pharmaceutical composition according to any of the above-mentioned embodiments is characterized in that it contains an interleukin-2 analogue having increased interleukin-2β receptor binding affinity compared to aldesleukin.

[0073] In the pharmaceutical composition according to any of the above-mentioned embodiments, the interleukin-2 analogue is characterized in that it is selected from the group consisting of SEQ ID NOs: 3-106.

[0074] In the pharmaceutical composition according to any of the above-mentioned embodiments, the interleukin-2 analogue is selected from the group consisting of SEQ ID NOs: 22, 42, 53, 87, 105 and 106.

[0075] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the long-acting conjugate is characterized by being represented by chemical formula (1).

[0076] X-La-F···(1)

[0077] Here, X is an interleukin 2 analogue having enhanced interleukin 2β receptor binding affinity compared to aldesleukin, L is a polyethylene glycol linker, a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others, F is an immunoglobulin Fc region in a dimeric form, and - indicates a covalent bond between X and L and between L and F.

[0078] In any of the above-mentioned specific examples of the pharmaceutical composition, the cancer is characterized in that it is any one selected from the group consisting of metastatic renal cell carcinoma, metastatic melanoma, colon cancer, liver cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, kidney cancer, lung cancer, skin cancer, melanoma, breast cancer, bladder cancer and gastric cancer.

[0079] In a pharmaceutical composition according to any of the above-mentioned embodiments, the composition is characterized by exhibiting increased serum exposure, tumor growth inhibition, and / or memory T cell responses compared to aldesleukin.

[0080] The pharmaceutical composition according to any of the above-mentioned embodiments, wherein the pharmaceutical composition is administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary or rectal administration routes.

[0081] In the pharmaceutical composition according to any of the above-mentioned embodiments, the pharmaceutical composition is characterized in that it is administered at a time interval ranging from one week to one month.

[0082] Yet another aspect of the present invention is a method for preventing or treating cancer, comprising the step of administering to an individual the interleukin-2 analogue, a long-acting conjugate thereof, or a composition containing same.

[0083] Yet another aspect of the present invention is the use of the interleukin-2 analogue, its long-acting conjugate, or a composition containing same for the prevention or treatment of cancer.

[0084] Yet another aspect of the present invention is the use of the interleukin-2 analog or its long-acting conjugate to provide a medicament for the prevention or treatment of cancer.

[0085] Hereinafter, the mode for carrying out the present invention will be described. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining the state of the art to which the present invention belongs and the contents of the present invention.

[0086] 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

[0087] In this specification, "Aib" is used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid," and 2-aminoisobutyric acid and aminoisobutyric acid are used interchangeably.

[0088] One aspect of the present invention provides a long-acting conjugate comprising an interleukin-2 analogue.

[0089] Specifically, the persistent conjugate may be represented by chemical formula (1).

[0090] X-La-F···(1)

[0091] Here, X is an interleukin 2 analog having enhanced interleukin 2β receptor binding affinity compared to aldesleukin, L is a polyethylene glycol linker, a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others, F is an immunoglobulin Fc region in a dimeric form, and - indicates a covalent bond between X and L and between L and F.

[0092] The interleukin 2 analogue of the long-acting conjugate of the present invention is characterized in that, when it is not part of a conjugate and exists alone, it has an altered binding ability to the interleukin 2 receptor, in particular an increased binding ability to the interleukin 2β receptor. Specifically, when it is not part of a conjugate and exists alone, the interleukin 2 analogue of the present invention may have an increased binding ability to the interleukin 2β receptor compared to natural interleukin 2 or a known aldesleukin, and more specifically, it may have an altered (increased or decreased) binding ability to the interleukin 2α receptor and may include a sequence in which at least one amino acid in natural interleukin 2 has been mutated.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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-2 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.

[0101] 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.

[0102] 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.

[0103] Therefore, the interleukin-2 analogue of the present invention having increased binding strength to the interleukin-2β receptor has the effect of increasing therapeutic effects such as tumor inhibition and tumor killing, and reducing side effects.

[0104] 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.

[0105] An example of the interleukin-2 analog of the present invention is one in which at least one amino acid has been added to the amino acid corresponding to position 133, but the present invention 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 containing chemical modifications may be added.

[0106] 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.

[0107] 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.

[0108] As 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 at least one of the amino acids corresponding to the 18th, 22nd, 38th, 42nd, 61st, 68th, 80th, 81st, 85th, 86th, and 92nd positions further replaced with another amino acid, but is not limited thereto.

[0109] 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, 42nd, 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. (bo) 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, 86th, and 92nd positions are replaced with other amino acids. (bp) 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, and 86th positions are replaced with other amino acids.

[0110] 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 94 with phenylalanine or valine (ae) Substitution of amino acid 95 with aspartic acid (af) Substitution of amino acid 96 with phenylalanine, valine, or isoleucine (ag) Substitution of amino acid 125 to serine (ah) Substitution of amino acid 126 to threonine

[0111] As yet another example, the interleukin 2 analogue may be any one selected from the following interleukin 2 analogues: (a) 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; (b) 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; (c) 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; (d) 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; (e) 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; (f) 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;

[0112] 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 18 with arginine (b) Substitution of amino acid 22 with glutamic acid (c) Substitution of amino acid 38 with alanine (d) Substitution of amino acid 42 with lysine (e) Substitution of amino acid 61 with aspartic acid (f) Substitution of amino acid 68 with aspartic acid (g) Substitution of amino acid 80 with phenylalanine (h) Substitution of amino acid 81 with glutamic acid (i) Substitution of amino acid 85 with valine (j) Substitution of amino acid 86 with valine (k) Substitution of amino acid 92 with phenylalanine (l) Substitution of amino acid 125 with serine

[0113] "Corresponding to" herein means the amino acid residue at the recited position in the peptide, or an amino acid residue similar, identical or equivalent to the recited residue in the peptide. Identifying the amino acid at the corresponding position determines the particular amino acid of the sequence to which the particular sequence refers.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] Furthermore, even if the term "interleukin 2 analog consisting of a specific sequence number" is used in this specification, it does not exclude meaningless sequence additions before or after the amino acid sequence of the sequence number, naturally occurring mutations, or silent mutations thereof, 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 invention.

[0119] 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.

[0120] As a specific example, the interleukin 2 analog of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 41, 52, 86, 104, and 105, but is not limited to these.

[0121] 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 is expressed as a percentage.

[0122] 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 will 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.

[0123] Homology and identity are often used interchangeably.

[0124] 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.

[0125] 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, pp. 1171-1175. 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 include (1) a binary 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 10 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (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.

[0126] The above also applies to other embodiments or aspects of the present invention, but is not limited thereto.

[0127] The interleukin 2 analog of the present invention is used as a new alternative to interleukin 2, which changes the in vitro activity by decreasing 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 is used as an effective therapeutic agent due to its activity against both receptors.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] Amino acid derivatives are similarly available, such as 4-imidazoacetic acid.

[0132] Furthermore, the interleukin 2 analogue of the present invention may be in a form in which its N-terminus and / or C-terminus, etc. are chemically modified in order to protect it from protein-cleaving enzymes in the body and improve its stability, or may be in a form in which it is protected by an organic group, or may be in a form in which an amino acid is added to the peptide terminus, etc.

[0133] 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.

[0134] The interleukin-2 analogs of the present invention can be synthesized by solid phase synthesis, produced by recombinant methods, or made commercially, but are not limited to these.

[0135] Furthermore, the interleukin 2 analog of the present invention can be synthesized by a method well known in the art depending on its length, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0136] Specifically, the interleukin 2 analogs of the present invention can be produced by standard synthesis methods, recombinant expression systems, or any other method known in the art. Thus, the interleukin 2 analogs of the present invention can be synthesized in many ways, including, for example, (a) synthesizing the peptide stepwise in solid or liquid phase methods or by fragment assembly, and isolating and purifying the final peptide product, (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture, (c) expressing a nucleic acid construct encoding the peptide in a cell-free test tube and recovering the expression product, or any combination of (a), (b), and (c) to obtain fragments of the peptide, and then recovering the peptide by ligating the fragments to obtain the peptide.

[0137] In the present invention, the binding strength of any interleukin-2 analogue (or a long-acting conjugate containing it) to a native interleukin-2 receptor can be measured using SPR (surface plasmon resonance) as a method for measuring affinity to the receptor.

[0138] Specifically, examples of SPR analysis include, but are not limited to, a method in which an interleukin-2 receptor is immobilized on a sensor chip using the principle of protein-ligand binding, an interleukin-2 analogue diluted in an experimental buffer solution by serial dilution is injected to induce binding with the immobilized receptor, and then only the experimental buffer solution is injected at the same flow rate to induce dissociation between the receptor and the interleukin-2 analogue, and the binding strength is measured; and a method in which an antibody against the human immunoglobulin Fc region is first immobilized on a sensor chip, then the interleukin-2 receptor bound to the Fc region is immobilized, an interleukin-2 analogue is injected, and the binding strength is measured.

[0139] More specifically, biotin-labeled human interleukin-2 receptor is immobilized on a streptavidin biosensor chip, and an interleukin-2 analogue long-acting conjugate diluted in HBS-EP+ buffer by two-fold serial dilution is injected for 3 minutes at a flow rate of 20 μL per minute, and then HBS-EP+ buffer alone is injected for 3 minutes at the same flow rate to induce dissociation between the interleukin-2 receptor and the interleukin-2 analogue long-acting conjugate. The binding strength can then be measured from the obtained binding constant and dissociation constant using a 1:1 binding fitting model using the Bioaevaluation program, but is not limited to this.

[0140] More specifically, the interleukin-2 analogs of the present invention may have decreased or increased interleukin-2α receptor binding affinity compared to native interleukin-2 or an interleukin-2 analog (aldesleukin; or interleukin-2 analog 1).

[0141] 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 analog whose binding strength has been changed compared to natural interleukin 2 or aldesleukin is included in the present invention.

[0142] 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 has been changed compared to that of natural interleukin 2 or aldesleukin is included in the present invention.

[0143] 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 has changed or increased compared to natural interleukin 2 or aldesleukin are included in the present invention.

[0144] 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 natural interleukin 2 or aldesleukin is included in the present invention.

[0145] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about."

[0146] The interleukin 2 analogues of the present invention are characterized by an altered binding affinity to the interleukin 2α receptor and an increased binding affinity to the interleukin 2β receptor, as compared to native interleukin 2 or aldesleukin.

[0147] In a specific embodiment of the present invention, in order to prepare the interleukin 2 analog of the present invention, an interleukin 2 analog was prepared by introducing a mutation into natural interleukin 2 (SEQ ID NO: 1). The interleukin 2 analog prepared 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.

[0148] 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.

[0149] 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, 98% or more, and 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, 98% or more, and less than 100% homology or identity to any of the sequences of SEQ ID NOs: 108 to 211, but is not limited thereto.

[0150] 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 11 and 12).

[0151] 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, adenine 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.

[0152] 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 11).

[0153] The homology or identity is as described above.

[0154] Furthermore, the interleukin 2 analog of the present invention has a half-life extended in vivo compared to natural interleukin or aldesleukin, but is not particularly limited thereto. For example, a biocompatible substance for extending the half-life (e.g., immunoglobulin Fc region) is bound to the interleukin 2 analog directly or via a linker, and the half-life is extended in the form of a long-acting conjugate, but is not limited thereto.

[0155] The long-acting conjugate of the present invention not only contains an interleukin-2 analog with improved binding strength to the interleukin-2β receptor, but also binds to the immunoglobulin Fc region as a representative carrier for extending its half-life, thereby extending the half-life of the interleukin-2 analog, increasing its exposure in the blood, and increasing the immune response in the body, thereby effectively achieving the inhibition and reduction of cancer cell growth.

[0156] Another embodiment of the present invention for carrying out the present invention provides a long-acting conjugate of an interleukin-2 analogue.

[0157] In the present invention, the long-acting interleukin-2 analogue conjugate may be in a form in which a biocompatible substance for extending the in vivo half-life is bound to the interleukin-2 analogue. The biocompatible substance in the present specification is mixed with a carrier.

[0158] The long-acting conjugate of the present invention has a longer-lasting efficacy than an interleukin-2 analog that is not conjugated to a carrier, and such a conjugate is referred to as a "long-acting conjugate" or "conjugate" in the present invention.

[0159] Furthermore, such a conjugate may be non-naturally occurring.

[0160] In one embodiment of the present invention, the persistent conjugate is a persistent conjugate represented by chemical formula (1).

[0161] X-La-F···(1)

[0162] Here, X is an interleukin 2 analog having enhanced interleukin 2β receptor binding affinity compared to aldesleukin, L is a polyethylene glycol linker, a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others, F is an immunoglobulin Fc region in a dimeric form, and the "-" indicates a covalent bond.

[0163] More specifically, X and L, and L and F may be linked to each other by a covalent bond, and the conjugate is a conjugate in which X, L, and F are linked to each other by a covalent bond in the order of chemical formula (1).

[0164] Furthermore, the F may be directly linked to X (that is, a is 0 in chemical formula (1)) or may be linked via a linker (L).

[0165] The interleukin 2 analog in the present invention is a component that forms a part of the conjugate. Specifically, it corresponds to X in chemical formula (1). The interleukin 2 analog is as described above.

[0166] F in said conjugate is an agent that extends the half-life of X, the interleukin 2 analogue, and is an integral part of the conjugate of the invention.

[0167] The F's may be bonded to X by a covalent chemical bond, or may be bonded to X via L by a covalent chemical bond.

[0168] Specifically, the F is an immunoglobulin Fc region, and the immunoglobulin Fc region is an IgG Fc region or a non-glycosylated IgG4 Fc region, but is not particularly limited thereto.

[0169] As a specific example of the present invention, the F (immunoglobulin Fc region) is a dimer consisting of two polypeptide chains, and has a structure in which one end of L is linked to only one of the two polypeptide chains, but is not limited to this.

[0170] At least one amino acid side chain in the peptides of the invention is conjugated to such biocompatible substances to improve solubility and / or half-life in vivo and / or increase bioavailability. Such modifications also reduce the clearance of therapeutic proteins and peptides.

[0171] The biocompatible materials mentioned above may be water soluble (amphiphilic or hydrophilic), non-toxic, and / or pharma- ceutical acceptable.

[0172] The F may be directly linked to X (i.e., a is 0 in chemical formula (1)) or may be linked via a polyethylene glycol linker (L).

[0173] In a specific embodiment, the long-acting conjugate of the present invention is, but is not limited to, an interleukin-2 analog linked to an immunoglobulin Fc region.

[0174] In the present invention, the "immunoglobulin Fc region" refers to a region excluding the variable regions of the heavy and light chains of an immunoglobulin, and including the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3). The immunoglobulin Fc region may be a component constituting a part of the conjugate of the present invention. Specifically, it corresponds to F in chemical formula (1).

[0175] As used herein, the Fc region includes not only the native sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, such as variants in which at least one amino acid residue in the native sequence is deleted, inserted, non-conservative or conservatively substituted, or a combination thereof to produce a sequence different from the native sequence. The derivatives, substitutions, and variants are premised on having the ability to bind to FcRn. In the present invention, F is a human immunoglobulin region, but is not limited thereto. As used herein, the term "biocompatible substance" or "carrier" refers to the Fc region.

[0176] The F (immunoglobulin Fc region) has a structure in which two polypeptide chains are linked by a disulfide bond, and is linked only via a nitrogen atom of one of the two chains, but is not limited thereto. The linkage via the nitrogen atom may be achieved by linkage to the ε-amino atom of lysine or the amino group at the N-terminus by reductive amination.

[0177] Reductive amination refers to a reaction in which an amine group or amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to produce an amine, which is then reduced to form an amine bond, and is an organic synthesis reaction well known in the art.

[0178] As a specific example of the long-acting conjugate of the present invention, the immunoglobulin Fc region may be linked to a linker via its N-terminal nitrogen atom.

[0179] Such an immunoglobulin Fc region includes, but is not limited to, a hinge portion in a heavy chain constant region.

[0180] The immunoglobulin Fc region of the present invention may comprise a specific hinge sequence at the N-terminus.

[0181] The term "hinge sequence" as used herein refers to a site located in a heavy chain that forms a dimer of an immunoglobulin Fc region through inter disulfide bonds.

[0182] In the present invention, the hinge sequence is a mutated hinge sequence having the following amino acid sequence, in which a portion of the hinge sequence has been deleted to have only one cysteine ​​residue, but is not limited thereto. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (sequence number 418).

[0183] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 418 is deleted, leaving only one cysteine ​​residue. The hinge sequence of the present invention is one that contains only one cysteine ​​residue and is composed of 3 to 12 amino acids, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 419) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 420) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 421) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 422) Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 423) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 424) Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 425) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 426) Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 427) Pro-Ser-Cys-Pro (SEQ ID NO: 428) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 429) Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 430) Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 431) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 432) Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 433) Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 434) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 435) Glu-Pro-Ser-Cys (SEQ ID NO: 436) Ser-Cys-Pro (SEQ ID NO: 437)

[0184] More specifically, the hinge sequence includes, but is not limited to, the amino acid sequence of SEQ ID NO: 428 (Pro-Ser-Cys-Pro) or SEQ ID NO: 437 (Ser-Cys-Pro).

[0185] In a more specific embodiment of the long-acting conjugate of the present invention, the N-terminus of the immunoglobulin Fc domain in the conjugate is proline, and the Fc domain is linked to a linker via the nitrogen atom of the proline.

[0186] In one embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region may be in the form of a dimer in which two chains of the immunoglobulin Fc region form a homodimer or a heterodimer due to the presence of a hinge sequence. The conjugate of the present invention represented by chemical formula (1) is in the form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0187] In the present invention, the "N-terminus" refers to the amino terminus of a protein or polypeptide, and includes the extreme amino terminus, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the extreme amino terminus. The immunoglobulin Fc region of the present invention includes a hinge sequence at the N-terminus, but is not limited thereto.

[0188] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes a part or the whole of heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) excluding the variable regions of the heavy and light chains of an immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of a natural one. Furthermore, it may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0189] For example, the immunoglobulin Fc region of the present invention is, but is not limited to, 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of at least one of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a portion of a hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0190] The immunoglobulin Fc region of the present invention is in the form of a dimer or multimer composed of single-chain immunoglobulins consisting of domains of the same origin, but is not limited thereto.

[0191] In one embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, and the Fc region dimer F and X are covalently linked via one and the same linker L containing an ethylene glycol repeat unit. In a specific example of this embodiment, X is covalently linked via the linker L to only one of the two polypeptide chains of the Fc region dimer F. In a more specific example of this embodiment, only one molecule of X is covalently linked via L to one of the polypeptide chains to which X is linked, among the two polypeptide chains of the Fc region dimer F. In the most specific example of this embodiment, F is a homodimer.

[0192] In another specific example, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains, but is not limited to this.

[0193] In another embodiment of the long-acting conjugate of the present invention, two molecules of X may be symmetrically bound to one Fc region in a dimeric form, where the immunoglobulin Fc region and X may be linked to each other via L. However, the present invention is not limited to the above example.

[0194] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means a sequence in which at least one amino acid residue of the naturally occurring amino acid sequence is different due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0195] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, are used as sites suitable for modification.

[0196] Various derivatives are also used, such as derivatives in which the site forming disulfide bonds has been removed, derivatives in which several amino acids have been deleted from the N-terminus of native Fc, and derivatives in which a methionine residue has been added to the N-terminus of native Fc. Furthermore, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in Patent Documents 2 and 3, etc.

[0197] Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (Non-Patent Document 13). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications may be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0198] The described Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have improved structural stability against heat, pH, and the like of the Fc region.

[0199] In addition, such an Fc region may be obtained from a natural source isolated from the living body of an animal such as a human or a cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, or may be a recombinant product obtained from a transformed animal cell or a microorganism, or a derivative thereof. Here, the method for obtaining it from a natural source may be a method for obtaining it by isolating the whole immunoglobulin from the living body of a human or animal and then treating it with a protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. These can be separated into Fc or pF'c using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0200] Furthermore, the immunoglobulin Fc region may have a natural sugar chain, an increased sugar chain compared to the natural one, a decreased sugar chain compared to the natural one, or a form in which the sugar chain has been removed. For increasing, decreasing, or removing such immunoglobulin Fc sugar chains, conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms are used. Here, an immunoglobulin Fc region from which the sugar chains have been removed from Fc has a significantly reduced binding ability to complement (c1q), and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. For these reasons, an immunoglobulin Fc region from which the sugar chains have been removed or which has been deglycosylated is in a form suitable for its original purpose as a drug carrier.

[0201] In the present invention, "deglycosylation" refers to an Fc region from which sugar chains have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0202] On the other hand, the immunoglobulin Fc region is of human origin or animal origin, such as bovine, goat, porcine, mouse, rabbit, hamster, rat, guinea pig, and in a more specific embodiment, of human origin.

[0203] The immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, or may be an Fc region derived from a combination of these or a hybrid of these. In a more specific embodiment, it is derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to extend the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0204] As a specific example, the immunoglobulin Fc region may be a fragment of human IgG4 Fc and may be a homodimer in which two monomers are linked by a disulfide bond (inter-chain form) between the third amino acid cysteines of each monomer, and here the homodimer has / may have disulfide bonds between the 35th and 95th cysteines and between the 141st and 199th cysteines in each monomer, i.e., two disulfide bonds (intra-chain form).

[0205] Each monomer is composed of 221 amino acids, and the amino acids forming the homodimer are composed of a total of 442 amino acids, but are not limited thereto. Specifically, the immunoglobulin Fc fragment is a homodimer formed by disulfide bonds between the cysteines at the third amino acids of each monomer, and the monomers of the homodimer independently form internal disulfide bonds between the 35th and 95th cysteines and between the 141st and 199th cysteines, but are not limited thereto.

[0206] F in formula (1) includes a monomer having the amino acid sequence of SEQ ID NO: 438, and F is a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 438, but is not limited thereto.

[0207] As an example, but not limited to, the immunoglobulin Fc region is a homodimer comprising the amino acid sequence of SEQ ID NO: 439 (consisting of 442 amino acids).

[0208] In one non-limiting example, the immunoglobulin Fc region and X are aglycosylated.

[0209] On the other hand, the term "combination" in the present invention means that, when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin is bound to a single-chain polypeptide of a different origin, i.e., a dimer or multimer can be prepared from at least two fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0210] In the present invention, the term "hybrid" means that sequences corresponding to immunoglobulin Fc fragments of at least two different origins are present in a single-chain immunoglobulin constant region. In the present invention, various forms of hybrids are possible. That is, hybrids of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include a hinge.

[0211] On the other hand, IgG is also divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations or hybridizations of these are also possible. Specifically, these are the IgG2 and IgG4 subclasses, and more specifically, the Fc region of IgG4, which has almost no effector functions such as complement dependent cytotoxicity (CDC).

[0212] Furthermore, the above-mentioned conjugates have improved efficacy over natural interleukin 2 or aldesleukin, or over X where F is not modified, and such conjugates include, but are not limited to, all of the above-mentioned forms as well as forms encapsulated in biodegradable nanoparticles.

[0213] In formula (1), L may be a polyethylene glycol linker.

[0214] The "polyethylene glycol linker" of the present invention includes a biocompatible polymer having at least two ethylene glycol repeating units bound thereto. The repeating units are linked to each other by any covalent bond except a peptide bond. The polyethylene glycol linker is one component that forms a part of the conjugate of the present invention, and corresponds to L in chemical formula (1). In the present specification, the linker is also referred to as a "non-peptidic linker" or a "non-peptidic polymer".

[0215] In the above La, a may be 1 or more, and when a is 2 or more, each L may be independent.

[0216] In a specific embodiment, the conjugate may be one in which F and X are covalently linked to each other via a non-peptide linker that includes reactive groups at both ends that are bonded to F (specifically, an immunoglobulin Fc region) and X (specifically, an interleukin 2 analog).

[0217] Specifically, the non-peptidic linker in the present invention contains a reactive group at its terminal and can form a conjugate by reacting with other components constituting the conjugate. When a non-peptidic linker having reactive functional groups at both terminals forms a conjugate by binding to X and F in chemical formula (1) via each reactive group, the non-peptidic linker or non-peptidic polymer is also called a non-peptidic polymer linker moiety or a non-peptidic linker linker moiety.

[0218] In a specific embodiment, the L (polyethylene glycol linker) is a linker containing an ethylene glycol repeat unit, such as, but not limited to, polyethylene glycol. The polyethylene glycol in this specification includes, but is not limited to, ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG). In addition, derivatives thereof known in the art and derivatives that can be easily prepared at the technical level in the art are also included in the present invention.

[0219] The polyethylene glycol linker may contain an ethylene glycol repeating unit and a functional group at its end that is used for preparing a conjugate until it is configured as a conjugate. The long-lasting conjugate according to the present invention has a form in which X and F are linked via the functional group, but is not limited thereto. The non-peptidic linker according to the present invention contains two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0220] Specifically, the linker is polyethylene glycol (PEG) represented by chemical formula (5), but is not limited thereto.

[0221] TIFF2024512418000002.tif2027...(5)

[0222] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited to these.

[0223] The PEG portion of the long-acting conjugate is -(CH2CH2O). n -Not only the structure, but also the linking elements and their -(CH2CH2O) n - This includes, but is not limited to, an intervening oxygen atom.

[0224] As a specific example, the ethylene glycol repeating unit is represented by [OCH2CH2]n, where the n value is a natural number and is determined so that the average molecular weight of the [OCH2CH2]n portion in the interleukin 2 analogue conjugate, for example the number average molecular weight, is greater than 0 to approximately 100 kDa, but is not limited thereto. As another specific example, the n value is a natural number, and the average molecular weight, for example, the number average molecular weight, of the [OCH2CH2]n portion in the interleukin 2 analogue conjugate is about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa, about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, About 3 to about 15 kDa, about 3 to about 13 kDa, about 3 to about 11 kDa, about 3 to about 10 kDa, about 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.4 kDa, about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa Da, about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9.5 to about 10.5 kDa, or about 10 kDa, but is not limited to these.

[0225] In one specific embodiment, the conjugate has a structure in which an interleukin-2 analog and an immunoglobulin Fc region (F) are covalently linked via a linker (L) containing an ethylene glycol repeat unit, but is not limited thereto.

[0226] In another specific embodiment of the long-acting conjugate, L may be a linker containing an ethylene glycol repeat unit, a may be 1, and F may be a dimeric immunoglobulin Fc region. More specifically, one molecule of X is covalently linked to any one of the Fc regions of the dimeric immunoglobulin Fc region via the linker containing an ethylene glycol repeat unit, but is not limited thereto. In yet another specific embodiment, one end of the linker containing an ethylene glycol repeat unit is linked only to any one of the two Fc region chains of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0227] The molecular weight of the polyethylene glycol linker used in the present invention is in the range of more than 0 to 200 kDa, specifically about 1 to 100 kDa, about 1 to 50 kDa, about 1 to 30 kDa, about 2 to 30 kDa, about 1 to 20 kDa, more specifically about 3.4 kDa to 10 kDa, and even more specifically about 3.4 kDa, but is not limited thereto. Furthermore, the non-peptidic linker of the present invention bound to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0228] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0229] Specifically, the non-peptide linker may have reactive groups at both ends when not bound to F and X, and be bound to F and X via the reactive groups.

[0230] In one specific example, both ends of the linker are bound to a thiol group, an amino group, or a hydroxyl group of an immunoglobulin Fc region, and a thiol group, an amino group, an azide group, or a hydroxyl group of an interleukin-2 analog (X), but this is not limited thereto.

[0231] Specifically, the linker has reactive groups at both ends which are bonded to an immunoglobulin Fc region and an interleukin 2 analog (X), respectively, more specifically, a reactive group bonded to at least one selected from the group consisting of a thiol group of cysteine ​​in the immunoglobulin Fc region, an amino group located at the N-terminus, lysine, arginine, glutamine and / or histidine, and a hydroxyl group located at the C-terminus, and a reactive group bonded to at least one selected from the group consisting of a thiol group of cysteine, an amino group of lysine, arginine, glutamine and / or histidine, an azide group of azidolysine, and a hydroxyl group of the interleukin 2 analog (X), but is not limited thereto.

[0232] More specifically, the reactive group of the linker is at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0233] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0234] In the above, examples of succinimide derivatives include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate.

[0235] The linker may be linked to an immunoglobulin Fc region, F, and an interleukin 2 analog, X, via the reactive groups described above, to form a linker linker.

[0236] Furthermore, the final products generated by reductive alkylation with aldehyde bonds are much more stable than those linked with amide bonds: the aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​e.g., pH 9.0.

[0237] The reactive groups at both ends of the linker may be the same or different. For example, the linker may have aldehyde groups at both ends, or may have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. However, the linker is not particularly limited thereto as long as F, specifically, the immunoglobulin Fc region and X are bound to each end of the linker.

[0238] For example, the linker may contain a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like at the other end.

[0239] When polyethylene glycol having hydroxy reactive groups at both ends is used as a linker, the hydroxy groups can be activated into various reactive groups as described above by known chemical reactions, or a commercially available polyethylene glycol having modified reactive groups can be used to prepare the long-acting interleukin-2 analogue conjugate of the present invention.

[0240] In a specific, but non-limiting, embodiment, the linker is linked to a cysteine ​​residue of X, more specifically to the -SH group of cysteine.

[0241] Specifically, the reactive group of the linker may be linked to the -SH group of the cysteine ​​residue. The reactive group is as described above. When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X by a thioether bond, and the aldehyde group can be linked to F, specifically, the -NH2 group of immunoglobulin Fc by reductive amination, but is not limited thereto.

[0242] In another specific, non-limiting embodiment, the linker is linked to a lysine residue of X, more specifically to the amino group of a lysine.

[0243] In the conjugate, the reactive group of the linker may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but is not limited thereto.

[0244] Furthermore, in said conjugate, the interleukin-2 analogue according to the present invention may be linked via its C-terminus to a linker having a reactive group, but this is just one example.

[0245] The "C-terminus" in the present invention means the carboxy terminus of a peptide, and for the purposes of the present invention, means the position to be bound to a linker. For example, but not limited to, it includes not only the most terminal amino acid residue of the C-terminus, but also all amino acid residues around the C-terminus, specifically the 1st to 20th amino acid residues from the most terminal.

[0246] In addition, the above-mentioned conjugates have improved efficacy over X in which F is not modified, and such conjugates include not only the above-mentioned forms but also forms encapsulated in biodegradable nanoparticles.

[0247] Yet another embodiment of the present invention for achieving the present invention provides a long-acting conjugate in which an interleukin-2 analogue comprising any one of amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3 to 106 is linked to an immunoglobulin Fc region via a linker.

[0248] The interleukin-2 analog, the linker and the long-acting conjugate containing the same are as described above.

[0249] Yet another embodiment of the present invention for realizing the present invention provides a long-acting conjugate of an interleukin-2 analogue represented by chemical formula (2).

[0250] XZ-Fc···(2)

[0251] Here, X is an interleukin 2 analogue comprising any one of the polypeptide sequences selected from the amino acid sequences of SEQ ID NOs: 3 to 106, Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa, Fc is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X and Z and between Z and Fc. The long-acting conjugate is one in which one end of Z is covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X is covalently linked to the opposite end of Z.

[0252] The interleukin-2 analog, linker, Fc region and long-acting conjugate containing them are as described above.

[0253] In a specific embodiment, 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 to these.

[0254] In another specific embodiment, 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, 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, but is not limited to these.

[0255] In yet another specific embodiment, 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: 22, 42, 53, 87, 105, and 106, but is not limited to these.

[0256] Furthermore, the interleukin 2 analogue further comprises at least one amino acid at the C-terminus, but is not limited thereto.

[0257] Yet another embodiment of the present invention for realizing the present invention provides a long-acting conjugate of an interleukin-2 analogue represented by chemical formula (3).

[0258] X'-Z-Fc (3)

[0259] Here, X' is an interleukin 2 analogue comprising an amino acid sequence represented by general formula 1, Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa, Fc is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X' and Z and between Z and Fc. The long-acting conjugate is one in which one end of Z is covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X' is covalently linked to the opposite end of Z.

[0260] [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)

[0261] 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).

[0262] The interleukin-2 analog, linker, Fc region and long-acting conjugate containing them are as described above.

[0263] In general formula 1, at least one amino acid is added to the threonine (T) corresponding to X133, but this is not limiting.

[0264] 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.

[0265] Such interleukin-2 analogs include, but are not limited to, aldesleukin or native interleukin-2, which have increased beta-receptor binding affinity.

[0266] In a specific embodiment, in general formula 1, X43 is lysine (K), X45 is tyrosine (Y), X61 is aspartic acid (D), glutamic acid (E) or glutamine (Q), X68 is glutamic acid (E), X74 is glutamine (Q), X80 is phenylalanine (F) or leucine (L), X85 is leucine (L), valine (V) or tyrosine (Y), X86 is isoleucine (I) or valine (V), and X92 is phenylalanine (F) or isoleucine (I), but is not limited thereto.

[0267] 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.

[0268] The interleukin-2 analogs of the present invention further comprise, but are not limited to, at least one amino acid at the C-terminus.

[0269] Yet another embodiment of the present invention for realizing the present invention provides a long-acting conjugate of an interleukin-2 analogue represented by chemical formula (4).

[0270] X''-Z-Fc...(4)

[0271] Here, X″ is an interleukin 2 analogue comprising an amino acid sequence represented by general formula 2, Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa, Fc is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X″ and Z and between Z and Fc. The long-acting conjugate is one in which one end of Z is covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X″ is covalently linked to the opposite end of Z.

[0272] [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)

[0273] 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).

[0274] The interleukin-2 analog, linker, Fc region and long-acting conjugate containing them are as described above.

[0275] 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.

[0276] 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.

[0277] Yet another embodiment of the present invention for carrying out the present invention provides a method for producing a long-acting conjugate comprising an interleukin-2 analogue.

[0278] The interleukin 2 analog and the long-acting conjugate containing the same are as described above.

[0279] Specifically, the method may include a step of linking an interleukin-2 analog having at least one amino acid mutation introduced into it selected from the group consisting of amino acids corresponding to the following positions in natural interleukin-2: 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, to a biocompatible substance (e.g., an immunoglobulin Fc region) via a linker (e.g., a non-peptidic polymer).

[0280] The method for producing a long-acting conjugate of the present invention may further include a 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 natural interleukin-2. However, the method is not limited to any particular step or its order as long as a long-acting conjugate in which the above-mentioned interleukin-2 analog according to the present invention is linked to a biocompatible substance (e.g., an immunoglobulin Fc region) is produced.

[0281] In addition, the method for producing a persistent conjugate of the present invention further includes, but is not limited to, 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.

[0282] The nucleic acid encoding the interleukin 2 analogue includes any one of the base sequences of SEQ ID NOs: 109 to 212, or is (essentially) composed of the base sequence, but is not limited thereto.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] Alternatively, an interleukin 2 analog prepared by peptide synthesis may be linked to a biocompatible substance (e.g., an immunoglobulin Fc region) via a non-peptide polymer. Since the interleukin 2 analog sequence of the present invention is provided, such peptide synthesis can be easily performed using known peptide synthesis methods.

[0292] Interleukin 2 analogues are as described above.

[0293] A further aspect of the present invention for carrying out the present invention provides a persistent conjugate produced by the above method.

[0294] Unless otherwise specified in the present specification, the detailed description and claims of the "interleukin 2 analog" or "conjugate" in which an interleukin 2 analog is covalently linked to a biocompatible substance according to the present invention apply to the interleukin 2 analog or conjugate itself, as well as to a category including all forms of salts of the interleukin 2 analog or conjugate (e.g., pharma- ceutically acceptable salts of the interleukin 2 analog) or solvates thereof. Thus, even if the specification only describes an "interleukin 2 analog" or "conjugate," the description also applies to a specific salt thereof, a specific solvate thereof, and a specific solvate of a specific salt thereof. These salt forms may be, for example, forms using any pharma- ceutically acceptable salt.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] Further, the term "solvate" in the present invention means a complex formed between the interleukin-2 analogue or a salt thereof according to the present invention and a solvent molecule.

[0299] Yet another embodiment of the present invention for realizing the present invention provides a composition comprising an interleukin 2 analog or a long-acting conjugate thereof. The interleukin 2 analog and the long-acting conjugate thereof are as described above. The composition according to the present invention may be a pharmaceutical composition, and in one specific form, it may be a pharmaceutical composition for use in preventing or treating cancer.

[0300] The composition according to the present invention may contain an interleukin 2 analog or a long-acting conjugate thereof, specifically, a pharmacologically effective amount of an interleukin 2 analog or a long-acting conjugate thereof, and may further contain a pharma- ceutically acceptable carrier.

[0301] Specific examples of the composition according to the present invention include interleukin-2 analogues having any amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 106, or long-acting conjugates containing the same, and more specifically, interleukin-2 analogues having any amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 42, 53, 87, 105 and 106, or long-acting conjugates containing the interleukin-2 analogues, but are not limited to these.

[0302] In the present invention, the term "pharmacologically effective amount" means a safe administration dose at which the interleukin 2 analog or its long-acting conjugate exerts a preventive or therapeutic effect against cancer and at the same time has no toxicity or side effects on the patient, and means an administration dose that shows activity on interleukin 2 receptors, specifically β and / or α receptors, but is not limited to these.

[0303] The composition according to the present invention may exhibit at least one of the following properties, but may be any property that exhibits an improved immune response, an anti-cancer effect, etc. based on increased binding ability to the interleukin 2β receptor. (i) Higher blood exposure compared to aldesleukin (ii) Tumor growth inhibition compared to aldesleukin (iii) Memory T cell generation response compared to aldesleukin

[0304] Interleukin 2, known as a T cell growth factor, is a protein involved in immune regulation, and has the activity of proliferating T cells, stimulating B cells, and acting on T cells to secrete gamma interferon. Based on the immune regulation activity of interleukin 2, cancer cells can be eliminated using the body's immune system, thereby providing preventive and therapeutic effects on cancer.

[0305] In particular, the interleukin 2 analog of the present invention has an increased binding strength to the interleukin 2β receptor, which plays a major role in signal transduction, and thus provides a more effective anti-cancer effect in the immune system of an individual. In addition, the long-acting conjugate containing the interleukin 2 analog has an increased binding strength to the interleukin 2β receptor, and as the duration increases, the blood exposure increases, showing excellent bioavailability, and ultimately has excellent tumor growth suppression and memory T cell generation capabilities, thereby providing an effective cancer prevention or treatment effect.

[0306] Specifically, the long-acting conjugates of interleukin-2 analogs of the present invention have completely lost their binding ability to interleukin-2α receptors compared to the binding ability of natural interleukin-2, aldesleukin, or long-acting conjugates containing them, or have interleukin-2α receptor binding ability 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 0.6 times or more, about 0.7 times or more, about 0.8 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; however, the numerical values ​​are not limited, and any binding ability that has been changed compared to natural interleukin-2 or aldesleukin is included in the present invention.

[0307] Specifically, the long-acting conjugate of an interleukin-2 analog of the present invention has 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, about 100 times, about 130 times, about 150 times, or about 200 times or more, compared to the interleukin-2β receptor binding affinity of natural interleukin-2, aldesleukin, or a long-acting conjugate containing the same; however, the numerical value is not limited, and any binding affinity that has been changed or increased compared to natural interleukin-2 or aldesleukin is included in the present invention.

[0308] In the present invention, the cancer is any one selected from the group consisting of colon cancer, liver cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, kidney cancer, lung cancer, skin cancer, melanoma, breast cancer, bladder cancer, and gastric cancer, or metastatic cancer, but is not limited thereto, and may also include metastatic renal cell carcinoma or metastatic melanoma.

[0309] In the present invention, "prevention" means any action of suppressing or delaying cancer or tumors by administering the interleukin 2 analog (e.g., the interleukin 2 analog itself or in the form of a long-acting conjugate in which a biocompatible substance is bound to it) or a composition containing it.

[0310] In the present invention, "treatment" means any action that improves or beneficially changes the symptoms of cancer by administering the interleukin 2 analog (e.g., the interleukin 2 analog itself or in the form of a long-acting conjugate in which a biocompatible substance is bound to it) or a composition containing it.

[0311] The use of the interleukin 2 analog or its long-acting conjugate of the present invention has the great advantage of reducing the number of administrations to chronic patients who must receive daily treatment due to the dramatic improvement in blood exposure, blood half-life, and in vivo sustained efficacy, thereby improving the quality of life of patients.

[0312] The pharmaceutical composition of the present invention may further comprise a pharma- ceutically acceptable carrier or diluent. Such a pharma- ceutically acceptable carrier or diluent may be non-naturally occurring.

[0313] In the present invention, "pharmacologically acceptable" means a sufficient amount to exert a therapeutic effect and to cause no side effects, and can be easily determined by a person skilled in the art based on factors known in the medical field, such as the type of cancer, the patient's age, weight, health condition, sex, sensitivity of the patient to the drug, administration route, administration method, number of doses, treatment period, combination, and drugs used concomitantly.

[0314] The pharmaceutical composition containing the interleukin-2 analog or its long-acting conjugate of the present invention may contain a pharma- ceutically acceptable excipient. The excipient may be, for oral administration, a binder, a lubricant, a disintegrant, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a flavoring, etc., for injection, a buffer, a preservative, an analgesic, a solubilizer, an isotonicity agent, a stabilizer, etc., for topical administration, a base, an excipient, a lubricant, a preservative, etc., but is not limited thereto.

[0315] The composition of the present invention can be prepared in various forms by mixing with the pharma- ceutically acceptable excipients as described above.For example, for oral administration, it can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it can be prepared in the form of disposable ampoules or multiple doses.Other forms include solutions, suspensions, tablets, pills, capsules, sustained release preparations, etc.

[0316] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Furthermore, the formulation may further contain a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, a preservative, etc.

[0317] In addition, the pharmaceutical composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0318] Furthermore, the composition can be formulated into a unit-dose formulation suitable for administration to the body of a patient by a conventional method in the pharmaceutical field, specifically, into a formulation form useful for administration of a protein pharmaceutical, and administered orally or via a parenteral administration route, including, but not limited to, dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, using an administration method conventionally used in the art.

[0319] Furthermore, the conjugate can be used by mixing it with various medicament-acceptable carriers such as physiological saline or organic solvents, and in order to improve stability and absorbability, carbohydrates such as glucose, sucrose, dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, other stabilizers, etc. can be used as drugs.

[0320] Yet another aspect of the present invention provides a method for preventing or treating cancer, comprising the step of administering an interleukin-2 analogue, a long-acting conjugate containing the same, or a pharmaceutical composition containing the same to an individual in need thereof.

[0321] The interleukin-2 analogue and / or the long-acting conjugate of the interleukin-2 analogue, the composition containing the same, and the cancer, prevention and treatment are as described above.

[0322] The individual in the present invention refers to an individual who has developed cancer or is suspected of having cancer, and refers to mammals including humans, mice, livestock, etc., and may be any individual that can be treated with the interleukin-2 analogue and / or conjugate of the present invention, or the composition containing same.

[0323] In the present invention, "administration" means introducing a predetermined substance (e.g., an interleukin 2 analog or a long-acting conjugate thereof) into a patient by any appropriate method, and the route of administration can be any common route that can deliver the substance to a target in the body, including, but not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0324] The method of the present invention may include administering a pharmaceutical composition comprising said interleukin 2 analog or its long-acting conjugate in a pharmacologic effective amount. The appropriate total daily dose may be determined by the attending physician within the scope of sound medical judgment and may be administered in one or several doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on a variety of factors, including the type and extent of the response to be achieved, whether or not other formulations are used, the specific composition, the patient's age, weight, general health, sex, diet, time of administration, route of administration, excretion rate of the composition, duration of treatment, drugs administered with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical arts.

[0325] In the method of the present invention, the dosage and frequency of administration are determined by the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, the severity of the disease, etc. Specifically, the composition of the present invention contains the interleukin 2 analog or a long-acting conjugate containing the same in a pharmacologic effective amount, but is not limited thereto.

[0326] The term "containing a pharmacologic effective amount of the interleukin-2 analog or long-acting conjugate" means that the interleukin-2 analog or long-acting conjugate is contained to such an extent that the desired pharmacological activity (e.g., prevention, amelioration, or treatment of cancer) can be obtained, and also means that the amount is pharmacologic acceptable and has little or no toxicity or side effects on the individual to which the interleukin-2 analog or long-acting conjugate is administered, but is not limited thereto. Such a pharmacologic effective amount is determined by comprehensively considering the number of administrations, the patient, the dosage form, etc.

[0327] The pharmaceutical composition of the present invention contains the above-mentioned ingredient (active ingredient) in an amount of 0.01 to 99% by weight by volume, but is not particularly limited thereto.

[0328] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredients depending on the severity of the disease. Specifically, the total dose of the interleukin 2 analog or its long-acting conjugate of the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day. However, the dose of the interleukin 2 analog or its conjugate is determined by taking into consideration various factors such as the age, body weight, health condition, sex, severity of the disease, diet, and excretion rate of the patient as well as the administration route and number of treatments of the pharmaceutical composition, and therefore, taking these factors into consideration, a person having ordinary knowledge in the art would be able to determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route, and administration method as long as it exhibits the effects of the present invention.

[0329] The pharmaceutical composition of the present invention has excellent in vivo persistence and potency, and therefore the number and frequency of administration of the pharmaceutical preparation of the present invention can be significantly reduced. The pharmaceutical composition is administered by intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary or rectal administration, but is not limited to a specific administration route as long as the desired pharmacological effect can be obtained.

[0330] For example, the pharmaceutical composition of the present invention may be administered once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, or may be administered one or more times at time intervals ranging from one week to one month, but is not limited thereto.

[0331] Yet another embodiment of the present invention is the use of said interleukin-2 analogue, its long-acting conjugate or a composition containing same in the manufacture of a medicament for the prevention or treatment of cancer.

[0332] The interleukin-2 analog and / or its long-acting conjugate, or the composition containing same, cancer, prevention, treatment, administration route and administration frequency are as described above.

[0333] Yet another embodiment of the present invention provides the use of an interleukin-2 analogue, a long-acting conjugate thereof, or a composition containing same for the prevention or treatment of cancer.

[0334] The interleukin-2 analog and / or its long-acting conjugate, or the composition containing same, cancer, prevention, treatment, administration route and administration frequency are as described above.

[0335] In this specification, unless otherwise specified, the terms "comprise," "have," "contain," and the like are intended to mean the inclusion of a specified integer or set of integers, but are not intended to exclude other integers or sets of integers.

[0336] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of explaining the present invention more specifically, and the present invention is not limited to these examples. EXAMPLES

[0337] Construction of expression vectors for native interleukin 2 and interleukin 2 analogues To prepare a natural interleukin-2 expression vector encoding 133 amino acids, a synthetic interleukin-2 based on the reported interleukin-2 sequence (NM_000586.3; SEQ ID NO: 1) was cloned into the 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.

[0338] The PCR conditions for amplifying the interleukin 2 analogs were 95°C for 30 seconds, 55°C for 60 seconds, and 65°C for 6.5 minutes, and this process was repeated for 16 cycles. To determine whether the amino acids at the desired sites were successfully modified, the mutagenesis products obtained under the above conditions were subjected to sequence analysis, and it was confirmed that the desired mutation positions of each interleukin 2 analog were found to have the mutations shown in Table 1 below, based on the natural ones. The expression vectors thus obtained were named pET22b-interleukin 2 analogs 1-105.

[0339] The modified sequence and analog name of each amino acid are shown in Table 1. To generate these interleukin 2 analogs, forward (F) and reverse (R) primers were synthesized, and then PCR was performed to amplify each analog gene.

[0340] In Table 1, Analog 1 is aldesleukin, and primers #1 to #204 correspond to SEQ ID NOs: 214 to 417, respectively, herein.

[0341] [Table 1] JPEG2024512418000004.jpg230150 JPEG2024512418000005.jpg229150 JPEG2024512418000006.jpg229150 JPEG2024512418000007.jpg228150 JPEG2024512418000008.jpg181150

[0342] DesA1 means that the first amino acid, alanine, of interleukin 2 has been deleted. The full-length protein sequences of the interleukin 2 analogs are shown in Table 2. The bold characters in Table 2 indicate the positions of the mutations.

[0343] [Table 2] JPEG2024512418000010.jpg226150 JPEG2024512418000011.jpg224150 JPEG2024512418000012.jpg224150 JPEG2024512418000013.jpg223150 JPEG2024512418000014.jpg225150 JPEG2024512418000015.jpg225150 JPEG2024512418000016.jpg225150 JPEG2024512418000017.jpg228150 JPEG2024512418000018.jpg227150 JPEG2024512418000019.jpg84150 EXAMPLES

[0344] 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 transfected with an expression Escherichia coli strain, E. coli BL21DE3 (E. coli BF - dcm ompT hsdS(r B - m B - ) gal λ(DE3; Novagen) was transformed. The transformation method recommended by Novagen was used. Single colonies transformed with each recombinant expression vector were obtained, inoculated into 2x Luria Broth containing ampicillin (50 μg / mL), and cultured at 37°C for 15 hours. The culture medium of the recombinant strain and 2x LB medium containing 30% glycerol were mixed at a 1:1 (v / v) ratio, and 1 mL of each was dispensed into cryotubes and stored at -150°C. These were used as cell stocks for the production of recombinant proteins.

[0345] For the expression of recombinant interleukin-2 analogs, one vial of each cell stock was dissolved and inoculated into 500 mL of 2×LB, and cultured at 37°C for 14–16 h with shaking. When the absorbance value at 600 nm reached 4.0 or higher, 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 the OD value, and when the absorbance value reached 70 or higher, 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

[0346] 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 medium 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 microfluidizer. The mixture was centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and 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 added to 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 added to the diluted solution to dilute 4.8 M Guanidine to 3.5 M Guanidine, and the pH was then 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 dissolved in 400 mL of a refolding buffer (6 mM guanidine, 100 mM Tris pH 8.0, 0.1 mM CuCl2). The mixed solution was stirred at 4°C for 15 to 24 hours to carry out the refolding process. EXAMPLES

[0347] 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 to elute the sample after the introduction of the refolding solution. 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, the sample was used in the experiment. EXAMPLES

[0348] Evaluation of receptor binding ability of interleukin 2 analogues Surface plasmon resonance (SPR, 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 1 (aldesleukin).

[0349] First, anti-human immunoglobulin antibody (Abcam, #ab97221) was immobilized on a CM5 chip (GE Healthcare) by amine coupling at about 5,000 RU (resonance unit), and then interleukin 2α receptor (SYMANSIS, #4102H) or interleukin 2β receptor (SYMANSIS, #4122H) bound to the human immunoglobulin Fc site by antigen-antibody binding reaction were bound to the immunoglobulin antibody and finally immobilized. Next, the recombinant interleukin 2 analogs prepared as described above were diluted in HBS-P+ buffer (Cytiva, BR100671) by 2-fold serial dilution method according to concentration, and flowed into 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 (Ka) and dissociation rate (Kd). The binding rate was measured by flowing the interleukin 2 analog at a flow rate of 10uL / min for 3 minutes, and the dissociation rate from each interleukin 2 receptor was measured by flowing only HBS-P+ 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.

[0350]

number

[0351] Table 3 summarizes the relative binding strength of interleukin 2 analogs to interleukin 2 α or β receptors compared to Analog 1 (aldesleukin). In Table 3, "undefined" means that no binding to the receptor was observed in surface plasmon resonance measurements, and the physical quantity cannot be defined for that receptor only.

[0352] [Table 3] JPEG2024512418000022.jpg225150 JPEG2024512418000023.jpg55150

[0353] 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 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.

[0354] 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. EXAMPLES

[0355] Ligation reaction of interleukin-2 analogs with polyethylene glycol (3.4K PEG) linker and purification of interleukin-2 analog conjugates In order to prepare a long-acting conjugate in which the interleukin 2 analog obtained in Example 4 is bound to the immunoglobulin Fc region, first, a conjugate was prepared in which the interleukin 2 analog was linked to one end of a polyethylene glycol (PEG) linker. To prepare the conjugate, the 21st, 41st, 52nd, 86th, 104th, and 105th interleukin 2 analogs were used, and polyethylene glycol (ALD(2)3.4K PEG from NOF Japan) with a molecular weight of 3.4 kDa and hydroxy hydrogen at both ends modified with propylaldehyde groups was used as the PEG linker, which was linked to the N-terminus of the interleukin 2 analog. The molar ratio of interleukin 2 analog:PEG linker was set to 1:15 to 1:20, and the concentration of the interleukin 2 analog was set to 1 mg / mL or less, and the reaction was carried out at 2 to 10°C for 1 hour. Here, the reaction was carried out in 100 mM potassium phosphate (pH 5.5) and 20 mM sodium cyanoborohydride (SCB) was added as a reducing agent. The reaction solution was changed to 20 mM triethylamine (pH 8.0) buffer using a desalting column, and then purified using a Fractogel (registered trademark) EMD TMAE (S) (Merck Millipore) or Source 15Q (Cytiva) column using a concentration gradient of triethylamine (pH 8.0) and sodium chloride to obtain an interleukin 2 analog-3.4K PEG conjugate. EXAMPLES

[0356] Preparation of long-acting conjugates of interleukin 2 analogues, 3.4K PEG and immunoglobulin Fc domain To prepare a long-acting interleukin 2 analog-3.4K PEG-immunoglobulin Fc region conjugate, the interleukin 2 analog-3.4K PEG conjugate obtained by the method of Example 6 and the immunoglobulin Fc region (SEQ ID NO: 438) were reacted at a molar ratio of 1:10, a total protein concentration of 30 mg / mL, and at 2 to 10° C. for 15 to 16 hours. Here, the reaction solution was 100 mM potassium phosphate (pH 6.0), and 20 mM sodium cyanoborohydride was added as a reducing agent.

[0357] The immunoglobulin region used here is a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 423 (consisting of 221 amino acids) via a disulfide bond between the third amino acid cysteine ​​of each monomer, and the monomers of the homodimer independently form internal disulfide bonds between the 35th and 95th cysteines, and between the 141st and 199th cysteines.

[0358] [Table 4]

[0359] After the reaction was completed, the unreacted immunoglobulin Fc region was removed from the reaction solution using Bis-Tris (pH 6.5) and sodium chloride with Butyl FF (Cytiva), and purified with sodium citrate buffer (pH 5.5) and ammonium sulfate with Source 15ISO (Cytiva) to obtain an interleukin 2 analog-3.4K PEG-immunoglobulin Fc region conjugate (sustained conjugate) in which the N-terminus of the interleukin 2 analog was linked to one end of a 3.4 kDa PEG linker and the opposite end of the 3.4 kDa PEG linker was linked to the nitrogen of the N-terminal proline of the Fc region. This sustained conjugate was analyzed by SDS-PAGE, RP-HPLC, and SE-HPLC (Figures 1 and 2). EXAMPLES

[0360] Evaluation of interleukin 2 receptor binding ability of interleukin 2 analogue conjugates To measure the receptor binding strength of the long-acting interleukin-2 analogue conjugate obtained in Example 7 to each of the interleukin-2α and β receptors, surface plasmon resonance (SPR, BIACORE T200, GE Healthcare) was used.

[0361] Specifically, biotin-labeled human interleukin 2 receptor α and β subunits (ACROBiosystems) were immobilized on a streptavidin biosensor chip (SA chip, Cytiva) at approximately 100 RU and 500 RU, respectively, and the interleukin 2 analogue long-acting conjugate or aldesleukin diluted in HBS-EP+ buffer (Cytiva, BR100669) by two-fold serial dilution was injected at a flow rate of 20 μL per minute. After a 3-minute binding process, HBS-EP+ buffer alone was injected at the same flow rate for 3 minutes to induce dissociation of the interleukin 2 analogue long-acting conjugate or aldesleukin at the interleukin 2 receptor. The binding strength was calculated from the obtained binding constant and dissociation constant. The binding strength evaluation was performed using a 1:1 binding fitting model in the Biaevaluation program.

[0362] As a result of the evaluation, it was confirmed that the interleukin 2 analogue conjugates have a unique binding ability to the interleukin 2 receptor, and a clear difference in binding ability was confirmed between each candidate substance, especially to the interleukin 2 receptor α subunit. More specifically, as a result of the evaluation, it was confirmed that the interleukin 2 analogue conjugates No. 21 and No. 52 did not bind to the interleukin 2 receptor α subunit, and the interleukin analogue conjugates No. 41, No. 86, No. 104 and No. 105 had relative binding abilities of 50.8%, 65.2%, 81.0% and 112.9%, respectively, compared to aldesleukin. It was confirmed that all interleukin 2 analogue conjugates, including the interleukin 2 analogue conjugates No. 21 and No. 52 conjugates, had higher binding abilities to the interleukin 2 receptor β subunit than aldesleukin.

[0363] Table 5 summarizes the relative binding strength (%) of long-acting interleukin 2 analogue conjugates prepared separately for α and β receptors, compared with the binding strength of aldesleukin as a standard. In Table 5, the long-acting conjugates are indicated by the numbers of the interleukin 2 analogues that constitute them (for example, the long-acting conjugate of interleukin 2 analogue no. 21 is indicated as "interleukin 2 analogue no. 21 conjugate").

[0364] [Table 5] EXAMPLES

[0365] Pharmacokinetic evaluation of long-acting interleukin-2 analogue conjugates. Based on the binding ability of the interleukin-2 analogs and long-acting conjugates containing them to the interleukin-2 receptor confirmed in Examples 5 and 7, it was confirmed whether drugs using interleukin-2 analogs and long-acting conjugates have excellent blood exposure and bioavailability.

[0366] Specifically, the present inventors compared and analyzed the pharmacokinetics of a long-acting interleukin-2 analogue conjugate and aldesleukin in ICR and C57BL / 6 strain normal mouse models.

[0367] More specifically, in the control group administered with aldesleukin and the experimental groups administered with interleukin 2 analogue conjugates (interleukin 2 analogue no. 21 conjugate, interleukin 2 analogue no. 41 conjugate, interleukin 2 analogue no. 52 conjugate, interleukin 2 analogue no. 86 conjugate, interleukin 2 analogue no. 104 conjugate and interleukin 2 analogue no. 105 conjugate), normal mice were subcutaneously, intravenously or intraperitoneally injected with aldesleukin. In the group administered with interleukin 2 analogue conjugate subcutaneously, blood was collected from the orbital vein of three animals at 1, 4, 8, 24, 48, 72 and 96 hours, and in the group administered with aldesleukin intravenously or intraperitoneally, blood was collected from three animals at 0.08, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 hours. The collected blood from each group was centrifuged to separate the serum, and the interleukin 2 analog conjugate and aldesleukin in the blood were quantified by ELISA using human interleukin 2 antibody. As a result, the interleukin 2 analog conjugate showed a higher blood exposure amount than aldesleukin in ICR and C57BL / 6 normal mice (Figure 3, Tables 6 and 7). The values ​​of the capacity, AUC, and concentration of the conjugate in the following table are the conjugate amount values ​​(mg, ng) based only on the weight occupied by the interleukin 2 analog moiety in the entire conjugate.

[0368] [Table 6]

[0369] [Table 7]

[0370] As can be seen from these tables, the interleukin 2 analogue conjugate according to the present invention, although administered subcutaneously, shows a higher blood distribution rate than aldesleukin administered intravenously or intraperitoneally. These results suggest that, even if the interleukin 2 analogue conjugate is administered in a smaller amount than human recombinant interleukin 2, a larger amount is exposed to the blood, and furthermore, convenience of administration can be ensured. EXAMPLES

[0371] Anticancer efficacy evaluation of long-acting conjugates of interleukin-2 analogues To examine the therapeutic efficacy of the interleukin-2 analog conjugates, the interleukin-2 analog no. 21 conjugate, the interleukin-2 analog no. 41 conjugate, the interleukin-2 analog no. 52 conjugate, and aldesleukin as a control substance were administered to a CT26 colon tumor syngeneic mouse model, and then the tumor size and individual mortality were evaluated.

[0372] Specifically, CT26 cells (ATCC) cultured in a cell culture flask were subcutaneously injected into the legs of BALB / c mice, and after a few days, when tumors were observed with the naked eye, seven mice were allocated to each group so that the tumors were of similar size. Each group was repeatedly administered 0.5 mg / kg of interleukin 2 analogue No. 21 conjugate, interleukin 2 analogue No. 41 conjugate, and interleukin 2 analogue No. 52 conjugate via subcutaneous route once a week, twice in total. Here, the dosage of the long-acting conjugate is indicated by the weight of only the interleukin 2 analogue part in the conjugate. The aldesleukin administration group was administered 3.0 mg / kg of Proleukin injection (Novartis) via intraperitoneal route once a day for five consecutive days, and after a two-day break, was further administered for five days on the same schedule.

[0373] Body weight and death of individual were observed for 32 days from the first administration date. The results confirmed excellent anticancer efficacy in all interleukin 2 analogue conjugate administration groups. In particular, complete remission was observed in all individuals in the group administered with interleukin 2 analogue no. 41 conjugate. In contrast, in the group administered with aldesleukin, 2 out of 7 animals died, and only 2 animals were observed to achieve complete remission. It is believed that the interleukin 2 analogue conjugate of the present invention strongly binds to the human interleukin 2 receptor β subunit due to the change in amino acid sequence, thereby exhibiting excellent anticancer efficacy (Figure 4). In addition, the structure of the long-acting conjugate linked to the human immunoglobulin Fc region allows high blood exposure, and the administration interval is increased, suggesting that changing the conventional injection therapy to a subcutaneous route will bring convenience to patients and medical providers. EXAMPLES

[0374] Evaluation of memory T cell responses following repeated tumor exposure Following the anticancer efficacy evaluation of Example 10, the memory T cell response due to repeated exposure to tumors was evaluated. Specifically, after the anticancer efficacy evaluation was completed, individuals confirmed to be in complete remission were re-exposed to CT26 cells by subcutaneous administration in the same manner as the first exposure on days 42 and 70 from the first drug administration, and then observed for tumor growth. Four and seven days after the second re-exposure, the number of memory T cells in the spleens of the mice was analyzed using a FACS Canto II (Becton Dickinson) device.

[0375] As a result, in all drug administration groups except the negative control group, not only was tumor growth suppressed by re-exposure, but a significant increase in memory T cells in the spleen was observed (Figure 5). In conclusion, the interleukin 2 analog conjugate according to the present invention can induce memory immune responses in a mouse model of allografted CT26 colon carcinoma in a single formulation, and maintains strong anti-cancer efficacy not only against initial tumor exposure, but also against tumor re-exposure. EXAMPLES

[0376] Evaluation of anticancer efficacy of long-acting conjugates of interleukin-2 analogues in malignant melanoma In order to evaluate the anticancer efficacy of the interleukin 2 analog conjugate according to the present invention, the interleukin 2 analog no. 86 conjugate and aldesleukin as a control substance were administered to a B16F10 melanoma tumor syngeneic mouse model in which B16F10 melanoma cells were allogeneically transplanted, and then the tumor size and individual survival rate were evaluated.

[0377] Specifically, B16F10 cells (ATCC) cultured in a cell culture flask were subcutaneously injected into the thighs of C57BL / 6 mice, and when tumors were observed with the naked eye several days later, nine mice were allocated to each group so that the tumors would be of similar size. Based on the weight of the interleukin 2 analog site in the long-acting conjugate, 0.08 mg / kg to 10 mg / kg of interleukin 2 analog 86 conjugate was administered subcutaneously once a week for a total of four times. In addition, the aldesleukin administration group, which served as a control group, was administered 3.0 mg / kg of Proleukin injection (Novartis) intraperitoneally once a day for five consecutive days, with a two-day rest period, for a total of four times.

[0378] The tumor size of each group on the 15th day after the first administration (FIG. 6A) and the survival rate of each individual for 60 days (FIG. 6B) were observed. As a result, it was confirmed that the group administered with the interleukin-2 analogue no. 86 conjugate of the present invention had smaller tumor size and better individual survival rate than the group administered with aldesleukin, and the anticancer efficacy of the long-acting conjugate of the present invention was confirmed to be dose-dependent. In particular, in the group administered with 4.0 mg / kg or more of the interleukin-2 analogue no. 86 conjugate per week, complete remission in which the tumor was completely removed was observed in some individuals. In contrast, no complete remission was observed in any individual in the group administered with 3.0 mg / kg of aldesleukin continuously for 5 days.

[0379] This means that the interleukin 2 analog conjugate of the present invention exhibits excellent anticancer efficacy by being designed to have the characteristic of binding to the human interleukin 2 receptor β subunit with relatively strong binding power compared to aldesleukin.In addition, excessive immune response is effectively controlled by the minimal binding power to the human interleukin 2 receptor α subunit that does not cause undesirable side effects, so no side effects were observed that required interruption of administration even in the high-dose group of 10 mg / kg or more based on the weight of the interleukin 2 analog moiety in the long-acting conjugate.

[0380] These experimental results suggest that the long-acting conjugates containing interleukin-2 analogs obtained by introducing amino acid mutations into natural interleukin-2 according to the present invention have altered binding affinity to interleukin-2α and interleukin-2β receptors, and thus may be useful in the development of various drugs, particularly anticancer drugs.

[0381] 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. A long-acting conjugate containing an interleukin 2 analog, the long-acting conjugate being represented by the following chemical formula (1): [Formula 1] wherein X is an interleukin 2 analog having increased interleukin 2β receptor binding affinity compared to aldesleukin; L is a polyethylene glycol linker; a is 0 or a natural number, but if a is 2 or more, each L is independent of the others; F is a dimeric form of an immunoglobulin Fc region, - indicates a covalent bond link between X and L, and between L and F; The interleukin 2 analogue 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.

2. The long-acting conjugate according to claim 1, wherein the interleukin 2 analogue has a binding ability to the interleukin 2α receptor different from that of aldesleukin.

3. The persistent conjugate of claim 1, wherein at least one amino acid is added to the amino acid corresponding to position 133.

4. The long-acting conjugate according to claim 1, wherein the interleukin-2 analog is an interleukin-2 analog in which the first amino acid is deleted and the 125th amino acid is replaced with another amino acid in natural interleukin-2.

5. The long-acting conjugate according to claim 4, wherein the interleukin-2 analogue further comprises 1 to 10 amino acid substitutions.

6. The long-acting conjugate of claim 5, wherein the interleukin 2 analogue further comprises at least one amino acid at positions 18, 19, 20, 22, 38, 42, 43, 45, 61, 68, 69, 74, 80, 81, 84, 85, 86, 88, 89, 91, 92, 94 and 96 being substituted with another amino acid.

7. The long-acting conjugate of claim 5, wherein the interleukin 2 analogue further has at least one amino acid at positions 18, 22, 38, 42, 61, 68, 80, 81, 85, 86 and 92 substituted with another amino acid.

8. The long-acting conjugate of claim 1 , wherein the interleukin 2 analog is any of the following analogs: (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 has been deleted and the amino acids at the 125th, 12th, 38th and 42nd positions have been 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 in natural interleukin 2 is deleted and the amino acids at the 125th, 38th, 81st and 92nd positions are replaced with other amino acids. (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. (a) 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, 42nd, 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 in natural interleukin 2 is deleted and the amino acids at the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th, and 92nd positions 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 in natural interleukin 2 is deleted and the amino acids at the 125th, 38th, 42nd, 43rd, 61st, 80th, 81st, 85th, 86th, and 92nd positions 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. (bo) 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, 86th, and 92nd positions are replaced with other amino acids. (bp) An interleukin 2 analog in which the first amino acid in natural interleukin 2 is deleted and the 125th, 80th, 81st, 85th, and 86th amino acids are replaced with other amino acids.

9. The persistent conjugate according to claim 8 , wherein the amino acid substitution is any one selected from the group consisting of the following amino acid substitutions: (a) Substitution of the 12th amino acid with valine or phenylalanine (b) substitution of the 18th amino acid with arginine (c) Substitution of the 19th amino acid with tyrosine, valine, phenylalanine, or arginine (d) substitution of the 20th amino acid with valine or phenylalanine (e) substitution of the 22nd amino acid with glutamic acid (f) substitution of amino acid 32 with cysteine (g) substitution of the 35th amino acid with cysteine ​​or glutamic acid (h) substitution of the 38th amino acid with alanine or aspartic acid (i) substitution of the 42nd amino acid with lysine, alanine, or tryptophan (j) substitution of the 43rd amino acid with cysteine, glutamic acid, or glutamine (k) substitution of 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 the amino acid at position 68 with aspartic acid or glutamine (p) substitution of amino acid 69 with glycine (q) substitution of the 74th amino acid with histidine or alanine (r) substitution of amino acid 76 with cysteine (s) substitution of the 80th amino acid with phenylalanine, tyrosine, valine, aspartic acid, or tryptophan (t) substitution of the 81st amino acid with aspartic acid, glutamic acid, or asparagine (u) substitution of the 82nd amino acid with glycine or valine (v) substitution of the amino acid at position 84 with glutamic acid, valine, or phenylalanine (w) substitution of the 85th amino acid with valine, alanine, glycine, tryptophan, tyrosine, threonine, isoleucine, glutamic acid, or phenylalanine (x) substitution of the amino acid at position 86 with valine, alanine, glycine, or leucine (y) substitution of amino acid 87 with cysteine (z) substitution of the amino acid at position 88 with glutamine, valine, or phenylalanine (aa) Substitution of amino acid 89 with phenylalanine (ab) substitution of the 91st amino acid with threonine, phenylalanine or glutamic acid (ac) substitution of the 92nd amino acid with phenylalanine, leucine, tyrosine, or tryptophan (ad) substitution of amino acid 94 with phenylalanine or valine (ae) Substitution of the 95th amino acid with aspartic acid (af) substitution of the 96th amino acid with phenylalanine, valine, or isoleucine (ag) substitution of amino acid 125 with serine (ah) substitution of amino acid 126 with threonine

10. The long-acting conjugate according to claim 8, wherein the interleukin 2 analogue 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 amino acids at the 125th, 38th, 42nd, 80th, 81st and 92nd positions 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 amino acids at the 125th, 80th, 81st, 85th, 86th, and 92nd positions 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 amino acids at the 125th, 18th, 22nd, 80th, 81st, 85th, 86th, and 92nd positions 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 amino acids at the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, and 92nd positions 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 amino acids at the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th, and 92nd positions 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 amino acids at the 125th, 18th, 22nd, 68th, 80th, 81st, 85th, 86th, and 92nd positions are replaced with other amino acids;

11. The persistent conjugate according to claim 10, wherein the amino acid substitution is any one selected from the group consisting of the following amino acid substitutions: (a) Substitution of the 18th amino acid with arginine (b) substitution of the 22nd amino acid with glutamic acid (c) substitution of the 38th amino acid with alanine (d) substitution of amino acid 42 with lysine (e) substitution of amino acid 61 with aspartic acid (f) substitution of amino acid 68 with aspartic acid (g) substitution of the 80th amino acid with phenylalanine (h) substitution of the 81st amino acid with glutamic acid (i) substitution of amino acid 85 with valine (j) substitution of amino acid 86 with valine (k) substitution of amino acid 92 with phenylalanine (l) substitution of amino acid 125 with serine

12. The long-acting conjugate according to claim 1 , wherein the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof.

13. The long-acting conjugate according to claim 1 , wherein the immunoglobulin Fc region is an IgG4 Fc region.

14. The long-acting conjugate of claim 1 , wherein the immunoglobulin Fc region is non-glycosylated.

15. 2. The persistent conjugate of claim 1, wherein L comprises an ethylene glycol repeating unit, and the chemical formula weight of the ethylene glycol repeating unit moiety is in the range of 1 to 100 kDa.

16. The persistent conjugate according to claim 1, wherein one molecule of X is covalently linked to any one of the Fc regions of the dimeric immunoglobulin Fc regions via the polyethylene glycol linker.

17. A long-acting conjugate of an interleukin-2 analogue represented by the following chemical formula (2): [Case 2] wherein X is an interleukin 2 analog comprising any polypeptide sequence selected from the amino acid sequences of SEQ ID NOs: 3 to 106; Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa; Fc is a dimeric form of an immunoglobulin Fc region; - indicates a covalent bond link between X and Z and between Z and Fc; The persistent conjugate has one end of Z covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X covalently linked to the end opposite to Z.

18. The long-acting conjugate of claim 17, wherein the interleukin 2 analogue comprises 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.

19. The long-acting conjugate of claim 17, wherein the interleukin 2 analogue 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.

20. The interleukin 2 analog of claim 17, comprising any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105 and 106.

21. The long-acting conjugate of claim 17, wherein the interleukin 2 analogue further comprises at least one amino acid at the C-terminus.

22. A long-acting conjugate of an interleukin-2 analogue represented by the following chemical formula (3): [C3] In the above formula, X' is an interleukin 2 analogue having an amino acid sequence represented by the following general formula 1: Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa; Fc is a dimeric form of an immunoglobulin Fc region; - indicates a covalent bond link between X' and Z and between Z and Fc; The persistent conjugate has one end of Z covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X' covalently linked to the end opposite to Z. [General formula 1] X1-P-T-S-S-S-TK-K-T-Q-L-Q-L-E-HL-X18-X19-D-L-X22-M-I-L-N-G-I-N-N-Y-K-N-P-K-L- T-X38-M-L-T-X42-X43-F-X45-M-P-K-K-A-T-E-L-K-HL-Q-CL-E-X61-E-L-K-PL-E-X68-V-L- NL -T-T-F-M-C-E-Y-A-D-ET-AT-T-V-E-F-L-N-R-W-I-T-F-S-Q-S-I-I-S-TLT (General formula 1, SEQ ID NO: 212) In the 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); X42 is alanine (A), phenylalanine (F), lysine (K) or tryptophan (W); X43 is glutamic acid (E), lysine (K) or glutamine (Q); X45 is alanine (A) or tyrosine (Y); X61 is aspartic acid (D), glutamic acid (E), glutamine (Q) or arginine (R); X68 is aspartic acid (D) or glutamic acid (E); 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); X96 is phenylalanine (F) or leucine (L).

23. In the 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); 23. The method of claim 22, wherein X92 is phenylalanine (F) or isoleucine (I).

24. 23. The interleukin 2 analog of claim 22, further comprising at least one amino acid at the C-terminus.

25. A long-acting conjugate of an interleukin-2 analogue represented by the following chemical formula (4): [C4] In the above formula, X″ is an interleukin 2 analogue having an amino acid sequence represented by the following general formula 2: Z is a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa; Fc is a dimeric form of an immunoglobulin Fc region; - indicates a covalent bond link between X″ and Z, and between Z and Fc; The persistent conjugate has one end of Z covalently linked to only one polypeptide chain of the dimeric Fc region, and one molecule of X″ covalently linked to the end opposite to Z. [General formula 2] X1-P-T-S-S-S-TK-K-T-Q-L-Q-L-EHL-X18-L-D-L-X22-M-I-L-N-G-I-N-N-Y-K-N-P-K -L-T-X38-M-L-T-X42-K-F-Y-M-P-KK-A-T-E-L-K-HL-Q-C-L-E-X61-E-L-K-PLE-X68- V-L-N-L-A-Q-S-K-N-F-H-F-X81-PRD-X85-X86-S-N-I-N-V-F-V-L-E-L-K-G-S-E-T-T- F-M-CEY-A-D-ET-AT-T-I-V-E-F-LN-R-W-IT-F-S-Q-S-I-I-S-TLT (General formula 2, SEQ ID NO: 213) 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); X86 is isoleucine (I) or valine (V).

26. The interleukin 2 analog of claim 25, comprising any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105 and 106.

27. A pharmaceutical composition for preventing or treating cancer, comprising a pharma- ceutical effective amount of the long-acting conjugate according to claim 1 or 17, and a pharma- ceutical acceptable excipient.

28. 28. The pharmaceutical composition of claim 27, wherein the interleukin 2 analog is selected from the group consisting of SEQ ID NOs: 22, 42, 53, 87, 105 and 106.

29. The pharmaceutical composition of claim 27, wherein the cancer is any one selected from the group consisting of metastatic renal cell carcinoma, metastatic melanoma, colon cancer, liver cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, kidney cancer, lung cancer, skin cancer, melanoma, breast cancer, bladder cancer and gastric cancer.

30. 28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition exhibits at least one of the following properties: (i) Higher blood exposure compared to aldesleukin (ii) Higher tumor growth inhibitory activity compared to aldesleukin (iii) Higher memory T cell generation response compared to aldesleukin

31. 28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary or rectal routes of administration.

32. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is administered at time intervals ranging from one week to one month.

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