Combination therapy of interleukin-2 (IL-2) analogs or conjugates thereof with immune checkpoint inhibitors for prevention or treatment of cancer

Abstract

The present invention relates to a combined use of an interleukin-2 analogue or a conjugate thereof and an immune checkpoint inhibitor in the prevention or treatment of cancer, and a pharmaceutical composition comprising the same.

Description

Technical Field

[0001] The present invention relates to a combination therapy of an interleukin-2 analogue or a conjugate thereof and an immune checkpoint inhibitor for preventing or treating cancer. Background Art

[0002] Interleukin-2 (IL-2) is an important immunostimulant with a molecular weight of approximately 15 kDa and a total of 133 amino acid residues. It activates various cells of the immune system, including T and B cells. IL-2's high efficacy as an immunostimulant has been used to treat a variety of immune-related diseases, including cancer and AIDS (Korean Patent Publication No. 10-2017-0070091). Currently, IL-2 (brand name: Proleukin) is an FDA-approved drug for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the severe toxicities associated with high-dose IL-2 therapy limit the number of patients who can receive this treatment, resulting in only a small number of eligible patients undergoing treatment. IL-2-related toxicities include severe fever, nausea, vomiting, vascular leakage, severe hypotension, pulmonary edema, and vascular leak syndrome, which can lead to liver damage.

[0003] The interleukin-2 receptor has three subunit receptors. The subunits are composed of an alpha chain (IL-2Rα, CD25), a beta chain (IL-2Rβ or CD122), and a gamma chain (IL-2Rγ or CD132). Interleukin-2 can exhibit various functions by binding to various combinations of receptor subunits. A single interleukin-2α receptor is called a low-affinity interleukin-2 receptor, and it does not participate in signal transduction. A complex of interleukin-2β and interleukin-2γ receptors binds to interleukin-2 with moderate affinity. A complex of interleukin-2α, interleukin-2β, and interleukin-2γ receptors binds to interleukin-2 with high affinity. The complex of interleukin-2β and interleukin-2γ receptors is required for efficient signal transduction through kinase activation in multiple signal transduction pathways. Specifically, interleukin-2β and interleukin-2γ bind to receptors on the CD8 + In addition, complexes of high-affinity interleukin-2α, interleukin-2β, and interleukin-2γ receptors are often found on CD4 + T regulatory cells (Tregs), and more recently they have also been found in activated T cells. + Since T cells or natural killer cells (NK cells) participate in the immune response in the body, research has been conducted to develop drugs by enhancing the activity of β receptors for immune activation.

[0004] Generally speaking, immune cells have proteins called immune checkpoints in their cell membranes that suppress unnecessary autoimmune responses and eliminate cancer cells by detecting tumor-specific antigens expressed due to changes in cancer cells (such as mutations). However, to evade these immune responses, cancer cells alter the function of immune checkpoints to suppress T cell function and prevent normal immune responses.

[0005] Immune checkpoint inhibitors are drugs that activate a patient's own immune system to induce immune cells in the body to suppress cancer cell growth. Unlike existing immunotherapies, immune checkpoint inhibitors bind to the interaction sites between cancer cells and T cells to block immune evasion signals, allowing T cells to destroy cancer cells without being hindered by immune evasion. Therefore, research into the use of immune checkpoint inhibitors in immunotherapy for cancer patients continues.

[0006] As mentioned above, research has been actively conducted on treatments in which the body's immune system is activated to act on cancer cells, thereby achieving an anti-cancer effect. However, the use of drugs that stimulate the immune system in anti-cancer treatments may cause side effects due to excessive immune cell activity, resulting in a need for the discovery of safe and effective anti-cancer drugs and therapies. Summary of the Invention

[0007]

Technical Issues

[0008]

Technical solution

[0009] Another aspect of the present invention is to provide a therapy for preventing or treating cancer, wherein an interleukin-2 analog or a long-acting conjugate thereof is used in combination with an immune checkpoint inhibitor.

[0010] Another aspect of the present invention is to provide a combination comprising: an interleukin-2 analog or a long-acting conjugate thereof; and an immune checkpoint inhibitor.

[0011] Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the composition.

[0012] Another aspect of the present invention is to provide a drug kit for preventing, alleviating or treating cancer, wherein the kit comprises: an interleukin-2 analogue or a long-acting conjugate thereof; and an immune checkpoint inhibitor.

[0013] Another aspect of the present invention is to provide a method for preventing or treating cancer, which comprises administering and / or using the combination, pharmaceutical composition or pharmaceutical kit to a subject in need thereof.

[0014] Another aspect of the present invention is to provide a method for preventing, alleviating or treating cancer, which comprises administering and / or using a composition comprising a pharmaceutically effective amount of an interleukin-2 analog or a long-acting conjugate thereof in combination with a composition comprising a pharmaceutically effective amount of an immune checkpoint inhibitor to a subject in need thereof.

[0015] Another aspect of the present invention is to provide use of the combination, pharmaceutical composition or pharmaceutical kit in preventing, alleviating or treating cancer and / or in preparing a medicament for preventing, alleviating or treating cancer.

[0016]

Beneficial effects

[0017] Figure 1 The SDS-PAGE results of interleukin-2 analogue long-acting conjugates (long-acting conjugates of analogues 21, 41 and 52) are shown.

[0018] Figures 2A to 2C The results of purity analysis of long-acting conjugates of interleukin-2 analogs (long-acting conjugates of analogs 21, 41, and 52) are shown.

[0019] Figure 3 The results of the evaluation of the anti-tumor efficacy of a combination therapy of a long-acting interleukin-2 analog conjugate with an immune checkpoint inhibitor (anti-PD-1 antibody) in an animal model of malignant melanoma are shown. These results confirmed that in a mouse model where aldesleukin or interleukin-2 analog conjugate 86 was administered alone, or aldesleukin and interleukin-2 analog conjugate 86 were administered in combination with an immune checkpoint inhibitor, tumor size ( Figure 3 A) and survival rate of mouse models ( Figure 3 B). Figure 3 In the present invention, the dosage of the long-acting conjugate is based only on the weight of the interleukin-2 analog site in the entire conjugate.

[0020] Figure 4The results of the evaluation of the anti-tumor efficacy of a combination therapy of a long-acting interleukin-2 analog conjugate with an immune checkpoint inhibitor (anti-PD-1 antibody) in an animal model of lung cancer are shown. These results verified that the tumor size in the mouse model was significantly reduced when aldesleukin or interleukin-2 analog conjugate 86 was administered alone, or when aldesleukin and interleukin-2 analog conjugate 86 were administered in combination with an immune checkpoint inhibitor. Figure 4 In the present invention, the dosage of the long-acting conjugate is based only on the weight of the interleukin-2 analog site in the entire conjugate.

[0021] Figure 5 The results of the analysis of the anti-tumor efficacy of the combination therapy of a long-acting conjugate of an interleukin-2 analog and an immune checkpoint inhibitor (anti-PD-1 antibody) in an animal model of lung cancer using the Bliss independence model are shown. Figure 5 In the present invention, the dosage of the long-acting conjugate is based only on the weight of the interleukin-2 analog site in the entire conjugate.

[0022] Figure 6 The results show the analysis of the anti-cancer efficacy and anti-relapse efficacy of the combination therapy of a long-acting conjugate of interleukin-2 analog and immune checkpoint inhibitor (anti-PD-1 antibody) in a pancreatic cancer mouse model through memory response. Figure 6 In the present invention, the dosage of the long-acting conjugate is based only on the weight of the interleukin-2 analog site in the entire conjugate. DETAILED DESCRIPTION

[0023] According to one aspect of the present invention, a composition comprising a novel interleukin-2 analog (or IL-2 analog) or a long-acting conjugate thereof is provided, wherein the composition is administered in combination with an immune checkpoint inhibitor. The interleukin-2 (IL-2) analog has increased binding affinity for the interleukin-2 beta receptor compared to native interleukin-2 or aldesleukin as an interleukin-2 analog, and may include a sequence having a modification of at least one amino acid in the native interleukin-2.

[0024] In one embodiment, the interleukin-2 analog may comprise an amino acid sequence selected from SEQ ID NOs: 3 to 106.

[0025] In another embodiment, the long-acting conjugate can be represented by the following Chemical Formula 1: [Chemical Formula 1] X - L - F Wherein: X is an interleukin-2 analogue comprising any one of the amino acid sequences selected from SEQ ID NO: 3 to 106; L is a polyethylene glycol linker; F is the dimeric immunoglobulin Fc region, and - symbols represent covalent bonds between X and L and between L and F, respectively, In the long-acting conjugate, one end of L is covalently linked to only one polypeptide chain of the Fc region of the dimer, and X is covalently linked to the other end of L.

[0026] In the composition according to any of the preceding embodiments, the long-acting conjugate may include as part thereof an interleukin-2 analogue having an altered binding affinity for the interleukin-2 alpha receptor and an increased binding affinity for the interleukin-2 beta receptor compared to the native interleukin-2 or aldesleukin.

[0027] In the composition according to any one of the preceding embodiments, the interleukin-2 analogue may have an increased binding affinity for the interleukin-2 beta receptor compared to aldesleukin.

[0028] In the composition according to any of the preceding embodiments, the interleukin-2 analog may include any one of the amino acid sequences selected from SEQ ID NO: 10, 13-15, 17, 20-22, 32, 35, 36, 42, 53, 54, 56, 58-60, 62, 71, 72, 74-78, 85, 87, 89, 91-94, 97-106, 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.

[0029] In the composition according to any of the preceding embodiments, the interleukin-2 analog may comprise any one of the amino acid sequences selected from SEQ ID NOs: 17, 22, 42, 53, 56, 58-60, 62, 71, 72, 74-77, 87, 89, 91-93, 98-101 and 103-106.

[0030] In the composition according to any of the preceding embodiments, the interleukin-2 analog may comprise any one of the amino acid sequences selected from SEQ ID NOs: 22, 42, 53, 87, 105 and 106.

[0031] In the composition according to any one of the preceding embodiments, the interleukin-2 analogue may further comprise at least one amino acid at the C-terminus.

[0032] In the composition according to any one of the preceding embodiments, the immune checkpoint inhibitor is a PD-1 antagonist.

[0033] In the composition according to any one of the aforementioned embodiments, the immune checkpoint inhibitor may be at least one selected from anti-PD-1 antibodies or antigen-binding fragments thereof.

[0034] In the composition according to any of the preceding embodiments, the anti-PD-1 antibody may be selected from nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514.

[0035] In the composition according to any of the preceding embodiments, the long-acting conjugate may comprise an IgG4 Fc region.

[0036] In the composition according to any of the preceding embodiments, the long-acting conjugate may include an aglycosylated immunoglobulin Fc region.

[0037] In the composition according to any of the preceding embodiments, the immunoglobulin Fc region may be derived from the aglycosylated Fc region of human IgG4.

[0038] In the composition according to any one of the aforementioned embodiments, the immunoglobulin Fc region has a structure in which two polypeptide chains are linked by a disulfide bond, wherein the two polypeptide chains are linked only through a nitrogen atom of one of the two chains.

[0039] In the composition according to any of the preceding embodiments, the immunoglobulin Fc region may comprise a monomer having the amino acid sequence of SEQ ID NO: 438.

[0040] In the composition according to any one of the preceding embodiments, the immunoglobulin Fc region is a homodimer of monomers of the amino acid sequence of SEQ ID NO: 438.

[0041] In the composition according to any one of the preceding embodiments, the immunoglobulin Fc region is linked via the nitrogen atom of the N-terminal proline.

[0042] In the composition according to any one of the preceding embodiments, in the long-acting conjugate, X may be covalently linked to one Fc region of the dimeric immunoglobulin Fc region via the polyethylene glycol linker.

[0043] In the composition according to any one of the aforementioned embodiments, one end of the linker may be linked to only one Fc region chain of the two Fc region chains of the dimeric immunoglobulin Fc region.

[0044] In the composition according to any of the preceding embodiments, the polyethylene glycol linker may have a molecular weight of 1 kDa to 100 kDa.

[0045] In the composition according to any one of the preceding embodiments, the composition may further comprise an excipient.

[0046] In the composition according to any of the preceding embodiments, the cancer can be selected from renal cell carcinoma, melanoma, colorectal cancer, liver cancer, uterine cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, breast cancer, bladder cancer, stomach cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, thyroid cancer, Any of the following: thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal pelvis cancer, CNS tumor, primary CNS lymphoma, spinal tumor, brain tumor, glioma (astrocytoma, glioblastoma, oligodendroglioma, ependymoma), blastoma, meningioma, brain stem glioma, pituitary adenoma, neuroblastoma, congenital tumor, craniopharyngioma and brain tumor.

[0047] In the composition according to any one of the preceding embodiments, the cancer may be hyporesponsive to a PD-1 antagonist.

[0048] In the composition according to any one of the preceding embodiments, the cancer with low responsiveness to a PD-1 antagonist may be an immune-infiltrating tumor (cold tumor).

[0049] In the composition according to any of the preceding embodiments, the composition may be administered via an intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary or intrarectal route.

[0050] In the composition according to any one of the preceding embodiments, the composition may be administered at time intervals ranging from 1 week to 1 month.

[0051] In the composition according to any one of the preceding embodiments, the composition may be administered in combination with a composition comprising an immune checkpoint inhibitor simultaneously, separately, sequentially or in the reverse order.

[0052] According to another aspect of the present invention, a method for preventing or treating cancer is provided, which comprises administering the interleukin-2 analog or its long-acting conjugate or a composition comprising the same, and an immune checkpoint inhibitor or a composition comprising the same to a subject in need.

[0053] According to another aspect of the present invention, provided is the combined use of the interleukin-2 analogue or its long-acting conjugate or a composition comprising the same and an immune checkpoint inhibitor or a composition comprising the same in preventing or treating cancer.

[0054] According to another aspect of the present invention, there is provided a use of the interleukin-2 analogue or its long-acting conjugate or a composition comprising the same, administered in combination with an immune checkpoint inhibitor, in the preparation of a medicament for preventing or treating cancer.

[0055] According to another aspect of the present invention, provided is the interleukin-2 analogue or its long-acting conjugate or a kit comprising the same, wherein the interleukin-2 analogue or its long-acting conjugate is administered in combination with an immune checkpoint inhibitor. DETAILED DESCRIPTION

[0056] Hereinafter, the detailed content for implementing the present invention will be described. The various descriptions and embodiments disclosed in this application may also be applicable to other descriptions and embodiments. That is to say, all combinations of the various elements disclosed in this disclosure fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the following specific description. In addition, throughout the specification, reference is made to multiple articles and patent documents and their citations are provided. The disclosures of the cited articles and patent documents are incorporated herein by reference in their entirety, and more clearly illustrate the level of the technical field to which the present invention belongs and the details of the present invention.

[0057] Throughout the specification, the conventional one-letter or three-letter codes for amino acids are used.Amino acids referred to herein in abbreviated form are described according to IUPAC-IUB rules.

[0058] Alanine A Arginine R Asparagine N Aspartic acid D Cysteine ​​C Glutamate E Glutamine Q Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V In one aspect of the present invention, a composition for preventing or treating cancer is provided, the composition comprising an interleukin-2 analog or a long-acting conjugate thereof, wherein the composition is administered in combination with an immune checkpoint inhibitor.

[0059] The interleukin-2 analogs of the present invention may have altered binding affinity for the interleukin-2 receptor, and in particular, increased binding affinity for the interleukin-2 β receptor. Specifically, compared to native interleukin-2 or known aldesleukin, the interleukin-2 analogs of the present invention may have increased binding affinity for the interleukin-2 β receptor, and more specifically, may have altered (increased or decreased) binding affinity for the interleukin-2 α receptor and may include sequences having at least one modified amino acid in native interleukin-2.

[0060] A specific aspect of the composition of the present invention may include an interleukin-2 analog comprising, consisting of, or consisting essentially of any one of the amino acid sequences selected from SEQ ID NOs: 3 to 106, but not limited thereto.

[0061] Specifically, the composition of the present invention can be a pharmaceutical composition comprising an interleukin-2 analogue or a long-acting conjugate thereof for preventing or treating cancer, and more specifically, can be a pharmaceutical composition comprising an interleukin-2 analogue or a long-acting conjugate thereof and a pharmaceutically acceptable excipient administered in combination with an immune checkpoint inhibitor, but is not limited thereto.

[0062] The pharmaceutical composition of the present invention can be administered in the following ways: a) as a mixture comprising (i) an interleukin-2 analogue or a long-acting conjugate thereof; and (ii) an immune checkpoint inhibitor; or b) in a form wherein (i) an interleukin-2 analogue or a long-acting conjugate thereof; and (ii) an immune checkpoint inhibitor are separated from each other, but not limited thereto.

[0063] For example, interleukin-2 analogs or their long-acting conjugates and immune checkpoint inhibitors can be formulated into one preparation or can be formulated separately. When interleukin-2 analogs or their long-acting conjugates and immune checkpoint inhibitors are separated from each other, interleukin-2 analogs or their long-acting conjugates and immune checkpoint inhibitors can be formulated into separate preparations and administered simultaneously, separately, sequentially or in reverse order.

[0064] In the present invention, combined administration refers not only to simultaneous administration, but also to a form of administration in which an interleukin-2 analogue or its long-acting conjugate and an immune checkpoint inhibitor act together on a subject so that each substance can function at a level equivalent to or higher than its natural function. Therefore, the term "combined" or "combination" as used herein should be understood to encompass administration of an interleukin-2 analogue or its long-acting conjugate and an immune checkpoint inhibitor in any manner, such as simultaneously, alone, successively or in reverse order. When administered successively, in reverse order or alone, the order of administration is not particularly limited, but the administration interval of the second component should be determined so as not to lose the beneficial effects of the combination.

[0065] The interleukin-2 analogue or its long-acting conjugate and immune checkpoint inhibitor of the present invention, or the composition comprising the same can be provided in the form of a set, but is not limited thereto. The term "set" as used herein may include a composition according to the present invention for the combined administration of an interleukin-2 analogue or its long-acting conjugate and an immune checkpoint inhibitor. Specifically, the set according to the present invention may include a single formulation of an interleukin-2 analogue or its long-acting conjugate and an immune checkpoint inhibitor, or a separate formulation of an interleukin-2 analogue or its long-acting conjugate and an immune checkpoint inhibitor, and also includes the substances required for the combined administration of the two components, but is not limited thereto.

[0066] As used herein, the term "interleukin-2 (IL-2)" refers to a class of cytokines that signal in the body's immune system and is intended to be an immunomodulator. Interleukin-2 is well known to be an important immunostimulator of approximately 15 kDa.

[0067] As used herein, the term "interleukin-2 analog" refers to an analog having at least one amino acid modified in the sequence of the natural form, and in particular, may be an interleukin-2 analog having an amino acid modified in interleukin-2, thereby increasing or decreasing the binding affinity for the receptor compared to natural interleukin-2. Specifically, the interleukin-2 analog of the present invention may be a non-naturally occurring analog.

[0068] The natural interleukin-2 may be human interleukin-2, the sequence of which may be obtained from a known database. Specifically, the sequence may be the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0069] In the present invention, the meaning that the natural interleukin-2 may include the amino acid sequence of SEQ ID NO: 1 is that the sequence of the natural interleukin-2 is not only identical to SEQ ID NO: 1, but also a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to SEQ ID NO: 1 also falls within the scope of the natural interleukin-2 of the present invention, wherein the amino acid modification position means that when a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology is aligned with SEQ ID NO: 1, the corresponding position in the amino acid sequence of SEQ ID NO: 1 is modified.

[0070] As used herein, the term "aldesleukin" may refer to a commercially available interleukin-2 analog, and the aldesleukin may be aldesleukin (brand name: Proleukin®), and specifically, may have the amino acid sequence of SEQ ID NO: 2. Herein, aldesleukin and "interleukin-2 analog 1" are used interchangeably. Compared to interleukin-2 analog 1, the interleukin-2 analog according to the present invention may have altered binding affinity for the interleukin-2α receptor and / or increased binding affinity for the interleukin-2β receptor.

[0071] Interleukin-2α receptor is not known to participate in the interleukin-2 signal transduction system, but it increases the binding affinity between interleukin-2 and other interleukin-2 receptors (beta or gamma receptors) by 10 to 100 times and is expressed on CD4 + Regulatory T cells, etc.

[0072] Since interleukin-2β receptors are mainly distributed in CD8 + IL-2β receptor activation plays an important role in the activation of immune responses and macrophages, so it can be predicted that the activation of interleukin-2β receptor will lead to tumor cell death and the activation of the body's immune response.

[0073] Therefore, the interleukin-2 analogs of the present invention having increased binding affinity for the interleukin-2 beta receptor can increase therapeutic effects, such as tumor inhibition and death, and reduce side effects.

[0074] As an example, the interleukin-2 analog may comprise, consist essentially of, or consist of an amino acid sequence selected from SEQ ID NOs: 3 to 106, but is not limited thereto.

[0075] In a specific embodiment, the interleukin-2 analog can comprise, consist essentially of, or consist of any one of the amino acid sequences selected from SEQ ID NO: 10, 13-15, 17, 20-22, 32, 35, 36, 42, 53, 54, 56, 58-60, 62, 71, 72, 74-78, 85, 87, 89, 91-94, 97-106, 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.

[0076] In another embodiment, the interleukin-2 analog may comprise, consist essentially of, or consist of any one of the amino acid sequences selected from SEQ ID NOs: 17, 22, 42, 53, 56, 58-60, 62, 71, 72, 74-77, 87, 89, 91-93, 98-101, and 103-106, but is not limited thereto. In another embodiment, the interleukin-2 analog may comprise, consist essentially of, or consist of any one of the amino acid sequences selected from SEQ ID NO: 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.

[0077] In another embodiment, the interleukin-2 analog may comprise, consist essentially of, or consist of any one of the amino acid sequences selected from SEQ ID NO: 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 thereto.

[0078] In another embodiment, the interleukin-2 analog may comprise, consist essentially of, or consist of any one of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105, and 106, but is not limited thereto.

[0079] In addition, the interleukin-2 analog may further include at least one amino acid at the C-terminus, but is not limited thereto.

[0080] Although expressed herein as "interleukin-2 analogs consisting of a specific sequence number", this term does not exclude the addition of nonsense sequences upstream or downstream of the amino acid sequence of the corresponding sequence number, or naturally occurring mutations or silent mutations thereof, as long as the analogs have the same or corresponding activity as the interleukin-2 analogs consisting of the amino acid sequence of the corresponding sequence number, and it is obvious that analogs with such sequence additions or mutations fall within the scope of the present invention.

[0081] The interleukin-2 analogs of the present invention may include amino acid sequences having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the amino acid sequences of SEQ ID NOs: 3 to 106, but are not limited thereto.

[0082] As used herein, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences, and the term can be expressed as a percentage.

[0083] The sequence homology or identity of conservative polynucleotides or polypeptides can be determined by standard comparison algorithms and can be used in conjunction with the default gap penalty set up by the program to be used. Basically, homologous or identical sequences can hybridize along their entire sequence or portions thereof under moderate or highly stringent conditions. Obviously, hybridization also includes hybridization of polynucleotides with polynucleotides that include universal codons or codons that take into account codon degeneracy.

[0084] The terms homology and identity are often used interchangeably.

[0085] Whether any two nucleotide or peptide sequences have homology, similarity or identity can be determined using any computer algorithm known in the art, such as the "FASTA" program, using default parameters, as disclosed in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, this can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the European Molecular Biology Open Software Suite (EMBOSS) package (Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or higher) (including the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information.

[0086] Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program known in Smith and Waterman, Adv. Appl. Math (1981) 2: 482 (e.g., Needleman et al., (1970), J Mol Biol. 48: 443). In short, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include (1) a unary comparison matrix (containing identity values ​​of 1 and non-identity values ​​of 0) and the weighted comparison matrix of Gribskov et al. (1986), Nucl. Acids Res. 14:6745, as disclosed in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, the terms "homology" or "identity" as used herein refer to the relatedness between sequences.

[0087] The interleukin-2 analogs of the present invention can be used as novel interleukin-2 substitutes that alter their in vitro activity by weakening or increasing their binding affinity for interleukin-2α and / or interleukin-2β receptors. Specifically, the interleukin-2 analogs of the present invention are useful as effective drugs due to their activity against both types of receptors, as they not only have increased binding affinity for β receptors but also have altered (i.e., increased or decreased) binding affinity for α receptors.

[0088] In the present invention, such modifications used to prepare interleukin-2 analogs encompass all modifications using L- or D-amino acids and / or non-natural amino acids; and / or modifications of the natural sequence, for example, modification of side chain functional groups, intramolecular covalent binding (e.g., cyclization between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, and the like.

[0089] Additionally, modifications encompass all modifications that add one or more amino acids to the amino terminus and / or carboxyl terminus of native interleukin-2.

[0090] Regarding amino acid substitutions or additions, not only the 20 amino acids commonly found in human proteins can be used, but also atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep Inc., and Genzyme Pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased from companies specializing in commercial peptide synthesis (e.g., American Peptide Company and Bachem in the United States, or Anygen in South Korea).

[0091] Amino acid derivatives can be obtained in a similar manner, and for example, 4-imidazoacetic acid or similar derivatives can be used.

[0092] To protect against in vivo proteolytic enzymes and improve stability, the interleukin-2 analogue according to the present invention may be in a modified form wherein its N-terminus and / or C-terminus are chemically modified or protected with an organic group, or amino acids may be added to the peptide terminus.

[0093] Specifically, chemically synthesized peptides have charged N-terminus and C-terminus, and thus, in order to eliminate these charges, the N-terminus may be acetylated and / or the C-terminus may be amidated, but are not particularly limited thereto.

[0094] The interleukin-2 analogs of the present invention can be synthesized by solid phase synthesis, produced by recombinant methods, or produced according to commercial requirements, but are not limited thereto.

[0095] In addition, the interleukin-2 analogs of the present invention can be synthesized by methods known in the art (eg, by an automatic peptide synthesizer) according to their length, or produced by genetic engineering techniques.

[0096] Specifically, the interleukin-2 analogs of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other methods known in the art. Thus, the interleukin-2 analogs according to the present invention can be synthesized by a variety of methods, including, for example, the following methods: (a) Methods for synthesizing peptides stepwise or by fragment assembly by solid-phase or liquid-phase methods, and for isolating and purifying the final peptide products; (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and collecting the expression product from the host cell culture; (c) a method for performing in vitro cell-free expression of a nucleic acid construct encoding a peptide and collecting the expression product therefrom; or A method of obtaining peptide fragments by any combination of (a), (b) and (c), ligating these fragments to obtain peptides, and then collecting the corresponding peptides.

[0097] In the present invention, the binding affinity of any interleukin-2 analog (or long-acting conjugate comprising the same) to the native interleukin-2 receptor can be measured using surface plasmon resonance (SPR), a method for measuring affinity for the receptor.

[0098] Specifically, in SPR analysis, the following methods are available: a method utilizing the principle of protein-ligand binding, wherein an interleukin-2 receptor is immobilized to a sensor chip, and an interleukin-2 analog diluted in an assay buffer using a serial dilution method is allowed to flow to induce binding to the immobilized receptor, and then, only the assay buffer is allowed to flow at the same flow rate to induce dissociation of the interleukin-2 analog from the receptor, thereby measuring binding affinity; or a method wherein an antibody to the Fc region of a human immunoglobulin is immobilized to a sensor chip, and then the interleukin-2 receptor bound to the Fc region is immobilized, and the interleukin-2 analog is allowed to flow to measure binding affinity, but the binding affinity measurement method is not limited thereto.

[0099] More specifically, a biotin-labeled human interleukin-2 receptor was immobilized on a streptavidin biosensor chip, and a long-acting conjugate of an interleukin-2 analog diluted into HBS-P + buffer using a two-fold serial dilution method was allowed to flow at a flow rate of 20 μL / min for 3 minutes. Then, only HBS-P + buffer was allowed to flow at the same flow rate to induce dissociation between the interleukin-2 receptor and the long-acting conjugate of the interleukin-2 analog. After that, the binding affinity was measured using the obtained association constant and dissociation constant according to a 1:1 binding fitting model using the Biaevaluation program, but the present invention is not limited thereto.

[0100] More specifically, the interleukin-2 analogs of the present invention may have reduced or increased binding affinity for the interleukin-2 alpha receptor compared to native interleukin-2 or aldesleukin (or interleukin-2 analog 1).

[0101] Specifically, the interleukin-2 analogs of the present invention may have a binding affinity of 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 binding affinity of natural interleukin-2 or aldesleukin for the interleukin-2α receptor, but the numerical value of the binding affinity is not limited, and as long as its binding affinity is changed compared to the binding affinity of natural interleukin-2 or aldesleukin, then the numerical value falls within the scope of the present invention.

[0102] Alternatively, relative to the binding affinity of aldesleukin for the interleukin-2α receptor (100%), the interleukin-2 analog of the present invention may have no binding affinity for the interleukin-2α receptor, or have a binding affinity for the interleukin-2α receptor 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, but the numerical value of the binding affinity is not limited, and as long as its binding affinity is changed compared to the binding affinity of native interleukin-2 or aldesleukin, then the numerical value falls within the scope of the present invention.

[0103] In addition, specifically, the interleukin-2 analogs of the present invention may specifically have a binding affinity for the interleukin-2 β receptor of 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 binding affinity of natural interleukin-2 or aldesleukin for the interleukin-2 β receptor, but the numerical value of the binding affinity is not limited, and as long as its binding affinity is changed compared to the binding affinity of natural interleukin-2 or aldesleukin, the numerical value falls within the scope of the present invention.

[0104] Alternatively, the interleukin-2 analogs of the present invention may have 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 1,000% or more, about 1,500% or more, about 3,000% or more, relative to the binding affinity of aldesleukin for the interleukin-2 beta receptor (100%). The binding affinity of the present invention may be about 5,000% or higher, about 7,000% or higher, about 10,000% or higher, about 12,000% or higher, about 15,000% or higher, about 20,000% or higher, or about 25,000% of the binding affinity to the interleukin-2 beta receptor, but the numerical value of the binding affinity is not limited, and as long as its binding affinity is changed compared to the binding affinity of native interleukin-2 or aldesleukin, then the numerical value falls within the scope of the present invention.

[0105] As used herein, the term "about" is meant to encompass ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​within the range that are equivalent to or approximately the value stated after the term, but is not limited thereto.

[0106] Compared to native interleukin-2 or aldesleukin, the interleukin-2 analogs of the present invention have altered binding affinity for the interleukin-2 alpha receptor and increased binding affinity for the interleukin-2 beta receptor.

[0107] In one embodiment of the present invention, to prepare the interleukin-2 analog of the present invention, the interleukin-2 analog is produced by introducing one or more mutations into native interleukin-2 (SEQ ID NO: 1). The interleukin-2 analog produced by the present invention may comprise an amino acid sequence of any one of SEQ ID NOs: 3 to 106, or may be encoded by a nucleotide sequence of any one of SEQ ID NOs: 108 to 211.

[0108] The nucleic acid encoding the interleukin-2 analog of the present invention may be modified so that modifications (deletions, substitutions, and / or additions of amino acids) are introduced into the amino acid(s) at specific positions(s) in the nucleotide sequence encoding the native interleukin-2 of SEQ ID NO: 1, and specifically, may include a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 3 to 106. As an example, the nucleic acid of the present invention may have or include the nucleotide sequence of any one of SEQ ID NOs: 108 to 211.

[0109] In the polynucleotides of the present invention, various modifications can be made in the coding region without changing the amino acid sequence of the interleukin-2 analog of the present invention, taking into account codon degeneracy or codon preference in the organism in which the nucleic acid of the present invention is to be expressed. Specifically, the nucleic acid of the present invention may have or include a nucleotide sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% and less than 100% homology or identity to the sequence of any one of SEQ ID NOs: 108 to 211, or may consist of or be primarily composed of a nucleotide sequence having 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% and less than 100% homology or identity to the sequence of any one of SEQ ID NOs: 108 to 211, but is not limited thereto.

[0110] In addition, the nucleic acids of the present invention may include, without limitation, probes, which may be prepared from known gene sequences (e.g., sequences that hybridize under stringent conditions to sequences that are complementary to all or part of the nucleic acid sequences of the present invention). The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; and F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8).

[0111] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine, while cytosine is complementary to guanine. Therefore, nucleic acids of the present invention can include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0112] The appropriate stringency for hybridizing polynucleotides depends on the length and complementarity of the polynucleotides, and these variables are well known in the art (eg, Sambrook et al., supra).

[0113] Homology and identity are as described above.

[0114] In addition, the interleukin-2 analogs of the present invention may have an increased in vivo half-life compared to native interleukin-2 or aldesleukin, but are not particularly limited thereto. For example, the interleukin-2 analogs of the present invention may include a long-acting conjugate form having an increased half-life by linking a biocompatible substance for increasing half-life (e.g., an immunoglobulin Fc region) to the interleukin-2 analog directly or via a linker, but are not limited thereto.

[0115] The long-acting conjugate according to the present invention not only includes an interleukin-2 analog with increased binding affinity to the interleukin-2β receptor, but also includes an immunoglobulin Fc region as a representative carrier to increase its half-life, thereby increasing the half-life of the interleukin-2 analog, increasing blood exposure, and increasing the in vivo immune response, thereby effectively achieving growth inhibition and reduction of cancer cells.

[0116] The above description may be applicable to other embodiments or aspects of the present invention, but is not limited thereto.

[0117] Another aspect of the compositions of the present invention may include, but is not limited to, long-acting conjugates of interleukin-2 analogs.

[0118] In the present invention, the long-acting conjugate of interleukin-2 analogues can be in the form of a biocompatible substance capable of increasing its half-life in vivo linked to the interleukin-2 analogue. In this article, biocompatible substance can be used interchangeably with carrier.

[0119] In the present invention, the long-acting conjugate can show an increased duration of efficacy compared to the interleukin-2 analog not conjugated to a carrier, and in the present invention, such a conjugate is referred to as a "long-acting conjugate" or "conjugate".

[0120] Such a conjugate may be one that does not occur in nature.

[0121] In one embodiment of the present invention, the long-acting conjugate is a long-acting conjugate represented by the following Chemical Formula 1: [Chemical Formula 1] X - L - F Wherein: X is an interleukin-2 analogue; L is a polyethylene glycol linker; F is the dimeric immunoglobulin Fc region, and - symbols represent covalent bonds between X and L and between L and F, respectively, In the long-acting conjugate, one end of L is covalently linked to only one polypeptide chain of the Fc region of the dimer, while X is covalently linked to the other end of L.

[0122] More specifically, in the long-acting conjugate, one molecule of X can be linked to one polypeptide chain of the Fc region via L. In the long-acting conjugate of the present invention, X and L, and L and F can be linked to each other via a covalent bond, respectively, wherein the conjugate can be a conjugate of the form of Chemical Formula 1 in which X, L and F are linked to each other via a covalent bond.

[0123] The interleukin-2 analog in the long-acting conjugate of the present invention has an altered binding affinity for the interleukin-2 receptor, and in particular, has an increased binding affinity for the interleukin-2 β receptor when present alone and not as part of the conjugate. Specifically, when present alone and not as part of the conjugate, the interleukin-2 analog of the present invention may have an increased binding affinity for the interleukin-2 β receptor compared to native interleukin-2 or known aldesleukin, and more specifically, may also have an altered (increased or decreased) binding affinity for the interleukin-2 α receptor.

[0124] In the present invention, the interleukin-2 analog may correspond to a part constituting the conjugate. Specifically, the interleukin-2 analog corresponds to X in Chemical Formula 1, and the interleukin-2 analog is as described above.

[0125] In the conjugate, F is a substance capable of increasing the half-life of X (ie, the interleukin-2 analog), and corresponds to one moiety constituting the conjugate of the present invention.

[0126] F and X may be linked to each other by a covalent chemical bond, and F and X may be linked to each other via L by a covalent chemical bond.

[0127] Specifically, F is an immunoglobulin Fc region, and the immunoglobulin Fc region may be an IgG Fc region or a non-glycosylated IgG4 Fc region, but is not particularly limited thereto.

[0128] As a specific example of the present invention, F (immunoglobulin Fc region) is a dimer composed of two polypeptide chains, and may have a structure in which one end of L is connected to only one of the two polypeptide chains, but is not limited thereto.

[0129] At least one amino acid side chain within the peptide of the present invention may be conjugated to a biocompatible substance to increase its in vivo solubility and / or half-life, and / or increase its bioavailability. These modifications may reduce the clearance rate of therapeutic proteins and peptides.

[0130] The biocompatible substances may be soluble (amphiphilic or hydrophilic) and / or non-toxic and / or pharmaceutically acceptable.

[0131] In a specific embodiment, the long-acting conjugate of the present invention may have an interleukin-2 analog and an immunoglobulin Fc region linked to each other, but is not limited thereto.

[0132] In the present invention, the term "immunoglobulin Fc region" refers to the region comprising the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3), excluding the heavy and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region can be part of the conjugate of the present invention. Specifically, the immunoglobulin Fc region corresponds to F in Chemical Formula 1.

[0133] In this article, the Fc region includes not only the native sequence obtained by papain digestion of immunoglobulins, but also its derivatives, for example, one or more amino acid residues in the native sequence are transformed by deletion, insertion, non-conservative or conservative substitution or a combination thereof and are therefore different from variants or analogs of the native sequence. It is assumed that the derivatives, substituents and variants retain the ability to bind to FcRn. In the present invention, F can be a human immunoglobulin region, but is not limited thereto. In this article, "biocompatible material" or "carrier" can refer to the Fc region.

[0134] The F (immunoglobulin Fc region) has a structure in which two polypeptide chains are linked to each other via a disulfide bond, but is not limited thereto, via a nitrogen atom in only one of the two chains. Linkage via a nitrogen atom can be achieved by reductive amination of the epsilon amino group or the N-terminal amino group of lysine.

[0135] The reductive amination reaction refers to a reaction in which an amine group or amino group of one reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to produce an amine, followed by a reduction reaction to form an amine bond, and is an organic synthesis reaction well known in the art.

[0136] In one embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region is linked to the linker via the nitrogen atom at its N-terminus.

[0137] The immunoglobulin Fc region may include, but is not limited to, a hinge region in a heavy chain constant region.

[0138] In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.

[0139] As used herein, the term "hinge sequence" refers to a site located in the heavy chain that forms a dimer of the immunoglobulin Fc region through an inter-disulfide bond.

[0140] In the present invention, the hinge sequence can be modified to have only one cysteine ​​residue by deleting a portion of the hinge sequence having the following amino acid sequence, but is not limited thereto: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 418).

[0141] The hinge sequence may include only one cysteine ​​residue by deleting the cysteine ​​residue at position 8 or position 11 in the hinge sequence of SEQ ID NO: 418. The hinge sequence of the present invention may consist of 3 to 12 amino acids including only one cysteine ​​residue, 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). 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) and Ser-Cys-Pro (SEQ ID NO: 437).

[0142] More specifically, the hinge sequence may include the amino acid sequence of SEQ ID NO: 428 (Pro-Ser-Cys-Pro) or SEQ ID NO: 437 (Ser-Cys-Pro), but is not limited thereto.

[0143] In a more specific embodiment of the long-acting conjugate of the present invention, in the conjugate, the N-terminus of the immunoglobulin Fc region is proline, and in the conjugate, the Fc region is linked to the linker via the nitrogen atom of the proline.

[0144] In one embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region may be in the form of a dimer, wherein due to the presence of the hinge sequence, the two chains of the immunoglobulin Fc region form a homodimer or heterodimer. The conjugate of Chemical Formula 1 of the present invention may be in the form of a linker connected to one chain of the immunoglobulin Fc region of the dimer, but is not limited thereto.

[0145] As used herein, the term "N-terminus" refers to the amino terminus of a protein or polypeptide, and includes the outermost end of the amino terminus, or includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids from the outermost end. In the immunoglobulin Fc region of the present invention, a hinge sequence may be included in the N-terminus, but is not limited thereto.

[0146] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), excluding the heavy and light chain variable regions of the immunoglobulin, as long as it has substantially the same or improved effects as compared to its native form. Alternatively, the immunoglobulin Fc region may be a region in which a portion of a relatively long amino acid sequence corresponding to CH2 and / or CH3 has been removed.

[0147] For example, the immunoglobulin Fc region of the present invention can be 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 one or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain and an immunoglobulin hinge region (or a portion of the hinge region), and 6) a dimer between each domain of the heavy chain constant region and the light chain constant region, but is not limited thereto.

[0148] In the present invention, the immunoglobulin Fc region may be a dimer or multimer composed of single-chain immunoglobulins composed of domains derived from the same source, but is not limited thereto.

[0149] In one embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region F is a dimer composed of two polypeptide chains, wherein the dimer Fc region F and X are covalently linked to each other via a linker L (comprising repeating ethylene glycol units). In a specific embodiment, X is covalently linked to only one of the two polypeptide chains of the dimer Fc region F via linker L. In a more specific embodiment, only one X molecule is covalently linked to the one polypeptide chain of the two polypeptide chains of the dimer Fc region F to which X is linked via L. In a most specific embodiment, F is a homodimer.

[0150] In another embodiment, the immunoglobulin Fc region F is a dimer composed of two polypeptide chains, wherein one end of L is connected to only one of the two polypeptide chains, but is not limited thereto.

[0151] In another embodiment of the long-acting conjugate of the present invention, two X molecules can be symmetrically linked to one Fc region in the form of a dimer. Specifically, the immunoglobulin Fc region and X can be linked to each other via L. However, this connection is not limited to the above embodiment.

[0152] The immunoglobulin Fc region of the present invention includes not only the natural amino acid sequence but also its derivatives. An amino acid sequence derivative means a different sequence due to deletion, insertion, non-conservative or conservative substitution or a combination thereof of one or more amino acid residues in the natural amino acid sequence.

[0153] For example, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331 in IgG Fc, which are known to be important for bonding, can be used as sites suitable for variation.

[0154] Furthermore, various types of derivatives can be obtained by, for example, removing sites capable of forming disulfide bonds, deleting certain amino acid residues at the N-terminus of the native Fc region, or adding a methionine residue to the N-terminus of the native Fc region. Furthermore, to eliminate effector functions, complement binding sites (e.g., C1q binding sites) can be removed, as can antibody-dependent cellular cytotoxicity (ADCC) sites. Techniques for preparing sequence derivatives of immunoglobulin Fc regions are disclosed in WO 97 / 34631 and WO 96 / 32478, among others.

[0155] Amino acid exchanges in proteins or peptides that do not alter the overall activity of the molecule are well known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). 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, amino acids can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation, amidation, or the like.

[0156] The above-mentioned Fc derivatives exhibit biological activities equivalent to those of the Fc region of the present invention, and may have increased structural stability to heat, pH, etc., compared to the Fc region.

[0157] Such Fc regions can be obtained from natural forms isolated from living organisms of humans or animals such as cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or recombinant forms or derivatives thereof obtained from transformed animal cells or microorganisms. Specifically, the Fc region can be obtained from natural immunoglobulins by isolating intact immunoglobulins from living humans or animals and then treating the separated immunoglobulins with proteases. When treated with papain, the separated immunoglobulins are digested into Fab and Fc, and when treated with pepsin, they are digested into pF'c and F(ab)2. These fragments can be separated from Fc and pF'c by size exclusion chromatography or similar methods. In a more specific embodiment, the immunoglobulin Fc region is a recombinant immunoglobulin Fc region in which the human Fc region is obtained from a microorganism.

[0158] Furthermore, the immunoglobulin Fc region may be in a form with native glycans, glycans increased compared to the native form, glycans decreased compared to the native form, or a deglycosylated form. The increase, reduction, or removal of immunoglobulin Fc glycans can be achieved by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In particular, immunoglobulin Fc regions obtained by glycan removal exhibit a dramatic decrease in binding affinity for complement C1q and a reduction or loss of antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity, thereby avoiding the induction of an unwanted immune response in vivo. Therefore, deglycosylated or non-glycosylated immunoglobulin Fc regions may be more suitable as drug carriers for the purposes of the present invention.

[0159] As used herein, the term "deglycosylated" indicates an Fc region from which glycans have been removed enzymatically, while the term "non-glycosylated" indicates an Fc region that has been enzymatically removed in prokaryotes, more specifically in Escherichia coli ( E. coli ) and is not glycosylated.

[0160] Meanwhile, the immunoglobulin Fc region may be derived from humans or other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and may be more specifically derived from humans.

[0161] In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, or a combination or hybrid thereof. In yet more specific embodiments, the immunoglobulin Fc region is derived from IgG or IgM, which are most abundant in human blood, and in yet more specific embodiments, the immunoglobulin Fc region is derived from IgG, which is known to increase the half-life of ligand-binding proteins. In yet more specific embodiments, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0162] In one embodiment, the immunoglobulin Fc fragment can be a human IgG4 Fc fragment in the form of a homodimer, wherein the two monomers are linked to each other via a disulfide bond (interchain form) formed between cysteine, the third amino acid of each monomer. Specifically, each monomer of the homodimer has / can have a disulfide bond formed between cysteine ​​at position 35 and cysteine ​​at position 95 and a disulfide bond formed between cysteine ​​at position 141 and cysteine ​​at position 199, i.e., two disulfide bonds (intrachain form).

[0163] Regarding the number of amino acids, each monomer may be composed of 221 amino acids, and the number of amino acids constituting the homodimer may be a total of 442, but is not limited thereto. Specifically, the immunoglobulin Fc fragment may be in the form of a homodimer, in which two monomers each having the amino acid sequence of SEQ ID NO: 438 (composed of 221 amino acids) are linked to each other via a disulfide bond between the amino acid cysteine ​​at position 3 of each monomer, wherein the monomers of the homodimer each independently have an intrachain disulfide bond formed between cysteines at positions 35 and 95 and an intrachain disulfide bond formed between cysteines at positions 141 and 199, but is not limited thereto.

[0164] In Chemical Formula 1, F may include a monomer of the amino acid sequence of SEQ ID NO: 438, and F may be a homodimer of the monomer of the amino acid sequence of SEQ ID NO: 438, but is not limited thereto.

[0165] As an example, the immunoglobulin Fc region may be a homodimer including the amino acid sequence of SEQ ID NO: 439 (consisting of 442 amino acids), but is not limited thereto.

[0166] In one embodiment, the immunoglobulin Fc region and X may be aglycosylated, but is not limited thereto.

[0167] As used herein, the term "combination" refers to a polypeptide encoding a single-chain immunoglobulin Fc region from the same source linked to a single-chain polypeptide from a different source to form a dimer or multimer. In other words, the dimer or multimer can be prepared from two or more fragments selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0168] As used herein, the term "hybrid" means that sequences encoding two or more immunoglobulin Fc fragments of different origin are present in a single-chain immunoglobulin constant region. In the present invention, several types of hybrids are possible. That is, the hybrid domain can be composed 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, and can include a hinge region.

[0169] Meanwhile, IgG can also be divided into IgG1, IgG2, IgG3 and IgG4 subclasses, and the present invention also includes combinations thereof or hybrids thereof. Specifically, IgG2 and IgG4 subclasses are preferred, and more specifically, the Fc fragment of IgG4 is preferred, which has almost no effector functions such as complement-dependent cytotoxicity (CDC).

[0170] In addition, the conjugate can have an increased duration of efficacy compared to native interleukin-2 or aldesleukin, or compared to X without F modification, and the conjugate can be in not only the above-mentioned form but also a form encapsulated in biodegradable nanoparticles, but is not limited thereto.

[0171] In the present invention, a "polyethylene glycol linker" includes a biocompatible polymer having two or more repeating units linked to each other. The repeating units are linked to each other by any covalent bond other than a peptide bond. The polyethylene glycol linker can be a component of the conjugate of the present invention. Herein, the polyethylene glycol linker is used interchangeably with a "non-peptidyl linker" or a "non-peptidyl polymer."

[0172] In one embodiment, the conjugate may be a conjugate in which F and X are covalently linked to each other via a non-peptidyl linker, and includes reactive groups capable of binding to F (specifically, an immunoglobulin Fc region) and X (specifically, an interleukin-2 analogue) at both ends.

[0173] Specifically, in the present invention, the non-peptidyl linker may include a reactive group at its end and thus form the conjugate by reacting with other components constituting the conjugate. When the non-peptidyl linker having reactive functional groups at both ends is bound to X and F in Chemical Formula 1 through their respective reactive groups to form a conjugate, the non-peptidyl linker or non-peptidyl polymer may be referred to as a non-peptidyl polymer linker portion or a non-peptidyl linker portion.

[0174] In one embodiment, L (polyethylene glycol linker) can be a linker containing ethylene glycol repeating units (e.g., polyethylene glycol), but is not limited thereto. As used herein, polyethylene glycol is a term encompassing all forms of ethylene glycol homopolymers, PEG copolymers, and monomethyl-substituted PEG polymers (mPEG), but is not particularly limited thereto. Furthermore, derivatives thereof known in the art and readily prepared within the capabilities of those skilled in the art are also encompassed within the scope of the present invention.

[0175] The polyethylene glycol linker may include a functional group used to prepare the conjugate before forming the conjugate at its end, and may include ethylene glycol repeating units. The long-acting conjugate according to the present invention may be in a form in which X and F are linked by a functional group, but is not limited thereto. In the present invention, the non-peptidyl linker may include two or three or more functional groups, and each functional group may be the same or different from each other, but is not limited thereto.

[0176] Specifically, the linker may include repeating units each represented by the following Chemical Formula 2. An example thereof may be polyethylene glycol (PEG), but is not limited thereto: [Chemical Formula 2] wherein n is 10 to 2,400, n is 10 to 480, or n is 50 to 250, but is not limited thereto.

[0177] In long-acting conjugates, the PEG moiety may include -(CH2CH2O) n - structure and inserted in the connecting element with -(CH2CH2O) n - oxygen atoms between, but not limited to.

[0178] In one embodiment, the ethylene glycol repeating unit can be represented by, for example, [OCH2CH2]n, wherein the value of n is a natural number and can be determined so that the average molecular weight of the [OCH2CH2]n moiety in the interleukin-2 analog conjugate is, for example, a number average molecular weight greater than 0 kDa to about 100 kDa, but is not limited thereto. As an example, the value of n is a natural number, and the average molecular weight of the [OCH2CH2]n sites in the interleukin-2 analog conjugate, for example, the number average molecular weight can be 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 2 to about 30 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 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, 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 are not limited thereto.

[0179] In a specific embodiment, the conjugate may have a structure in which the interleukin-2 analog and the immunoglobulin Fc region (F) are covalently linked via a linker (L) comprising ethylene glycol repeating units, but is not limited thereto.

[0180] In another embodiment, in the long-acting conjugate, L may be a linker comprising repeating ethylene glycol units, and F may be a dimerized immunoglobulin Fc region. More specifically, one molecule of X may be covalently linked to one of the Fc regions in the dimerized Fc region via a linker comprising repeating ethylene glycol units, but the present invention is not limited thereto. In another embodiment, one end of the linker comprising repeating ethylene glycol units may be linked to only one of the two Fc region chains in the dimerized immunoglobulin Fc region, but the immunoglobulin Fc region is not limited thereto.

[0181] The molecular weight of the polyethylene glycol linker that can be used in the present invention can be in the range of greater than 0 kDa to 200 kDa, specifically in the range of about 1 kDa to 100 kDa, about 1 kDa to 50 kDa, about 1 kDa to 30 kDa, about 2 kDa to 30 kDa, about 1 kDa to 20 kDa, more specifically in the range of about 3.4 kDa to 10 kDa, and even more specifically 3.4 kDa, but is not limited thereto. In addition, the non-peptidyl linker of the present invention that is linked to the polypeptide corresponding to F can use not only one type of polymer but also a combination of different types of polymers.

[0182] As used herein, the term "about" is meant to encompass ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​within the range that are equivalent to or approximately the value stated after the term, but is not limited thereto.

[0183] Specifically, while not binding to F and X, the non-peptidyl linker may have reactive groups at both ends thereof and may bind to F and X through these reactive groups.

[0184] In one embodiment, both ends of the linker can be 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 are not limited thereto.

[0185] Specifically, the linker may include reactive groups at both ends thereof that are capable of binding to the immunoglobulin Fc region and the interleukin-2 analog (X), respectively. Specifically, the linker may include reactive groups that are capable of binding to the thiol group of cysteine ​​in the immunoglobulin Fc region; the amino group at the N-terminus, lysine, arginine, glutamine, and / or histidine; and / or the hydroxyl group at the C-terminus; and the thiol group of cysteine; the amino group of lysine, arginine, glutamine, and / or histidine; the azide group of azide-lysine; and / or the hydroxyl group of the interleukin-2 analog (X), but are not limited thereto.

[0186] More specifically, the reactive group of the linker may be one or more selected from an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0187] In the above description, examples of the aldehyde group may include a propionaldehyde group or a butyraldehyde group, but are not limited thereto.

[0188] In the above description, examples of the succinimide derivative may include succinimidyl valerate, succinimidyl methylbutyrate, succinimidyl methylpropionate, succinimidyl butyrate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidylcarboxymethyl, or succinimidyl carbonate, but are not limited thereto.

[0189] The linker can be linked to the immunoglobulin Fc region F and the interleukin-2 analog X via these reactive groups, thereby converting it into a linker moiety.

[0190] Furthermore, the final product produced by reductive alkylation via an aldehyde bond is more stable than the bond formed via an amide bond. The aldehyde-reactive group selectively reacts with the N-terminus at low pH while forming a covalent bond with a lysine residue at high pH (e.g., at pH 9.0).

[0191] In addition, the reactive groups at both ends of the linker may be the same or different, and for example, an aldehyde group may be provided at both ends, or a maleimide group may be provided at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group may be provided at the other end. However, these reactive groups are not particularly limited thereto, as long as F, specifically an immunoglobulin Fc region, and X can be linked to the ends of the linker, respectively.

[0192] For example, the linker may include a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group as a reactive group at the other end.

[0193] When polyethylene glycol having hydroxyl reactive groups at both ends is used as a linker, the interleukin-2 analog long-acting conjugate of the present invention can be produced by activating the hydroxyl groups into various reactive groups through known chemical reactions or by utilizing commercially available polyethylene glycol having modified reactive groups.

[0194] In a specific embodiment, the linker can be attached to the cysteine ​​residue of X, more specifically the -SH group of cysteine, but is not limited thereto.

[0195] Specifically, the reactive group of the linker can be linked to the -SH group of a cysteine ​​residue, and all of the aforementioned reactive groups are applicable to the reactive group. When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X via a thioether bond, while the aldehyde group can be linked to the -NH2 group of F (specifically, immunoglobulin Fc) via reductive amination, but is not limited thereto.

[0196] In another embodiment, the linker can be attached to a lysine residue of X, more specifically, the amino group of lysine, but not limited thereto.

[0197] In the conjugate, the reactive group of the linker can be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but is not limited thereto.

[0198] Furthermore, in the conjugate, the interleukin analog according to the present invention may be linked to a linker having a reactive group via its C-terminus, but this is merely an example.

[0199] As used herein, the term "C-terminus" refers to the carboxyl terminus of a peptide, and for the purposes of the present invention, the term refers to a site capable of binding to a linker. For example, the C-terminus may include not only the outermost amino acid residue of the C-terminus, but also amino acid residues near the C-terminus, and specifically, including amino acid residues 1 to 20 from the outermost end, but is not limited thereto.

[0200] Furthermore, the above conjugate may have an increased duration of efficacy compared to X without F modification, and such a conjugate may be in not only the above form but also a form encapsulated in biodegradable nanoparticles.

[0201] Specifically, the interleukin-2 analog long-acting conjugate of the present invention may not have a binding affinity for the interleukin-2α receptor, or may have a binding affinity for the interleukin-2α receptor of 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 compared to natural interleukin-2, aldesleukin or a long-acting conjugate thereof , 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 of the binding affinity, but the numerical value of the binding affinity is not limited, and as long as its binding affinity is changed compared to the binding affinity of natural interleukin-2 or aldesleukin, then the numerical value falls within the scope of the present invention.

[0202] In addition, specifically, the interleukin-2 analog long-acting conjugate of the present invention may have a binding affinity for the interleukin-2 β receptor of 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 or more, about 130 times or more, about 150 times or more, or about 200 times or more compared to the binding affinity of natural interleukin-2, aldesleukin, or a long-acting conjugate including the same, but the numerical value of the binding affinity is not limited, and as long as its binding affinity is changed or increased compared to the binding affinity of natural interleukin-2 or aldesleukin, the numerical value falls within the scope of the present invention.

[0203] Unless otherwise specified herein, descriptions or claims regarding an "interleukin-2 analog" or "conjugate" according to the present invention—where the interleukin-2 analog is covalently linked to a biocompatible substance—may apply to forms encompassing not only the corresponding interleukin-2 analog or conjugate, but also salts (e.g., pharmaceutically acceptable salts of the interleukin-2 analog) or solvates thereof. Therefore, even if only an "interleukin-2 analog" or "conjugate" is described herein, the corresponding description equally applies to its specific salt, specific solvate, and specific solvate of its specific salt. These salts can be, for example, in the form of any pharmaceutically acceptable salt. The type of salt is not particularly limited. However, the salt is preferably, but not particularly limited to, a salt that is safe and effective for a subject (e.g., a mammal).

[0204] The type of salt is not particularly limited. However, the salt is preferably a salt that is safe and effective for a subject (eg, a mammal), but is not particularly limited thereto.

[0205] The term "pharmaceutically acceptable" refers to substances that are, within the scope of medical and pharmaceutical judgment, effective for the intended purpose without causing undue toxicity, irritation, allergic response, and the like.

[0206] As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. 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, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals (such as sodium and potassium), alkaline earth metals (such as magnesium), ammonium, and the like.

[0207] The term "solvate" as used herein refers to a complex formed by an interleukin-2 analogue or a salt thereof according to the present invention and solvent molecules.

[0208] The composition according to the present invention may include an interleukin-2 analogue or a long-acting conjugate thereof, and more specifically, the composition may be a pharmaceutical composition for cancer prevention or treatment when it is administered in combination with an immune checkpoint inhibitor or a composition comprising the same. Interleukin-2 analogues and long-acting conjugates thereof are as described above. More specifically, the composition according to the present invention may include a pharmacologically effective amount of an interleukin-2 analogue or a long-acting conjugate thereof, and further include a pharmaceutically acceptable carrier when it is administered in combination with a pharmacologically effective amount of an immune checkpoint inhibitor or a composition comprising the same.

[0209] Optionally, the composition of the present invention may further comprise an immune checkpoint inhibitor, but is not limited thereto.

[0210] In a specific embodiment, the composition according to the present invention may include an interleukin-2 analogue comprising an amino acid sequence selected from any one of SEQ ID NOs: 3 to 106, or a long-acting conjugate thereof, and more specifically, the composition according to the present invention may include an interleukin-2 analogue comprising an amino acid sequence selected from any one of SEQ ID NOs: 22, 42, 53, 87, 105 and 106, or a long-acting conjugate thereof, and may be administered in combination with an immune checkpoint inhibitor or a composition comprising the same.

[0211] The composition comprising an interleukin-2 analog or a long-acting conjugate of an interleukin-2 analog of the present invention can be administered simultaneously, sequentially, or in the reverse order with the composition comprising an immune checkpoint inhibitor, but is not limited thereto.

[0212] As used herein, the term "pharmaceutically effective amount" refers to a safe dose at which an interleukin-2 analog or a long-acting conjugate thereof and an immune checkpoint inhibitor exhibit cancer prevention or treatment effects in patients without exhibiting toxicity or side effects. Specifically, the term may refer to a dose that exhibits significant activity at interleukin-2 receptors (e.g., β and / or α receptors) or a dose that activates T cells, but is not limited thereto.

[0213] The composition according to the present invention may exhibit one or more of the following properties, but is not limited thereto, as long as the composition exhibits increased immune response, anti-cancer effect, and the like.

[0214] (i) higher blood exposure compared to aldesleukin alone or in combination with an immune checkpoint inhibitor; (ii) superior tumor growth inhibition compared to aldesleukin alone or in combination with an immune checkpoint inhibitor; (iii) superior memory T cell responses compared to aldesleukin alone or in combination with an immune checkpoint inhibitor; (iv) superior inhibition of immune checkpoint protein activation compared to administration of aldesleukin alone or in combination with an immune checkpoint inhibitor; and (v) Superior T cell activation compared to aldesleukin alone or in combination with immune checkpoint inhibitors.

[0215] Interleukin-2, also known as T cell growth factor, is a protein involved in immune regulation and has the activity of proliferating T cells, stimulating B cells, and secreting interferon-gamma by acting on T cells. Based on the immunomodulatory activity of interleukin-2, cancer prevention or treatment can be achieved by harnessing the body's immune system to eliminate cancer cells.

[0216] Specifically, the interleukin-2 analogs of the present invention have increased binding affinity for the interleukin-2 beta receptor, which plays a major role in signal transduction, thereby resulting in a more effective anti-cancer effect in the subject's immune system. In addition, the long-acting conjugates comprising the interleukin-2 analogs have increased binding affinity for the interleukin-2 beta receptor, increased duration of efficacy, and high blood exposure, resulting in excellent bioavailability, thereby exhibiting excellent tumor growth inhibition ability, thereby exhibiting effective cancer prevention or treatment effects. In addition, the long-acting conjugates have excellent memory T cell generation ability, and thus can exhibit cancer recurrence inhibition effects through the subject's immune memory response, thereby producing safe and effective cancer drugs.

[0217] When an immune checkpoint inhibitor is used together with the interleukin-2 analog according to the present invention, the immune checkpoint inhibitor activates T cells, thereby allowing the T cells to attack cancer cells that evade the immune system through immune checkpoints, thereby achieving excellent effects resulting from the combined administration.

[0218] As used herein, the term "immune checkpoint inhibitor" refers to a drug that activates T cells to attack cancer cells by binding to immune checkpoint proteins involved in suppressing T cells.

[0219] Immune checkpoint inhibitors are drugs that prevent immune checkpoint proteins (e.g., PD-1) from binding to cancer cell ligands (e.g., PD-L1), thereby maintaining the original functions of immune cells (e.g., T cells).

[0220] Immune checkpoint inhibitors may include peptides, antibodies or antigen-binding fragments thereof, nucleic acid molecules, and small molecules, but are not limited thereto, as long as they can have an antagonistic effect.

[0221] Examples of the immune checkpoint inhibitors of the present invention may include anti-PD-1 antibodies or antigen-binding fragments thereof.

[0222] Programmed cell death-1 (PD-1)—one of the most well-known immune checkpoint proteins—is located outside immune cells and specifically binds to the programmed death-ligand 1 (PD-L1) protein on various cells in the body. The binding of PD-L1 to PD-1 inactivates immune cell function, suppressing the immune response and thus preventing the onset of unnecessary autoimmune responses. However, large amounts of PD-L1 expressed on the surface of cancer cells bind to PD-1 on immune cells, inhibiting T cell function and preventing the body's immune response. Specifically, cancer cells survive by evading the immune response.

[0223] The immune checkpoint inhibitor of the present invention antagonizes immune checkpoint proteins used by cancer cells for immune evasion, thereby appropriately ensuring immune responses that may be disabled by cancer cells, thereby utilizing the immune response in the body to produce anti-cancer effects.

[0224] In the present invention, the anti-programmed cell death-1 (PD-1) antibody may be selected from nivolumab, pembrolizumab, cemiplizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalizumab, dotalimumab, INCMGA00012, AMP-224 and AMP-514, but is not limited thereto.

[0225] The pharmaceutical composition of the present invention comprising an interleukin-2 analogue or a long-acting conjugate thereof for preventing or treating cancer is administered in combination with an immune checkpoint inhibitor or a composition comprising the same, thereby exhibiting excellent anti-cancer efficacy and reducing side effects through the synergistic effect of the interleukin-2 analogue or its long-acting conjugate and the immune checkpoint inhibitor.

[0226] Immune checkpoint inhibitors are known to exhibit different levels of responsiveness depending on the phenotype of the tumor microenvironment (TME), and these immune checkpoint inhibitors have limitations as cancer drugs because of very low responsiveness in immune-infiltrated tumors (cold tumors).

[0227] The interleukin-2 analogs or their long-acting conjugates of the present invention can alter the tumor microenvironment, which is hyporesponsive to immune checkpoint inhibitors, thereby triggering a transition from immune-infiltrated tumors to infiltrated tumors (hot tumors). Therefore, the combined administration of immune checkpoint inhibitors and interleukin-2 analogs or their long-acting conjugates can achieve excellent therapeutic effects.

[0228] Programmed cell death-1 (PD-1)—one of the immune checkpoint proteins—is located outside immune cells and specifically binds to the programmed death ligand 1 (PD-L1) protein on various cells in the human body.

[0229] The pharmaceutical composition for preventing or treating cancer of the present invention comprising an interleukin-2 analog or a long-acting conjugate thereof can more effectively treat cancers that have low responsiveness to PD-1 antagonists, but is not limited thereto.

[0230] In the present invention, examples of cancer may include renal cell carcinoma, melanoma, colorectal cancer, liver cancer, uterine cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, breast cancer, bladder cancer, stomach cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal pelvis cancer, CNS tumors, primary CNS lymphoma, spinal tumors, brain tumors, gliomas (astrocytomas, glioblastomas, oligodendrogliomas, ependymomas), blastomas, meningiomas, brain stem gliomas, pituitary adenomas, neuroblastomas, congenital tumors, craniopharyngiomas and brain tumors, but are not limited to these.

[0231] Specifically, the cancer may be any one selected from colorectal cancer, liver cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, kidney cancer, lung cancer, skin cancer, melanoma, breast cancer, bladder cancer, and gastric cancer, but is not limited thereto. Alternatively, the cancer according to the present invention may be a primary cancer, a recurrent cancer, or a metastatic cancer, but is not limited thereto. In addition, the cancer may also include metastatic renal cell carcinoma or metastatic melanoma. More specifically, the cancer may be a cancer that is hyporesponsive to a PD-1 antagonist.

[0232] Although not particularly limited, the pharmaceutical composition of the present invention may contain 0.01% to 99% w / v of the interleukin-2 analog or its long-acting conjugate.

[0233] Specifically, the composition of the present invention may include an interleukin-2 analog or a long-acting conjugate thereof, and an immune checkpoint inhibitor, each of which is administered in an amount of 0.00001 mg / mL to 100 mg / mL, but is not limited thereto.

[0234] More specifically, in the composition of the present invention, the long-acting conjugate of the interleukin-2 analogue may be included at 0.0001 mg / kg to 2120 mg / kg, and the immune checkpoint inhibitor may be administered in combination with the long-acting conjugate of the interleukin-2 analogue at 0.0001 mg / kg to 1000 mg / kg, but is not limited thereto. Alternatively, in the composition of the present invention, the interleukin-2 analogue may be included at 0.00005 mg / kg to 500 mg / kg, and the immune checkpoint inhibitor may be administered in combination with the interleukin-2 analogue at 0.0001 mg / kg to 1000 mg / kg, but is not limited thereto.

[0235] However, the dose of the immune checkpoint inhibitor can be varied to exhibit the superior effects of combined administration of the interleukin-2 analog or its long-acting conjugate of the present invention.

[0236] For example, the amount of the immune checkpoint inhibitor contained in the composition of the present invention can be expressed based on the PD-1 inhibitor, and when another immune checkpoint inhibitor is contained, the composition may contain the other immune checkpoint inhibitor in an amount corresponding to the dose of the PD-1 inhibitor, but is not limited thereto.

[0237] In addition, the interleukin-2 analogue or its long-acting conjugate and the immune checkpoint inhibitor contained in the composition of the present invention can also be administered in appropriately divided doses, and the number of administrations can be the same or different from each other. For example, when the interleukin-2 analogue or its long-acting conjugate of the composition is administered once a week, the immune checkpoint inhibitor can be administered once, twice or more times a week, but is not limited thereto.

[0238] As used herein, the term "prevention" refers to any effect of inhibiting or delaying cancer or tumors by administering: an interleukin-2 analog (e.g., an interleukin-2 analog itself or a long-acting conjugate of an interleukin-2 analog with a biocompatible substance) or a composition comprising the same; and an immune checkpoint inhibitor or a composition comprising the same.

[0239] As used herein, the term "treatment" refers to any effect that alleviates or favorably alters the symptoms of cancer by administering: an interleukin-2 analog (e.g., an interleukin-2 analog itself or a long-acting conjugate of an interleukin-2 analog with a biocompatible substance) or a composition comprising the same; and an immune checkpoint inhibitor or a composition comprising the same.

[0240] The use of the interleukin-2 analogue or its long-acting conjugate and immune checkpoint inhibitor of the present invention can significantly increase blood exposure, blood half-life and duration of in vivo efficacy, thereby reducing the number of administrations and improving the quality of life of patients.

[0241] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable carrier or diluent may not be naturally occurring.

[0242] The term "pharmaceutically acceptable" as used herein means an amount sufficient to exhibit therapeutic effects without causing side effects, and such amount can be easily determined by those skilled in the art based on factors well known in the medical field such as the type of disease, the patient's age, weight, health condition and sex, the patient's sensitivity to drugs, administration routes, administration methods, number of administrations, duration of treatment, combined or concurrently used drugs.

[0243] The pharmaceutical composition of the present invention comprising an interleukin-2 analog or a long-acting conjugate thereof or an immune checkpoint inhibitor may contain a pharmaceutically acceptable excipient. Although there are no particular limitations on the excipient, binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, and the like may be used for oral administration, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, and the like may be mixed for injection, and bases, excipients, lubricants, preservatives, and the like may be used for topical administration.

[0244] The compositions of the present invention can be formulated in various ways by mixing with the pharmaceutically acceptable excipients described above. For example, for oral administration, the compositions of the present invention can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, or the like, while for injection, the compositions can be formulated into single-dose ampoules or multiple-dose forms. The compositions can be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or the like.

[0245] Meanwhile, examples of carriers, excipients and diluents suitable for formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the composition may also include fillers, anticoagulants, lubricants, wetting agents, flavorings, preservatives, etc.

[0246] Furthermore, the pharmaceutical composition of the present invention may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal liquid medicines, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0247] In addition, the composition can also be formulated into a single dosage form suitable for the patient's body, and specifically, can be formulated into a preparation for protein drug administration, and can be administered by an administration method commonly used in the art, through an oral route or a parenteral route including cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, intrapulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal route, but not limited thereto.

[0248] In addition, the conjugate can be used by mixing with various carriers approved as drugs, such as physiological saline or organic solvents, and in order to improve stability or absorbability, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as drugs.

[0249] According to another aspect of the present invention, a method for preventing or treating cancer is provided, comprising administering to a subject in need thereof a pharmaceutical composition comprising an interleukin-2 analog or a long-acting conjugate thereof in combination with an immune checkpoint inhibitor or a composition comprising the same. In the method, the pharmaceutical composition comprising an interleukin-2 analog or a long-acting conjugate thereof and the immune checkpoint inhibitor or a composition comprising the same may be administered simultaneously, sequentially, or in reverse order, but is not limited thereto.

[0250] Interleukin-2 analogs and / or long-acting conjugates of interleukin-2 analogs, immune checkpoint inhibitors, compositions comprising the same, cancer, prevention and treatment are as described above.

[0251] According to another aspect of the present invention, there is provided a method for preventing or treating cancer, the method comprising administering to a subject an interleukin-2 analog or a long-acting conjugate comprising the same in combination with an immune checkpoint inhibitor.

[0252] The method may include administering to the subject a composition comprising an interleukin-2 analog or a long-acting conjugate thereof and an immune checkpoint inhibitor, or administering an interleukin-2 analog or a long-acting conjugate thereof and an immune checkpoint inhibitor as separate preparations, but is not limited thereto.

[0253] In the method of the present invention, (i) the interleukin-2 analog or the long-acting conjugate comprising the same and (ii) the immune checkpoint inhibitor can be administered as one preparation, or can be administered as separate preparations simultaneously, sequentially, or in reverse order, but the present invention is not limited thereto.

[0254] The interleukin-2 analog or the long-acting conjugate comprising the same, the immune checkpoint inhibitor, the composition, the cancer, the prevention and the treatment are as described above.

[0255] In the present invention, the subject refers to a subject having cancer or suspected of having cancer, meaning mammals, including humans, rats, livestock, etc., but any subject that can be treated with the interleukin-2 analogs and / or conjugates, immune checkpoint inhibitors or compositions comprising the same of the present invention can be included without limitation.

[0256] As used herein, the term "administer" refers to introducing a predetermined substance (e.g., an interleukin-2 analog, a long-acting conjugate thereof, or an immune checkpoint inhibitor) into a patient by any appropriate method. The route of administration of the substance may include, but is not particularly limited to, any general route by which the substance can reach a target in the body, and for example, the route of administration may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, or the like.

[0257] The method of the present invention may include administering a pharmaceutically effective amount of a pharmaceutical composition comprising an interleukin-2 analog or a long-acting conjugate thereof and administering a pharmaceutically effective amount of an immune checkpoint inhibitor or a pharmaceutical composition comprising the same. A suitable total daily dose may be determined by the practitioner within an appropriate medical decision and may be administered once or several times in divided doses. For the purposes of the present invention, a specific therapeutically effective dose for a specific patient may preferably be applied differently, depending on various factors well known in the medical field, including the type and extent of the response to be achieved, the specific composition (including whether other agents are used together in some cases), the patient's age, weight, general health, sex and diet, the time of administration of the composition, the route of administration, the excretion rate, the duration of treatment, other drugs used in combination with or concurrently with the composition of the present invention, and similar factors well known in the medical field.

[0258] In the methods of the present invention, the dosage and frequency of administration are determined based on the type of drug (which is the active ingredient) and several related factors, such as the disease to be treated, the route of administration, the patient's age, sex, and weight, and the severity of the disease. Specifically, the compositions of the present invention may include a pharmaceutically effective amount of an interleukin-2 analog or a long-acting conjugate thereof and an immune checkpoint inhibitor, but are not limited thereto.

[0259] The term "pharmaceutically effective amount" refers to the degree to which the interleukin-2 analog or its long-acting conjugate and the immune checkpoint inhibitor can achieve the desired pharmaceutical activity (e.g., prevention, alleviation, or treatment of cancer) due to the interleukin-2 analog or its long-acting conjugate and the immune checkpoint inhibitor. It may also refer to a level at which toxicity or side effects are absent or minimal in the subject to be administered, up to a pharmaceutically acceptable level, but is not limited thereto. This pharmaceutically effective amount can be determined by comprehensively considering the number of administrations, the patient, the formulation, and the like.

[0260] Although not particularly limited, the pharmaceutical composition of the present invention may contain the ingredient (active ingredient) in an amount of 0.01% to 99% (w / v).

[0261] The total effective amount of the composition of the present invention can be administered to the patient in a single dose, or it can be administered in multiple doses for a long period of time according to a graded treatment regimen. The pharmaceutical composition of the present invention may include an active ingredient, the content of which may vary according to the severity of the disease. Specifically, the preferred total daily dose of the interleukin-2 analogue of the present invention or its long-acting conjugate and immune checkpoint inhibitor administered in combination can be about 0.0001 mg to 500 mg per 1 kg of patient body weight. However, with regard to the dosage of interleukin-2 analogues or its conjugates and immune checkpoint inhibitors, the effective dose for the patient is determined by taking into account various factors including the patient's age, weight, health status, sex, severity of the disease, diet and excretion rate in addition to the route of administration of the pharmaceutical composition and the number of times it is used for treatment. Therefore, taking these into account, those skilled in the art can determine an appropriate effective dose according to the specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to formulation, route of administration and mode of administration, as long as the pharmaceutical composition exhibits the effects of the present invention.

[0262] The pharmaceutical composition of the present invention can have excellent in vivo duration and titer, thereby significantly reducing the number and frequency of administration of the pharmaceutical preparation of the present invention. The pharmaceutical composition can be administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary or rectal routes, but administration is not limited to a specific route of administration, as long as the desired pharmacological effect can be achieved.

[0263] As an example, the pharmaceutical composition of the present invention can be administered once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, or can be administered once or more at intervals ranging from 1 week to 1 month, but the frequency is not limited thereto.

[0264] Specifically, the interleukin-2 analog or its long-acting conjugate and the immune checkpoint inhibitor can be administered in an amount of about 0.00001 mg or more or 0.0000001 mg or more per kg of patient body weight per week, respectively, and when the two substances are used in combination, the total amount is about 0.00001 mg or more per kg of patient body weight per week, but is not limited thereto.

[0265] Specifically, in the method of the present invention, 0.00001 mg to 175 mg of the interleukin-2 analog long-acting conjugate and 0.0000001 mg to 100 mg of the immune checkpoint inhibitor can be administered per kg of patient body weight per week, but is not limited thereto. Alternatively, in the method of the present invention, 0.000001 mg to 50 mg of the interleukin-2 analog and 0.0000001 mg to 100 mg of the immune checkpoint inhibitor can be administered per kg of patient body weight per week, but is not limited thereto.

[0266] Alternatively, in the method of the present invention, the interleukin-2 analog or its long-acting conjugate may be administered at 0.001 nmol / kg to 33,000 nmol / kg, and the immune checkpoint inhibitor may be administered at 0.0001 nmol / kg to 7,000 nmol / kg, but are not limited thereto.

[0267] In addition, the interleukin-2 analog or its long-acting conjugate and immune checkpoint inhibitor of the present invention can be administered in appropriate divided doses, and the number of administrations can be the same or different. For example, when the interleukin-2 analog or its long-acting conjugate is administered once a week, the immune checkpoint inhibitor can be administered once, twice or more times a week, but is not limited thereto.

[0268] However, the dose of the immune checkpoint inhibitor can be varied to exhibit the superior effects of combined administration of the interleukin-2 analog or its long-acting conjugate of the present invention.

[0269] For example, the dosage of an immune checkpoint inhibitor may be expressed based on a PD-1 inhibitor, and the composition may include another immune checkpoint inhibitor in an amount corresponding to the dosage of the PD-1 inhibitor, but is not limited thereto.

[0270] In another aspect of the present invention, there is provided use of an interleukin-2 analogue or a long-acting conjugate thereof or a composition comprising the same, administered in combination with an immune checkpoint inhibitor, in the preparation of a medicament for preventing or treating cancer.

[0271] In another aspect of the present invention, provided is the combined use of an interleukin-2 analog or a long-acting conjugate thereof and an immune checkpoint inhibitor in preventing or treating cancer.

[0272] The interleukin-2 analogs and / or long-acting conjugates thereof, immune checkpoint inhibitors, compositions comprising the same, cancer, prevention, treatment, administration routes, and administration times are as described above.

[0273] In another aspect of the present invention, there is provided use of an interleukin-2 analogue or a long-acting conjugate thereof or a composition comprising the same, administered in combination with an immune checkpoint inhibitor, in the prevention or treatment of cancer.

[0274] The interleukin-2 analogs and / or long-acting conjugates thereof, immune checkpoint inhibitors, compositions comprising the same, cancer, prevention, treatment, administration routes, and administration times are as described above.

[0275] Unless the context requires otherwise, expressions such as “comprises,” “comprising,” “including,” “containing,” etc., should be understood to mean that they include the stated integer(s) or groups of integers but not excluding other integers or groups of integers.

[0276] Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments. These exemplary embodiments are given to specifically illustrate the present invention, and the scope of the present invention is not limited to these exemplary embodiments.

[0277] Example Example 1: Construction of natural interleukin-2 and interleukin-2 analog expression vectors To generate an expression vector encoding the 133-amino acid natural interleukin-2, 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). Furthermore, novel interleukin-2 analogs with amino acid modifications to interleukin-2 were generated using interleukin-2 as a template.

[0278] The PCR conditions for amplifying interleukin-2 analogs were 16 cycles of 95°C for 30 seconds, 55°C for 60 seconds, and 65°C for 6.5 minutes. The mutagenesis products obtained under these conditions were sequenced, and it was confirmed that each interleukin-2 analog had the modifications shown in Table 1 below at the expected modification positions based on the native form. The expression vectors thus obtained were designated pET22b-interleukin-2 analogs 1 to 105.

[0279] The altered amino acid sequence of each analog and the name of the analog are shown in the following Table 1. To prepare these interleukin-2 analogs, PCR was performed using a forward (F) primer and a reverse (R) primer to amplify each analog gene.

[0280] In Table 1 below, analog 1 is aldesleukin, and primer numbers 1 to 204 correspond to SEQ ID NOs: 214 to 417, respectively.

[0281] Table 1 Types, modification positions, and altered sequences of interleukin-2 analogs In the table, "desA1" indicates the deletion of alanine, the amino acid at position 1 in interleukin-2. Table 2 below shows the full-length protein sequences of interleukin-2 analogs. Bold text in Table 2 below indicates the modified positions.

[0282] Table 2 Amino acid sequences of interleukin-2 analogs Example 2: Expression of interleukin-2 analogs The recombinant interleukin-2 analogs were expressed under the control of the T7 promoter using the expression vectors constructed in Example 1. Each recombinant interleukin-2 analog expression vector was used to transform the expression Escherichia coli strain, namely Escherichia coli BL21DE3 (Escherichia coli BF - dcmompThsdS (r B - m B - ) gal λ(DE3); Novagen). Novagen's recommended transformation method was used. A single colony transformed with each recombinant expression vector was inoculated into 2X Luria broth containing 50 μg / mL of ampicillin and cultured at 37°C for 15 hours. The recombinant strain culture and 2X LB medium containing 30% glycerol were mixed at a 1:1 (v / v) ratio, and 1 mL of this mixture was dispensed into each cryovial, which was then stored at -150°C. This mixture was used as the cell stock for producing each recombinant protein.

[0283] To express these recombinant interleukin-2 analogs, one vial of each cell stock was lysed and inoculated into 500 mL of 2X LB and cultured with shaking at 37°C for 14-16 hours. The culture was terminated when the absorbance at 600 nm reached 4.0 or higher, and the resulting culture was used as a seed culture. Initial fermentation was initiated by inoculating the seed culture into 1.6 L of fermentation medium using a 5-L fermentor (Bioflo-320, NBS, USA). Culture conditions included a temperature of 37°C, an air volume of 2.0 L / min (1 vvm), an agitation rate of 650 rpm, and a pH of 6.70 maintained using 30% aqueous ammonia. During the fermentation process, additional culture medium (feed solution) was added when nutrients in the culture medium became limiting, followed by fed-batch culture. Growth of the strain was monitored by OD, and IPTG was introduced at a final concentration of 500 μM when the absorbance reached 70 or higher. The culture was further continued for about 23-25 ​​hours after the introduction of IPTG, and after the culture was terminated, the recombinant strain was harvested by using a centrifuge and stored at -80°C until use.

[0284] Example 3: Extraction and refolding of interleukin-2 analogs To convert each interleukin-2 analog from the interleukin-2 analog-expressing E. coli obtained in Example 2 into a soluble form, the cells were lysed and renatured. Cell pellets corresponding to a 100 mL culture were suspended in 1-200 mL of lysis buffer (20 mM Tris-HCl, pH 9.0, 1 mM EDTA, pH 9.0, 0.2 M NaCl, 0.5% Triton X-100) and lysed using a microfluidizer at 15,000 psi. After centrifugation at 13,900 g for 30 minutes, the supernatant was discarded and the pellet was washed with 400 mL of first wash buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 9.0). After centrifugation under the same conditions as above, the supernatant was discarded and the pellet was washed with 400 mL of a second wash buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 2% Triton X-100). After centrifugation under the same conditions as above, the supernatant was discarded and the pellet was washed with 400 mL of a third wash buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1% sodium deoxycholate). After centrifugation under the same conditions as above, the supernatant was discarded and the pellet was washed with 400 mL of a fourth wash buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1 M NaCl). After centrifugation and washing under the same conditions, the E. coli inclusion body pellet was obtained. 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 the 6 M guanidine to 4.8 M guanidine. The solution was then centrifuged at 13,900 g for 30 minutes, and the pellet was discarded to obtain only the solution. An additional 185.7 mL of distilled water was added to the diluted solution to dilute the 4.8 M guanidine to 3.5 M guanidine. 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 containing the precipitated impurities was centrifuged at 13,900 g for 30 minutes, and the supernatant was discarded. The pellet was then washed with a final wash buffer (3.5 M guanidine, 20 mM sodium acetate pH 5.0, 5 mM DTT). The pellet was recovered by centrifugation under the same conditions as above.Dissolve the washed interleukin-2 analog in 400 mL of refolding buffer (6 mM guanidine, 100 mM Tris pH 8.0, 0.1 mM CuCl2) and stir the solution at 4°C for 15-24 hours for refolding.

[0285] Example 4: Size Exclusion Column Chromatography The renatured interleukin-2 analog solution obtained in Example 3 was concentrated to less than 1 mL and applied to a size exclusion column for purification. Prior to introducing the renatured solution, the column was equilibrated with a buffer (2 M guanidine, 100 mM Tris pH 8.0), and after the introduction of the renatured solution, elution was performed by passing the buffer through the column. Since the eluted sample contained guanidine, it was exchanged with a stabilizing solution (10 mM sodium acetate pH 4.5, 5% trehalose), and its purity was determined by RP-HPLC and peptide mapping analysis. When the measured purity reached 80% or higher, the sample was used in experiments.

[0286] Example 5: Evaluation of the binding affinity of interleukin-2 analogs to receptors Surface plasmon resonance (SPR, BIACORE T200, GE Healthcare) was used to measure the binding affinity of the interleukin-2 analogs obtained in Example 4 for each of the interleukin-2 α receptor and the interleukin-2 β receptor. The binding affinity of the prepared analogs for the α receptor and the β receptor was measured, and the binding affinity of each prepared analog was compared with the binding affinity of interleukin-2 analog 1 (aldesleukin).

[0287] First, an anti-human immunoglobulin antibody (Abcam, #ab97221) was immobilized to a CM5 chip (GE Healthcare) at approximately 5,000 resonance units (RU) via amine coupling. Interleukin-2α receptor (SYMANSIS, #4102H) or interleukin-2β receptor (SYMANSIS, #4122H), each linked to the Fc region of a human immunoglobulin, was then bound to the immunoglobulin antibody using an antigen-antibody binding reaction, ultimately immobilizing it. Thereafter, the recombinant interleukin-2 analog prepared above was serially diluted to various concentrations using HBS-P+ buffer (Cytiva, BR100671), and the diluted samples were passed through the CM5 chip, ultimately immobilizing the interleukin-2 receptor, to measure the binding affinity of each interleukin-2 receptor. The association rate (k) was used to determine the binding affinity of each interleukin-2 receptor. a ) and dissociation rate (k dBinding affinity was measured by flowing the interleukin-2 analog at a flow rate of 10 μL / min for 3 minutes to measure the association rate, while the dissociation rate from each interleukin-2 receptor was measured by flowing only HBS-P + buffer for the same period of time and at the same flow rate. After the measurement, the binding affinity for the receptor was evaluated based on a 1:1 binding fit model in the Biaevaluation program.

[0288] Relative binding affinity (K D ) (%) = dissociation constant (K) of analogue 1 (aldesleukin) d ) / dissociation constant of the analogue (K d ) × 100 In Table 3 below, "undefined" indicates that binding to the corresponding receptor was not observed in the surface plasmon resonance measurement, and thus the corresponding physical quantity of the corresponding receptor cannot be defined.

[0289] Table 3 Relative binding affinities of interleukin-2 analogs compared with interleukin-2 analog 1 (aldesleukin) for the interleukin-2α receptor or interleukin-2β receptor As clearly shown in the test results (Table 3), the interleukin-2 analogs of the present invention exhibit varying levels of binding affinity for the interleukin-2α receptor compared to native interleukin-2 or aldesleukin, for example, exhibiting no binding affinity, reduced binding affinity, or increased binding affinity compared to interleukin-2 analog 1. However, the interleukin-2 analogs of the present invention exhibited stronger binding affinity for the interleukin-2β receptor than native interleukin-2 or aldesleukin. These results demonstrate that the amino acid sequence of an interleukin-2 analog influences its binding affinity for the interleukin-2α receptor or interleukin-2β receptor. These results indicate that binding affinity for the interleukin-2 receptor can be altered by substituting amino acids at specific positions.

[0290] These experimental results indicate that the interleukin-2 analogs according to the present invention have altered binding affinities to the interleukin-2α receptor and the interleukin-2β receptor and can therefore be used to develop various drugs using the same.

[0291] Example 6: Linking reaction of interleukin-2 analogs with polyethylene glycol (3.4K PEG) linkers and their expression Purification of interleukin-2 analogue ligation products In order to prepare the long-acting conjugates of each interleukin-2 analog obtained in Example 4 linked to the Fc region of an immunoglobulin, a ligation product in which the interleukin-2 analog was linked to one end of a polyethylene glycol (PEG) linker was prepared. To prepare the ligation product, interleukin-2 analogs 21, 41, 52, 86, 104, and 105 were used, and polyethylene glycol (ALD(2)3.4K PEG, NOF, Japan) having a molecular weight of 3.4 kDa and having hydroxyl hydrogen modified with propionaldehyde groups at both ends was used as a PEG linker. The PEG linker was linked to the N-terminus of each interleukin-2 analog. The molar ratio of the interleukin-2 analog to the PEG linker was 1:15 to 1:20, and the reaction was carried out at 2°C to 10°C for 1 hour, while the concentration of the interleukin-2 analog was 1 mg / mL or less. Specifically, the reaction was carried out in 100 mM potassium phosphate (pH 5.5) with the addition of 20 mM sodium cyanoborohydride (SCB) as a reducing agent. The reaction solution was exchanged into 20 mM triethylamine (pH 8.0) buffer using a desalting column, and then purified using a Fractogel® EMD TMAE (S) (Merck Millipore) or Source 15Q (Cytiva) column using a concentration gradient of triethylamine (pH 8.0) and sodium chloride to obtain the respective interleukin-2 analog-3.4K PEG-linked products.

[0292] Example 7: Preparation of long-acting conjugates of interleukin-2 analogues, 3.4K PEG, and immunoglobulin Fc region To prepare long-acting interleukin-2 analog-3.4K PEG-immunoglobulin Fc region conjugates, the molar ratio of each interleukin-2 analog-3.4K PEG-linked product obtained by the method of Example 6 to the immunoglobulin Fc region (SEQ ID NO: 438) was set to 1:10, and the total protein concentration was set to 30 mg / mL. The reaction was carried out at 2-10°C for 15 to 16 hours. Specifically, the reaction was carried out at 100 mM potassium phosphate (pH 6.0), and 20 mM sodium cyanoborohydride was added as a reducing agent.

[0293] Specifically, in the immunoglobulin region used, two monomers having the amino acid sequence of SEQ ID NO: 438 (consisting of 221 amino acids) form a homodimer via a disulfide bond between the cysteine ​​residue at amino acid position 3 of each monomer, and the monomers of the homodimer each independently have an internal disulfide bond between cysteine ​​at position 35 and cysteine ​​at position 95 and an internal disulfide bond between cysteine ​​at position 141 and cysteine ​​at position 199.

[0294] Table 4 Amino acid sequence of immunoglobulin Fc After the reaction was completed, unreacted immunoglobulin Fc region was removed from the reaction solution using Bis-Tris (pH 6.5) and sodium chloride, followed by Butyl FF (Cytiva), and the reaction solution was purified using Source15ISO (Cytiva) using sodium citrate buffer (pH 5.5) and ammonium sulfate, thereby obtaining an interleukin-2 analog-3.4K PEG-immunoglobulin Fc region conjugate (long-acting conjugate), wherein 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. These long-acting conjugates ( Figure 1 and Figure 2).

[0295] Example 8: Evaluation of the Binding Affinity of Interleukin-2 Analog Conjugates to Interleukin-2 Receptor In order to measure the binding affinity of each interleukin-2 analog long-acting conjugate obtained in Example 7 to the interleukin-2α receptor and the interleukin-2β receptor, surface plasmon resonance (SPR, BIACORE T200, GE Healthcare) was used.

[0296] Specifically, biotin-labeled human interleukin-2 receptor α and β subunits (ACROBiosystems) were immobilized to a streptavidin biosensor chip (SA chip, Cytiva) at approximately 100 RU and 500 RU, respectively. A long-acting conjugate of an interleukin-2 analog and aldesleukin diluted in HBS-EP+ buffer (Cytiva, BR100669) by a two-fold dilution method were flowed at a flow rate of 20 μL / min. After a 3-minute binding period, only HBS-EP+ buffer was flowed at the same flow rate for 3 minutes to induce dissociation of the long-acting conjugate of the interleukin-2 analog or aldesleukin from the interleukin-2 receptor. Binding affinity was calculated using the association and dissociation constants obtained. Binding affinity was evaluated using a 1:1 binding fit model in the Biaevaluation program.

[0297] The intrinsic IL-2 receptor binding affinities of the evaluated IL-2 analog conjugates were verified, with significant differences in binding affinity between the candidate substances confirmed for the IL-2 receptor α subunit. A detailed examination of these results revealed that IL-2 analog conjugates 21 and 52 did not bind to the IL-2 receptor α subunit, while IL-2 analog conjugates 41, 86, 104, and 105 exhibited relative binding affinities of 50.8%, 65.2%, 81.0%, and 112.9%, respectively, compared to aldesleukin. Regarding the IL-2 receptor β subunit, all IL-2 analog conjugates, including IL-2 analog conjugates 21 and 52, exhibited higher binding affinities than aldesleukin.

[0298] Table 5 below summarizes the relative binding affinity (%) of the prepared interleukin-2 analog long-acting conjugates for each of the α receptor and β receptor, compared to the binding affinity of aldesleukin. In Table 5, each corresponding long-acting conjugate is indicated by the number of the interleukin-2 analog that constitutes it (e.g., the long-acting conjugate of interleukin-2 analog 21 is indicated as "interleukin-2 analog conjugate 21").

[0299] Table 5 Binding affinity of interleukin-2 analog conjugates to interleukin-2 receptor Example 9: Combined administration of interleukin-2 analog long-acting conjugate and immunotherapy in malignant melanoma Checkpoint inhibitors to evaluate anticancer efficacy To investigate the anticancer efficacy of the interleukin-2 analog conjugate when administered in combination with an immune checkpoint inhibitor, a malignant melanoma mouse model allogeneic with B16F10 melanoma cells (B16F10 melanoma syngeneic mouse model) was administered interleukin-2 analog conjugate 86 or aldesleukin (control) alone or in combination with a mouse anti-PD-1 antibody (Bio X Cell, catalog # BP0146), and tumor size and subject survival were evaluated.

[0300] Specifically, B16F10 cells (ATCC) were subcutaneously injected into the thighs of C57BL / 6 mice. After several days, when tumors were observed macroscopically, eight mice were assigned to each group to achieve similar tumor sizes. Interleukin-2 analog conjugate 86 was administered subcutaneously at a dose of 0.08 mg / kg or 6.0 mg / kg (based on the weight of the interleukin-2 analog site of the interleukin-2 analog long-acting conjugate) once weekly for a total of four weeks. A control group (aldesleukin group) received intraperitoneal administration of 3.0 mg / kg of Proleukin (Novartis) once daily for five consecutive days, followed by a two-day rest period. This cycle was repeated for a total of four weeks. The group receiving anti-PD-1 antibody alone received intraperitoneal administration of 10.0 mg / kg of mouse PD-1 antibody (Bio X Cell) twice weekly for a total of four weeks. The anti-PD-1 antibody combination group was administered with the same dose (10.0 mg / kg) of anti-PD-1 antibody as the interleukin-2 analog conjugate 86 (0.08 mg / kg) group, the interleukin-2 analog conjugate 86 (6.0 mg / kg) group, and the aldesleukin (3.0 mg / kg) group. 3 The time is set as a humanitarian endpoint.

[0301] On the 10th day after administration, the tumor size of each group was observed ( Figure 3 A) and 35-day survival rate of subjects ( Figure 3 B). As a result, interleukin-2 analog conjugate 86 (6.0 mg / kg) demonstrated superior tumor suppression and survival compared to the control group, despite weekly administration as monotherapy. In particular, complete remissions (CRs)—indicating complete tumor elimination—were observed in two of eight mice in the interleukin-2 analog conjugate 86 (6.0 mg / kg) administration group, but no CRs were observed in the control group.

[0302] In combination therapy with an anti-PD-1 antibody, the interleukin-2 analog conjugate 86 and anti-PD-1 antibody combination group demonstrated higher tumor suppressive activity and survival rates than the aldesleukin and anti-PD-1 antibody combination group. In particular, complete responses (CRs) were observed in two of eight mice in the interleukin-2 analog conjugate 86 (0.08 mg / kg, a very low dose compared to aldesleukin) and anti-PD-1 antibody combination group. In the interleukin-2 analog conjugate 86 (6.0 mg / kg) and anti-PD-1 antibody combination group, 100% survival was observed, with complete responses observed in seven of eight mice, demonstrating superior anticancer efficacy. However, the aldesleukin and anti-PD-1 antibody combination group did not demonstrate complete responses.

[0303] Example 10: Long-acting conjugates of interleukin-2 analogs by combined administration in a Lewis lung cancer mouse model Drugs and immune checkpoint inhibitors to evaluate anticancer efficacy To investigate the anti-cancer efficacy in a lung cancer mouse model, interleukin-2 analog conjugate 86 or aldesleukin were administered alone or in combination with a mouse anti-PD-1 antibody (Bio X Cell, catalog # BP0146) to a Lewis lung cancer mouse model allogeneic with Lewis lung cancer (LL / S) cells (LL / 2 tumor syngeneic mouse model), and the anti-tumor efficacy was evaluated.

[0304] LL / 2 cells (ATCC) were subcutaneously injected into the thighs of C57BL / 6 mice. After several days, when tumors were observed with the naked eye, 10 mice were assigned to each group so that tumors were of similar size. These groups were divided into a single-drug administration group and a combination of the drug and an immune checkpoint inhibitor. The single-drug administration groups were designated as those administering interleukin-2 analog conjugate 86, aldesleukin, and an anti-PD-1 antibody. Specifically, the interleukin-2 analog conjugate 86 administration group received 6.0 mg / kg (based on IL-2) of the conjugate via subcutaneous administration once weekly for a total of two times. The control group received 3.0 mg / kg of aldesleukin via intraperitoneal administration once daily for five consecutive days, followed by a two-day rest period, for a total of two cycles. The immune checkpoint inhibitor monotherapy group received anti-PD-1 antibodies via intraperitoneal administration every three to four days for two weeks, for a total of twice weekly.

[0305] An immune checkpoint inhibitor combination administration group set separately from the single drug administration group was prepared by further administering an anti-PD-1 antibody on the basis of the interleukin-2 analog conjugate 86 group and the control group, wherein the administration concentration and interval were set to be the same as those of the single drug administration group.

[0306] On day 12 of administration, the tumor size of each group was observed, and Bliss independence analysis was performed to evaluate the interaction between the drugs administered in combination and the immune checkpoint inhibitors.

[0307] As a result, interleukin-2 analog conjugate 86 showed excellent antitumor efficacy by single administration in Lewis lung cancer mouse model ( Figure 4 ), and showed significant synergistic effects by combining with immune checkpoint inhibitors, unlike aldesleukin ( Figure 5 A and Figure 5 B).

[0308] It has been reported that the Lewis lung cancer mouse model, a representative immune-infiltrated tumor model (cold tumor), did not show significant efficacy when treated with immune checkpoint inhibitors (including anti-PD-1 antibodies) alone (Maryland Franklin, 2019, Labcorp). In addition, in this example, the use of immune checkpoint inhibitors alone did not show significant anti-cancer efficacy ( Figure 4 ), but the combined administration of interleukin-2 analog conjugate 86 according to the present invention and immune checkpoint inhibitors showed a synergistic effect. These results confirm that the interleukin-2 analog conjugate of the present invention triggers the transformation of immune-infiltrated tumors into infiltrating tumors (hot tumors) in the tumor microenvironment (TME) of the Lewis lung cancer model, indicating that the interleukin-2 analog conjugate exhibits excellent effects when administered in combination with immune checkpoint inhibitors.

[0309] Example 11: Inhibition of pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) by combined administration of interleukin- 2 analog long-acting conjugates and anti-PD-1 antibodies to evaluate anticancer efficacy and validate memory responses in cancer cure models To evaluate the anticancer efficacy of a long-acting interleukin-2 analog conjugate alone and in combination with an anti-PD-1 antibody against pancreatic cancer (pancreatic ductal adenocarcinoma), one of the most aggressive malignancies, a syngeneic tumor mouse model was established by subcutaneously injecting mouse-derived pancreatic ductal adenocarcinoma cells (panc02) into the flank of 6-week-old female C57BL / 6 mice. After 14 days, the tumor volume (~80 mm 3 ) Seven animals were randomly assigned to each group.

[0310] For testing, each group was divided into a group receiving interleukin-2 analog conjugate 86 alone, a group receiving anti-PD-1 antibody alone, and a group receiving a dual-drug combination. Each drug was administered for a total of 6 weeks according to the respective administration schedule. The anti-tumor efficacy of each experimental group was evaluated by tumor growth inhibition (TGI) using the following expression. Detailed drug dosage, administration route, number of administrations, and anti-tumor efficacy for each group are shown in Table 6.

[0311] % TGI = (1 – (increase in tumor volume from day 0 to day X in the experimental group / increase in tumor volume from day 0 to day X in the negative control group)) x 100 Table 6 *Analysis was performed based on day 32 after drug administration (the last measurement day before tumor necrosis).

[0312] Antitumor efficacy of interleukin-2 analog conjugate 86 was observed at a dose of 1.7 mg / kg and increased in a dose-dependent manner up to a maximum of 25 mg / kg. Compared to the negative control group, no antitumor efficacy was observed in the interleukin-2 analog conjugate 86 (0.3 mg / kg) administration group and the anti-PD-1 antibody administration group, but a significant level of tumor inhibition was observed when the two drugs were administered in combination at the same dose. In particular, complete responses were observed in the interleukin-2 analog conjugate 86 (8.5 mg / kg or higher) administration group alone, and complete responses were observed in all subjects in the interleukin-2 analog conjugate 86 (25 mg / kg) administration group alone or in the combination group with the anti-PD-1 antibody.

[0313] On the 89th day from the first day of drug administration, the subjects who had been confirmed to have shown complete remission were injected subcutaneously with panc02 tumor cells, which were the same as the tumor cells of the primary cancer, into the flank to study the effect of preventing relapse through memory response. As a result, in the group that was administered with interleukin-2 analog conjugate 86 (25 mg / kg) alone or in the group that was administered with the conjugate and anti-PD-1 antibody in combination, the effect of preventing relapse through memory response was confirmed to be 70% or higher ( Figure 6 These results suggest that interleukin-2 analog conjugate 86 can both treat primary pancreatic cancer and prevent recurrence of the same tumor.

[0314] The above experimental results show that the interleukin-2 analogue long-acting conjugate according to the present invention exhibits strong anti-cancer efficacy and low side effect risk by changing the binding affinity for interleukin-2α receptor and interleukin-2β receptor, and achieves high blood exposure by the structure of the long-acting conjugate connected to the human immunoglobulin Fc region, thereby increasing the administration interval compared to existing interleukin-2 therapy, and enhancing the in vivo immune response with high activity at low doses. In addition, the interleukin-2 analogue conjugate according to the present invention can exhibit excellent anti-cancer efficacy through synergistic effects with immune checkpoint inhibitors, so that the interleukin-2 analogue conjugate can be used in safe and effective anti-cancer treatment when administered in combination with immune checkpoint inhibitors, and the interleukin-2 analogue according to the present invention or a long-acting conjugate containing the same can suppress cancer recurrence by eliciting an immune memory response in a subject.

[0315] Although the present invention has been described with reference to specific illustrative embodiments, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the technical spirit or essential features of the present invention. Therefore, the above exemplary embodiments should be understood to be exemplary rather than limiting. The scope of the present invention should be understood to be intended to be within the meaning and scope of the appended claims rather than the detailed description, as well as all changes or modifications derived from equivalent concepts that fall within the scope of the present invention.

[0316] Description of Government Funded Research This research was supported by the National New Drug Development Project Support of the KOREA DRUGDEVELOPMENT FUND (RS-2022-00165557) funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare of the Republic of Korea.

Claims

1. A pharmaceutical composition for preventing or treating cancer, the composition comprising an interleukin-2 analogue, the interleukin-2 analogue comprising any one of the amino acid sequences selected from SEQ ID NOs: 3 to 106, wherein the composition is administered in combination with an immune checkpoint inhibitor.

2. The composition according to claim 1, wherein the interleukin-2 analog is in the form of a long-acting conjugate, and the long-acting conjugate is represented by the following Chemical Formula 1: [Chemical Formula 1] X - L - F in: X is an interleukin-2 analog comprising any one of the amino acid sequences selected from SEQ ID NOs: 3 to 106; L is a polyethylene glycol linker; F is the dimeric immunoglobulin Fc region, and - symbols represent covalent bonds between X and L and between L and F, respectively, In the long-acting conjugate, one end of L is covalently linked to only one polypeptide chain of the Fc region of the dimer, and X is covalently linked to the other end of L.

3. The composition of claim 1 or 2, wherein the interleukin-2 analog comprises any one of the amino acid sequences selected from SEQ ID NO: 10, 13-15, 17, 20-22, 32, 35, 36, 42, 53, 54, 56, 58-60, 62, 71, 72, 74-78, 85, 87, 89, 91-94, and 97-106.

4. The composition of claim 1 or 2, wherein the interleukin-2 analog comprises any one of the amino acid sequences selected from SEQ ID NOs: 17, 22, 42, 53, 56, 58-60, 62, 71, 72, 74-77, 87, 89, 91-93, 98-101, and 103-106.

5. The composition according to claim 1 or 2, wherein the interleukin-2 analog comprises any one of the amino acid sequences selected from SEQ ID NO: 22, 42, 53, 87, 105 and 106.

6. The composition according to claim 1 or 2, wherein the interleukin-2 analog further comprises at least one amino acid at the C-terminus.

7. The composition of claim 1 or 2, wherein the interleukin-2 analog has increased binding affinity for the interleukin-2 beta receptor compared to aldesleukin.

8. The composition of claim 2, wherein the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE or IgM, or a combination or hybrid thereof.

9. The composition of claim 2, wherein the immunoglobulin Fc region is an IgG4 Fc region.

10. The composition of claim 2, wherein the immunoglobulin Fc region is aglycosylated. The composition according to claim 2 , wherein in the long-acting conjugate, X is covalently linked to one Fc region of the dimeric immunoglobulin Fc region via the polyethylene glycol linker.

12. The composition of claim 2, wherein the polyethylene glycol linker has a molecular weight of 1 to 100 kDa.

13. The composition according to claim 1 or 2, further comprising a pharmaceutically acceptable excipient.

14. The composition of claim 1 or 2, wherein the cancer is selected from renal cell carcinoma, melanoma, colorectal cancer, liver cancer, uterine cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, breast cancer, bladder cancer, stomach cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, small intestine cancer, endocrine gland cancer, thyroid cancer, Any of the following: thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal pelvis cancer, CNS tumor, primary CNS lymphoma, spinal tumor, brain tumor, glioma (astrocytoma, glioblastoma, oligodendroglioma, ependymoma), blastoma, meningioma, brain stem glioma, pituitary adenoma, neuroblastoma, congenital tumor, craniopharyngioma and brain tumor.

15. The composition of claim 1 or 2, wherein the cancer is hyporesponsive to a PD-1 antagonist.

16. The composition of claim 1 or 2, wherein the composition is administered via an intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary or intrarectal route.

17. The composition of claim 1 or 2, wherein the composition is administered at intervals ranging from 1 week to 1 month.

18. The composition of claim 1 or 2, wherein the composition is administered in combination with a composition comprising an immune checkpoint inhibitor simultaneously, separately, sequentially, or in the reverse order.

19. The composition of claim 1 or 2, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

20. The composition according to claim 19, wherein the immune checkpoint inhibitor is at least one selected from anti-PD-1 antibodies or antigen-binding fragments thereof.

21. The composition of claim 20, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, cemiplizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalizumab, dotalimumab, INCMGA00012, AMP-224, and AMP-514.

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