Combination therapy of interleukin-2 analogs or their conjugates with immune checkpoint inhibitors for the prevention or treatment of cancer

Abstract

The present invention aims to provide a pharmaceutical composition for cancer prevention or treatment comprising an interleukin-2 analog or a sustained-release conjugate thereof, characterized in that it is administered in combination with an immune checkpoint inhibitor. [Solution] The above problem is solved by a pharmaceutical composition for cancer prevention or treatment containing an interleukin-2 analog or a sustained-release conjugate thereof containing a sequence predetermined by the present invention.

Description

[Technical Field]

[0001] This invention relates to a combination therapy for the prevention or treatment of cancer using an interleukin-2 analog or a conjugate thereof and an immune checkpoint inhibitor. [Background technology]

[0002] Interleukin 2 is an important immunostimulant with a molecular weight of approximately 15 kDa, composed of 133 amino acid residues, that activates various cells of the immune system, including T cells and B cells. The high efficacy of interleukin 2 as an immunostimulant makes it useful in the treatment of various immune-related diseases, including cancer and AIDS (Patent Document 1). Currently, interleukin 2 (Proleukin®) is a drug approved by the FDA for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, high-dose interleukin 2 therapy is associated with serious toxicity, so the number of patients to whom it can be applied is limited, and in practice, this therapy is only performed on a small number of appropriate patients. Toxicity associated with interleukin 2 includes vascular leak syndrome, which causes high fever, nausea, vomiting, vascular leak, severe hypotension, pulmonary edema, and liver damage.

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

[0004] On the other hand, immune cells generally possess proteins called immune checkpoints on their cell membranes, which suppress unwanted autoimmune responses and eliminate cancer cells by detecting tumor-specific antigens expressed due to mutations and other changes that occur in cancer cells. However, cancer cells, in order to evade this immune response, alter the function of the immune checkpoints, thereby suppressing the function of T cells and preventing the immune response from occurring properly.

[0005] Immune checkpoint inhibitors are therapeutic agents that activate a patient's autoimmune system, inducing immune cells in the body to suppress the growth of cancer cells. Unlike conventional immunotherapies, they work by binding to the binding site between cancer cells and T cells, blocking immune evasion signals, allowing T cells to destroy cancer cells without being affected by immune evasion. Therefore, research into using immune checkpoint inhibitors in immunotherapy for cancer patients is ongoing.

[0006] Thus, while there is much research being done on treatment methods that activate the body's immune system and allow immune cells to act on cancer cells to achieve anti-cancer effects, using drugs that stimulate the immune system in anti-cancer treatment can lead to side effects due to the overactivation of immune cells. Therefore, there is a need to find safe and effective anti-cancer agents and therapies. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Korean Published Patent No. 10-2017-0070091 [Patent Document 2] International Publication No. 97 / 034631 [Patent Document 3] International Publication No. 96 / 032478 [Non-patent literature]

[0008] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-Patent Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

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Non-licensed literature 9

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Non-licensed Document 12

[0009] There is a need to develop a safe therapeutic agent and a treatment method using the same, which activate the immune system and exhibit an anti - cancer effect. [[Means for Solving the Problems]]

[0010] An object of the present invention is to provide a pharmaceutical composition for cancer prevention or treatment containing an interleukin - 2 analog or its sustained - release conjugate, which is characterized by being co - administered with an immune checkpoint inhibitor.

[0011] Another object of the present invention is to provide a therapy for cancer prevention or treatment by co - using an interleukin - 2 analog or its sustained - release conjugate and an immune checkpoint inhibitor.

[0012] Furthermore, an object of the present invention is to provide a combination containing an interleukin - 2 analog or its sustained - release conjugate and an immune checkpoint inhibitor.

[0013] Furthermore, an object of the present invention is to provide a pharmaceutical composition for cancer prevention, improvement or treatment containing the above combination. 90]]

[0014] Furthermore, the present invention aims to provide a pharmaceutical kit for the prevention, improvement, or treatment of cancer, comprising an interleukin-2 analog or a sustained-release conjugate thereof and an immune checkpoint inhibitor.

[0015] Furthermore, the present invention aims to provide a method for the prevention or treatment of cancer, comprising the step of administering and / or using the aforementioned combination, pharmaceutical composition, or pharmaceutical kit to an individual requiring the aforementioned combination, pharmaceutical composition, or pharmaceutical kit.

[0016] Furthermore, the present invention aims to provide a method for preventing, improving, or treating cancer, comprising the step of co-administering and / or co-using a composition containing a pharmaceutically effective amount of an interleukin-2 analog or a sustained-release conjugate thereof to an individual requiring a pharmaceutically effective amount of an immune checkpoint inhibitor.

[0017] Furthermore, the present invention aims to provide the aforementioned combination, pharmaceutical composition, or pharmaceutical kit for use in the prevention, improvement, or treatment of cancer, and / or for the manufacture of agents for the prevention, improvement, or treatment of cancer. [Effects of the Invention]

[0018] The pharmaceutical composition for cancer prevention or treatment according to the present invention, which includes an interleukin-2 analog or a sustained-release conjugate thereof, is administered in combination with an immune checkpoint inhibitor and exerts a tumor growth inhibitory effect while reducing side effects, making it an excellent anticancer treatment agent. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows the SDS-PAGE results for persistent interleukin-2 analog conjugates (analogs 21, 41, and 52). [Figure 2a] This figure shows the purity analysis results of the interleukin-2 analog sustained conjugate (analog 21). [Figure 2b]This figure shows the purity analysis results of the interleukin-2 analog persistent conjugate (analog 41). [Figure 2c] This figure shows the purity analysis results of the interleukin-2 analog persistent conjugate (analog 52). [Figure 3] This figure shows the results of evaluating the antitumor efficacy of combination therapy with a sustained-release interleukin-2 analog conjugate and an immune checkpoint inhibitor (anti-PD-1 antibody) in an animal model of malignant melanoma. The figure shows the results of examining tumor size (Figure 3A) and survival rate (Figure 3B) in mouse models administered either aldesleukin or interleukin-2 analog 86 conjugate alone, or aldesleukin and interleukin-2 analog 86 conjugate in combination with an immune checkpoint inhibitor. In Figure 3, the dose of the sustained-release conjugate is shown based only on the weight occupied by the interleukin-2 analog site in the entire conjugate. [Figure 4] This figure shows the results of evaluating the antitumor efficacy of combination therapy with a sustained-release interleukin-2 analog conjugate and an immune checkpoint inhibitor (anti-PD-1 antibody) in an animal model of lung cancer. The figure shows the results of examining tumor size in mouse models administered either aldesleukin or interleukin-2 analog 86 conjugate alone, or aldesleukin and interleukin-2 analog 86 conjugate in combination with an immune checkpoint inhibitor. In Figure 4, the dose of the sustained-release conjugate is shown based solely on the weight of the interleukin-2 analog site in the entire conjugate. [Figure 5] This figure shows the results of an analysis of the antitumor efficacy of combination therapy with a sustained-release interleukin-2 analog conjugate and an immune checkpoint inhibitor (anti-PD-1 antibody) in an animal model of lung cancer, using the Bliss independent model. In Figure 5, the dose of the sustained-release conjugate is shown based solely on the weight of the interleukin-2 analog site within the entire conjugate. [Figure 6]This figure shows the results of confirming the anticancer efficacy and recurrence prevention efficacy of combination therapy with a sustained-release interleukin-2 analog conjugate and an immune checkpoint inhibitor (anti-PD-1 antibody) in a mouse model of pancreatic cancer. In Figure 6, the dose of the sustained-release conjugate is shown based solely on the weight of the interleukin-2 analog site in the entire conjugate. [Modes for carrying out the invention]

[0020] One aspect of the present invention is a composition comprising a novel interleukin 2 analog (or IL-2 analog) or a sustained-release conjugate thereof, characterized in that it is administered in combination with an immune checkpoint inhibitor. The interleukin 2 (IL-2) analog is an interleukin 2 analog having increased interleukin 2β receptor binding affinity compared to natural interleukin 2 or aldesleukin, which is an interleukin 2 analog, and may contain a sequence in which at least one amino acid is mutated in natural interleukin 2.

[0021] As a specific example, the interleukin-2 analog is characterized by containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 106.

[0022] As another specific example, the persistent conjugate is characterized by being a persistent conjugate represented by chemical formula (1).

[0023] [ka]

[0024] Here, X is an interleukin-2 analog containing any sequence selected from the amino acid sequences of SEQ ID NOs: 3 to 106, L is a polyethylene glycol linker, F is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X and L, and between L and F. The persistent conjugate is such that one end of L is covalently bonded to only one polypeptide chain of the dimeric Fc region, and X is covalently bonded to the opposite end of L.

[0025] In a composition according to any of the above-described specific examples, the sustained-release conjugate is characterized in that, compared to natural interleukin-2 or aldesleukin, the interleukin-2α receptor binding affinity is altered and the interleukin-2β receptor binding affinity is increased, and the conjugate includes, as part of the conjugate, an interleukin-2 analog.

[0026] In a composition according to any of the above-described examples, the interleukin-2 analog is characterized by having increased binding affinity to the interleukin-2β receptor compared to aldesleukin.

[0027] In a composition according to any of the above-described examples, the interleukin-2 analog is characterized by containing any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13-15, 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.

[0028] In a composition according to any of the above-described examples, the interleukin-2 analog is characterized by containing any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 17, 22, 42, 53, 56, 58-60, 62, 71, 72, 74-77, 87, 89, 91-93, 98-101, and 103-106.

[0029] In a composition according to any of the above-described examples, the interleukin-2 analog is characterized by containing any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105, and 106.

[0030] In a composition according to any of the above-described examples, the interleukin-2 analog is further characterized by comprising at least one amino acid at its C-terminus.

[0031] In a composition according to any of the above-mentioned specific examples, the immune checkpoint inhibitor is characterized by being a PD-1 antagonist.

[0032] In a composition according to any of the above-described examples, the immune checkpoint inhibitor is characterized by being at least one selected from an anti-PD-1 antibody or its antigen-binding fragment.

[0033] In a composition according to any of the above-mentioned specific examples, the anti-PD-1 antibody is characterized by being selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514.

[0034] In a composition according to any of the above-mentioned specific examples, the persistent conjugate is characterized by containing an IgG4 Fc region.

[0035] In a composition according to any of the above-described examples, the persistent conjugate is characterized by comprising a non-glycosylated immunoglobulin Fc region.

[0036] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by being derived from a non-glycosylated Fc region of human IgG4.

[0037] In a composition according to any of the above-described examples, the immunoglobulin Fc region is characterized in that it has a structure in which two polypeptide chains are linked by a disulfide bond, and that the linkage is via a nitrogen atom of only one of the two chains.

[0038] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by comprising a monomer having the amino acid sequence of SEQ ID NO: 438.

[0039] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized by being a homodimer consisting of monomers of the amino acid sequence of SEQ ID NO: 438.

[0040] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that it is linked via the nitrogen atom of its N-terminal proline.

[0041] In a composition according to any of the above-described examples, the persistent conjugate is characterized in that X is covalently linked to any of the Fc regions of the immunoglobulin Fc region of the dimeric form via the polyethylene glycol linker.

[0042] A composition according to any of the above-described examples is characterized in that one end of the linker is linked to only one of the two Fc region chains of the immunoglobulin Fc region in the dimeric form.

[0043] In a composition according to any of the above-described examples, the polyethylene glycol linker is characterized by having a molecular weight of 1 kDa to 100 kDa.

[0044] In a composition according to any of the above-mentioned specific examples, the composition is characterized by further comprising a pharmaceutically acceptable excipient.

[0045] In a composition according to any of the above-mentioned specific examples, the cancer is 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, gastric cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, perianal gland cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin lymphoma, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute It is characterized by being one of the following selected from the group consisting of leukemia, lymphocytic lymphoma, renal and pelvic cancer, CNS tumors, primary CNS lymphoma, spinal cord tumors, brain tumors, gliomas (astrocytoma, glioblastoma, oligodendroglioma, ependymoma), germ cell tumors, meningiomas, brainstem gliomas, pituitary adenomas, schwannomas, congenital tumors, craniopharyngiomas, and brain tumors.

[0046] In a composition according to any of the above-mentioned specific examples, the cancer is characterized by being a carcinoma that is less responsive to a PD-1 antagonist.

[0047] In a composition according to any of the above-mentioned specific examples, the cancer that is less responsive to the PD-1 antagonist is characterized by being an immune-nonpenetrating tumor (cold tumor).

[0048] In a composition according to any of the above-described specific examples, the composition is characterized by being administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, local, intranasal, intrapulmonary, or rectal route.

[0049] In a composition according to any of the above-mentioned specific examples, the composition is characterized by being administered at time intervals ranging from one week to one month.

[0050] In a composition according to any of the above-mentioned specific examples, the composition is characterized by being administered in combination with an immune checkpoint inhibitor or a composition containing the same, simultaneously, sequentially, or in reverse order.

[0051] Another aspect of the present invention is a method for preventing or treating cancer, comprising the steps of administering to an individual an interleukin-2 analog, a sustained-release conjugate thereof, or a composition comprising the same, and an immune checkpoint inhibitor or a composition comprising the same.

[0052] A further aspect of the present invention is the combined use of the interleukin-2 analog, its sustained-release conjugate, or a composition comprising the same with an immune checkpoint inhibitor or a composition comprising the same for the prevention or treatment of cancer.

[0053] A further aspect of the present invention is the use of the interleukin-2 analog, its sustained-release conjugate, or a composition comprising the same, to be administered in combination with an immune checkpoint inhibitor, as a drug for the prevention or treatment of cancer.

[0054] A further aspect of the present invention is a pharmaceutical kit comprising the interleukin-2 analog, its sustained-release conjugate, or the interleukin-2 analog or its sustained-release conjugate in combination with an immune checkpoint inhibitor.

[0055] The following describes embodiments for carrying out the present invention. Note that each description and embodiment disclosed herein applies to other descriptions and embodiments. That is, any combination of the various elements disclosed herein is included in the present invention. Furthermore, the present invention is not limited to the following specific descriptions. In addition, numerous papers and patent documents are referenced throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated in their entirety as references in this specification, thereby more clearly explaining the level of the art to which the present invention belongs and the content of the present invention.

[0056] Throughout this specification, the usual one-letter and three-letter codes for amino acids are used. Furthermore, amino acids referred to by abbreviations in this specification are described according to IUPAC-IUB nomenclature. Alanine A Arginine R Asparagine N Aspartic acid D Cysteine ​​C Glutamic acid E Glutamine Q Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

[0057] One aspect of the present invention provides a pharmaceutical composition for cancer prevention or treatment comprising an interleukin-2 analog or a sustained-release conjugate thereof, which is administered in combination with an immune checkpoint inhibitor.

[0058] The interleukin-2 analog of the present invention is characterized by altered binding affinity to the interleukin-2 receptor, particularly increased binding affinity to the interleukin-2β receptor. Specifically, the interleukin-2 analog of the present invention may have increased binding affinity to the interleukin-2β receptor compared to natural interleukin-2 or known aldesleukins, and more specifically, may also have altered (increased or decreased) binding affinity to the interleukin-2α receptor, and may contain a sequence in which at least one amino acid is mutated compared to natural interleukin-2.

[0059] One specific example of the composition of the present invention includes, but is not limited to, an interleukin-2 analog comprising any sequence selected from the amino acid sequences of SEQ ID NOs: 3 to 106, substantially composed of, or comprising said sequences.

[0060] Specifically, the compositions of the present invention are pharmaceutical compositions for cancer prevention or treatment comprising an interleukin-2 analog or a sustained-release conjugate thereof, and more specifically, pharmaceutical compositions for cancer prevention or treatment comprising an interleukin-2 analog or a sustained-release conjugate thereof and a pharmaceutically acceptable excipient, and administered in combination with an immune checkpoint inhibitor, but are not limited thereto.

[0061] The pharmaceutical composition of the present invention may be administered as a mixture of (i) an interleukin-2 analog or a sustained-release conjugate thereof and (ii) an immune checkpoint inhibitor, or (b) an interleukin-2 analog or a sustained-release conjugate thereof and (ii) an immune checkpoint inhibitor in separate forms, but is not limited to these.

[0062] For example, the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor may be formulated into a single formulation, or they may be formulated separately. If the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor are separated, they may be formulated into separate formulations and administered simultaneously, individually, sequentially, or in reverse order.

[0063] In this invention, "combined administration" should be understood not only as simultaneous administration, but also as a form of administration in which the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor act together on the individual, and each substance acts at a level equivalent to or greater than its intrinsic function. Therefore, "combined administration" in this invention should be understood to include simultaneous, individual, sequential, or reversed administration of the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor. In the case of sequential, reversed, or individual administration, the order of administration is not particularly limited, but the interval between secondary component administrations must not lose the advantageous effects of the combined administration.

[0064] The interleukin-2 analog or its sustained-release conjugate and / or immune checkpoint inhibitor of the present invention, or compositions comprising them, are provided in the form of a kit, but are not limited thereto. The “kit” in the present invention may include the composition according to the present invention for co-administration of the interleukin-2 analog or its sustained-release conjugate and / or immune checkpoint inhibitor. Specifically, the kit according to the present invention may include a single formulation of the interleukin-2 analog or its sustained-release conjugate and / or an immune checkpoint inhibitor, or it may include individual formulations of the interleukin-2 analog or its sustained-release conjugate and / or an immune checkpoint inhibitor, or it may further include substances necessary for the co-administration of the two substances, but is not limited thereto.

[0065] In this invention, "interleukin 2 (IL-2)" refers to an immunomodulator, a type of cytokine that transmits signals in the in vivo immune system. Interleukin 2 is generally known as an important immunostimulant with a molecular weight of approximately 15 kDa.

[0066] In this invention, "interleukin-2 analog" means an interleukin-2 analog in which at least one amino acid has been mutated in the natural sequence. In particular, in this invention, it may be an interleukin-2 analog in which the amino acid of natural interleukin-2 has been mutated, resulting in a decreased or increased binding affinity to the interleukin-2 receptor compared to the natural interleukin-2. Specifically, the interleukin-2 analog of this invention may be non-naturally occurring.

[0067] The aforementioned natural interleukin-2 may also be human interleukin-2, and its sequence can be obtained from a known database or the like. Specifically, it is the amino acid sequence of Sequence ID No. 1, but is not limited to this.

[0068] In this invention, the statement that natural interleukin-2 is the amino acid sequence of SEQ ID NO: 1 means that not only the same sequence as SEQ ID NO: 1, but also sequences with homology of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to SEQ ID NO: 1 are included in the category of natural interleukin-2 of this invention. The amino acid mutation site means that when sequences with homology of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more are aligned with SEQ ID NO: 1, the mutation occurs at the position corresponding to the amino acid sequence of SEQ ID NO: 1.

[0069] In this invention, "aldesleukin" refers to aldesleukin (trademark name: Proleukin®), a commercially available interleukin-2 analog, and specifically refers to one having the amino acid sequence of SEQ ID NO: 2. In this invention, it is used interchangeably with "interleukin-2 analog 1". The interleukin-2 analog according to the present invention may have altered interleukin-2α receptor binding affinity and / or increased interleukin-2β receptor binding affinity compared to interleukin-2 analog 1.

[0070] Although the interleukin 2α receptor is known not to be involved in the interleukin 2 signaling system, it increases the binding affinity of other interleukin 2 receptors (β or γ) to interleukin 2 by 10 to 100 times, and CD4 + It is expressed in regulatory T cells, etc.

[0071] Interleukin-2β receptors are CD8 + Interleukin-2β receptors are primarily distributed in T cells or natural killer cells (NK cells) and their main function is to activate immune responses and macrophage activity. Therefore, activation of interleukin-2β receptors is expected to induce tumor apoptosis and activation of the body's immune response.

[0072] Therefore, the interleukin-2 analog of the present invention, with its increased binding affinity to the interleukin-2β receptor, enhances therapeutic effects such as tumor suppression and tumor death, while reducing side effects.

[0073] As an example, the interleukin-2 analog may include, but is not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 106, or may substantially consist of the aforementioned amino acid sequence.

[0074] In one specific embodiment, the interleukin-2 analog includes, but is not limited to, any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 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, or is substantially composed of, or consists of, the aforementioned sequences.

[0075] In other specific embodiments, the interleukin-2 analog includes, but is not limited to, any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 17, 22, 42, 53, 56, 58-60, 62, 71, 72, 74-77, 87, 89, 91-93, 98-101, and 103-106, or is substantially composed of, or consists of, the aforementioned sequences.

[0076] In yet another specific embodiment, the interleukin-2 analog includes, is substantially composed of, or consists of, any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 15, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 56, 58, 59, 60, 62, 71, 72, 74, 75, 76, 77, 78, 85, 87, 89, 91, 92, 93, 94, 95, 98, 99, 100, 101, 103, 104, 105, and 106.

[0077] In yet another specific embodiment, the interleukin-2 analog comprises, substantially comprises, or comprises any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 87, 89, 91, 92, 93, 94, 98, 99, 100, 101, 103, 104, and 105, or substantially comprises, or comprises, the sequences thereof.

[0078] In yet another specific embodiment, the interleukin-2 analog comprises, substantially consists of, or consists of, any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105, and 106.

[0079] Furthermore, the interleukin-2 analog further contains at least one amino acid at its C-terminus, but is not limited to this.

[0080] Furthermore, even if the Specified Specification refers to an "interleukin-2 analog consisting of a specific sequence number," it does not exclude meaningless sequence additions before or after the amino acid sequence of the said sequence number, naturally occurring mutations, or silent mutations thereof, as long as they have the same or equivalent activity as the interleukin-2 analog consisting of the amino acid sequence of the said sequence number. It goes without saying that compounds having such sequence additions or mutations are also included in the present invention.

[0081] The interleukin-2 analog of the present invention includes, but is not limited to, amino acid sequences having 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the amino acid sequences of SEQ ID NOs.

[0082] In this invention, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are related to each other, and is expressed as a percentage.

[0083] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderate to high stringent conditions. Hybridization includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy in the polynucleotide.

[0084] Homology and identity are often used interchangeably.

[0085] Whether any two nucleotide or peptide sequences are homologous, similar, or identical can be determined, for example, using default parameters as described in Non-Patent Document 1 and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information.

[0086] The homology, similarity, or identity of nucleotide sequences or peptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) as the total number of symbols in the shorter of two sequences divided by the number of similar sequence symbols. Default parameters for the GAP program include (1) a binary comparison matrix (where identity is 1 and non-identity is 0) and a weighted comparison matrix from Non-Patent Document 10 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix), as disclosed in Non-Patent Document 9; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5); and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this invention refers to the relevance between sequences.

[0087] The interleukin-2 analog of the present invention is used as a novel interleukin-2 substitute that alters the in vitro activity of interleukin-2 by decreasing or increasing its binding affinity to α and / or β receptors. In particular, since it not only increases the binding affinity to β receptors but also alters (increases or decreases) the binding affinity to α receptors, it can be used as an effective therapeutic agent due to its activity on both receptors.

[0088] Such modifications for the production of interleukin-2 analogs in the present invention include modifications using L-type or D-type amino acids and / or unnatural amino acids, and / or modifications of the natural sequence, such as modifications of side-chain functional groups, intramolecular covalent bonds, for example, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexanolysis, biotinylation, and other modifications.

[0089] Furthermore, it includes all forms of natural interleukin-2 in which at least one amino acid is added to the N and / or C-terminus.

[0090] As mentioned above, the amino acids that can be substituted or added include not only the 20 amino acids commonly found in human proteins, but also abnormal or non-natural amino acids. Suppliers of abnormal amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids, as well as typical peptide sequences, can be synthesized and purchased from private peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in South Korea.

[0091] Amino acid derivatives can also be obtained in a similar manner, with 4-imidazoacetic acid being one example.

[0092] Furthermore, the interleukin-2 analog according to the present invention may be in a form in which its N-terminus and / or C-terminus are chemically modified to protect it from protein-cleaving enzymes in living organisms and improve its stability, or it may be in a form protected by an organic group, or it may be in a form modified by adding amino acids to the peptide terminus, etc.

[0093] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, so acetylation of the N-terminus and / or amidation of the C-terminus are performed to remove the charge, but the process is not limited to these methods.

[0094] The interleukin-2 analog of the present invention can be synthesized by solid-phase synthesis, produced by recombinant methods, or manufactured by commercial commission, but is not limited to these methods.

[0095] Furthermore, depending on its length, the interleukin-2 analog of the present invention can be synthesized by methods well known in this field, such as an automated peptide synthesizer, or produced by genetic engineering techniques.

[0096] Specifically, the interleukin-2 analog of the present invention can be prepared by standard synthesis methods, recombinant expression systems, or any other methods of the art. Therefore, the interleukin-2 analog according to the present invention can be prepared by many methods, including, for example, (a) a method of synthesizing a peptide stepwise by solid-phase or liquid-phase method or by fragment assembly, and separating and purifying the final peptide product; (b) a method of expressing a nucleic acid preparation encoding the peptide in host cells and recovering the expression product from a host cell culture; (c) a method of expressing a nucleic acid preparation encoding the peptide in a cell-free test tube and recovering the expression product; or a method of obtaining peptide fragments by any combination of (a), (b), and (c), and then recovering the peptide by linking the fragments to obtain a peptide.

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

[0098] Specifically, SPR analysis can be performed using the principle of protein-ligand binding to immobilize an interleukin-2 receptor on a sensor chip, injecting an interleukin-2 analog diluted in experimental buffer using a serial dilution method to induce binding to the immobilized receptor, and then injecting only the experimental buffer at the same flow rate to induce dissociation between the receptor and the interleukin-2 analog, and measuring the binding strength. Alternatively, one method can be performed by first immobilizing an antibody against the human immunoglobulin Fc region on a sensor chip, then immobilizing an interleukin-2 receptor to which the Fc region is bound, injecting an interleukin-2 analog, and measuring the binding strength, but is not limited to these methods.

[0099] More specifically, a biotin-labeled human interleukin-2 receptor is immobilized on a streptavidin biosensor chip, and a persistent interleukin-2 analog conjugate diluted in HBS-EP+ buffer using a 2-fold serial dilution method is injected at a flow rate of 20 μL / min for 3 minutes. Subsequently, HBS-EP+ buffer alone is injected at the same flow rate for 3 minutes to induce dissociation between the interleukin-2 receptor and the persistent interleukin-2 analog conjugate. The binding strength can then be measured using a Bioaevaluation program with a 1:1 binding fitting model based on the obtained binding and dissociation constants, but this method is not limited to this.

[0100] More specifically, the interleukin-2 analog of the present invention may have a reduced or increased interleukin-2α receptor binding affinity compared to natural interleukin-2 or interleukin-2 analog (aldesleukin; or interleukin-2 analog 1).

[0101] More specifically, the interleukin-2 analog of the present invention has an interleukin-2α receptor binding affinity of approximately 0.001 times or more, approximately 0.005 times or more, approximately 0.01 times or more, approximately 0.05 times or more, approximately 0.1 times or more, approximately 0.3 times or more, approximately 0.5 times or more, approximately 0.7 times or more, approximately 0.9 times or more, approximately 1.1 times or more, approximately 1.3 times or more, approximately 1.5 times or more, and approximately 1.7 times or more compared to the interleukin-2α receptor binding affinity of natural interleukin-2 or aldesleukin. However, these values ​​are not limited, and any altered binding affinity compared to natural interleukin-2 or aldesleukin is included in the present invention.

[0102] Alternatively, using the interleukin-2α receptor binding affinity of aldethleukin (100%) as a baseline, the interleukin-2 analogs of the present invention may have completely lost their binding affinity, or possess a binding affinity of approximately 1% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 30% or more, approximately 50% or more, approximately 70% or more, approximately 90% or more, approximately 100% or more, approximately 150% or more, or approximately 200% or more. However, these values ​​are not limited, and any compound whose binding affinity has changed compared to natural interleukin-2 or aldethleukin is included in the present invention.

[0103] Specifically, the interleukin-2 analog of the present invention has an interleukin-2β receptor binding affinity of approximately 0.1 times, 0.3 times, 0.5 times, 0.7 times, 1.0 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times compared to the interleukin-2β receptor binding affinity of natural interleukin-2 or aldesleukin. However, these values ​​are not limited, and any substance whose binding affinity has changed or increased compared to natural interleukin-2 or aldesleukin is included in the present invention.

[0104] Alternatively, using the interleukin-2β receptor binding affinity of aldethleukin (100%) as a baseline, the interleukin-2 analogs of the present invention have a binding affinity of approximately 5% or more, approximately 9% or more, approximately 10% or more, approximately 20% or more, approximately 30% or more, approximately 50% or more, approximately 100% or more, approximately 200% or more, approximately 500% or more, approximately 700% or more, approximately 1000% or more, approximately 1500% or more, approximately 3000% or more, approximately 5000% or more, approximately 7000% or more, approximately 10000% or more, approximately 12000% or more, approximately 15000% or more, approximately 20000% or more, and approximately 25000% or more. However, these values ​​are not limited, and any compound with increased binding affinity compared to natural interleukin-2 or aldethleukin is included in the present invention.

[0105] In this invention, "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and any numerical value within a range equivalent to or similar to the numerical value following the term "approximately" is acceptable.

[0106] The interleukin-2 analog of the present invention is characterized by a change in binding affinity to the interleukin-2α receptor and an increase in binding affinity to the interleukin-2β receptor compared to natural interleukin-2 or aldesleukin.

[0107] In a specific embodiment of the present invention, in order to produce the interleukin-2 analog of the present invention, an interleukin-2 analog was prepared by introducing a mutation based on natural interleukin-2 (SEQ ID NO: 1). The interleukin-2 analog prepared in the present invention may contain any of the amino acid sequences of SEQ ID NOs: 3 to 106, or may be encoded by any of the nucleotide sequences of SEQ ID NOs: 108 to 211.

[0108] The nucleic acid encoding the interleukin-2 analog of the present invention may be modified to introduce mutations (deletion, substitution, and / or addition of amino acids) at specific positions in the base sequence encoding the natural interleukin-2 of SEQ ID NO: 1. Specifically, it may include a base sequence encoding any of the amino acid sequences of SEQ ID NOs: 3 to 106. For example, the nucleic acid of the present invention may have any of the base sequences of SEQ ID NOs: 108 to 211, or may include the aforementioned base sequences.

[0109] The base sequences of the present invention can be modified in various ways in the coding region, within the limits that the amino acid sequence of the interleukin-2 analog of the present invention does not change, by codon degeneracy or by taking into consideration codons preferred in organisms that intend to express the nucleic acid of the present invention. Specifically, the nucleic acids of the present invention have a base sequence that is homologous or identical to any of the sequences of SEQ ID NOs. 108 to 211 by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100%, or consist of a base sequence that is homologous or identical to any of the sequences of SEQ ID NOs. 108 to 211 by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100%, or are substantially composed of the above base sequences.

[0110] Furthermore, the nucleic acid of the present invention may be any probe prepared from a known gene sequence, for example, any sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the nucleic acid sequence of the present invention. The "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12).

[0111] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the nucleic acids of the present invention may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments whose entire sequences are complementary.

[0112] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 11).

[0113] The similarity or identity is as described above.

[0114] Furthermore, the interleukin-2 analog of the present invention has an extended half-life in the body compared to natural interleukins or aldesleukins, but is not limited to these. For example, a biocompatible substance for extending the half-life (e.g., an immunoglobulin Fc region) may be directly or via a linker to be bound to the interleukin-2 analog, and the invention may be limited to this form of a sustained-release conjugate with an extended half-life.

[0115] The sustained-release conjugate according to the present invention not only contains an interleukin-2 analog with improved binding affinity to the interleukin-2β receptor, but also binds to the immunoglobulin Fc region as a representative carrier to extend its half-life. This extends the half-life of the interleukin-2 analog, increases blood exposure, and enhances the in vivo immune response, thereby effectively achieving the suppression and reduction of cancer cell growth.

[0116] The above provisions apply to other specific examples or embodiments of the present invention, but are not limited thereto.

[0117] Other specific examples of the compositions of the present invention include, but are not limited to, persistent conjugates of the interleukin-2 analog.

[0118] In the present invention, the interleukin-2 analog sustained-release conjugate may be in which an interleukin-2 analog is conjugated with a biocompatible substance for extending its in vivo half-life. The biocompatible substance in this specification is used in combination with a carrier.

[0119] In the present invention, the sustained-release conjugate has improved efficacy persistence compared to an interleukin-2 analog without a carrier bound, and in the present invention, such a conjugate is referred to as a "sustained-release conjugate" or simply a "conjugate".

[0120] Furthermore, such compound forms may not exist naturally.

[0121] In one specific example of the present invention, the persistent conjugate is a persistent conjugate represented by chemical formula (1).

[0122] [ka]

[0123] Here, X is the interleukin-2 analog, L is a polyethylene glycol linker, F is a dimeric immunoglobulin Fc region, and - indicates a covalent bond between X and L, and between L and F. The persistent conjugate is formed in which one end of L is covalently bonded to only one polypeptide chain of the dimeric Fc region, and X is covalently bonded to the opposite end of L.

[0124] More specifically, the persistent conjugate may be one in which one molecule of X is linked to one polypeptide chain in the Fc region via L. In addition, in the persistent conjugate of the present invention, X and L, and L and F may be linked to each other by covalent bonds, and the conjugate may be one in which X, L, and F are linked by covalent bonds in the order of chemical formula (1).

[0125] The persistent-release conjugate interleukin-2 analog of the present invention is characterized by a change in its binding affinity to the interleukin-2 receptor when it exists independently, not as part of a conjugate, and particularly by an increased binding affinity to the interleukin-2β receptor. Specifically, the interleukin-2 analog of the present invention may, when it exists independently, not as part of a conjugate, exhibit an increased binding affinity to the interleukin-2β receptor compared to natural interleukin-2 or known aldesleukins, and more specifically, may also exhibit a change (increase or decrease) in its binding affinity to the interleukin-2α receptor.

[0126] In the present invention, the interleukin-2 analog is a component that forms part of the compound. Specifically, it corresponds to X in chemical formula (1). The interleukin-2 analog is as described above.

[0127] In the aforementioned compound, F is a substance that extends the half-life of X, i.e., the interleukin-2 analog, and is one component that forms part of the compound of the present invention.

[0128] The aforementioned F may be bonded to X by covalent chemical bonds, or it may be bonded to X by covalent chemical bonds via L.

[0129] Specifically, F is an immunoglobulin Fc region, and the immunoglobulin Fc region is an IgG Fc region or a non-glycosylated IgG4 Fc region, but is not limited to these.

[0130] As a specific example of the present invention, the F (immunoglobulin Fc region) is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains, but the invention is not limited to this.

[0131] At least one amino acid side chain in the peptide of the present invention is conjugated to such a biocompatible material to improve solubility and / or half-life in vivo and / or increase bioavailability. Furthermore, such modifications reduce the clearance of therapeutic proteins and peptides.

[0132] The aforementioned biocompatible substances may be water-soluble (amphiphilic or hydrophilic), non-toxic, and / or pharmaceutically acceptable.

[0133] In one specific embodiment, the persistent conjugate of the present invention is a link between an interleukin-2 analog and an immunoglobulin Fc region, but is not limited thereto.

[0134] In the present invention, the "immunoglobulin Fc region" refers to the region of immunoglobulin excluding the variable regions of the heavy and light chains, and including the heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3). The immunoglobulin Fc region may be a component that forms part of the conjugate of the present invention. Specifically, it corresponds to F in chemical formula (1).

[0135] In this specification, the Fc region includes not only the natural sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, such as modified sequences that differ from the natural sequence, for example, by deletion, insertion, non-conservative or conservative substitution, or combination thereof, of at least one amino acid residue of the natural sequence. The derivatives, substituted and modified sequences are assumed to have the ability to bind to FcRn. In the present invention, F is a human immunoglobulin region, but is not limited thereto. In this specification, "biocompatible substance" or "carrier" means the Fc region.

[0136] The aforementioned F (immunoglobulin Fc region) is a structure in which two polypeptide chains are linked by a disulfide bond, and is linked only via the nitrogen atom of one of the two chains, but is not limited to this. The linkage via the nitrogen atom may also be linked to the ε-amino atom or the N-terminal amino group of lysine by reductive amination.

[0137] Reductive amination is a reaction in which an amine group or amino group in one reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) in another reactant to produce an amine, which is then reduced to form an amine bond. This is a well-known organic synthesis reaction in the field.

[0138] As one specific example of the persistent conjugate of the present invention, the persistent conjugate may be one in which the immunoglobulin Fc region is linked to a linker via its N-terminal nitrogen atom.

[0139] Such immunoglobulin Fc regions include, but are not limited to, a hinge region within the heavy chain constant region.

[0140] The immunoglobulin Fc region in the present invention may include a specific hinge sequence at its N-terminus.

[0141] In this invention, "hinge arrangement" refers to a site located in the heavy chain that forms a dimer of the immunoglobulin Fc region via an interdisulfide bond.

[0142] In the present invention, the hinge sequence is a mutated hinge sequence having the following amino acid sequence, but with a portion of it deleted, resulting in a single cysteine ​​residue. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (Sequence ID 418)

[0143] The aforementioned hinge sequence may contain only one cysteine ​​residue, with the 8th or 11th cysteine ​​residue in the hinge sequence of Sequence ID No. 418 being deleted. The hinge sequence of the present invention consists of 3 to 12 amino acids, containing 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 (Sequence ID 419) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (Sequence ID 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 (Sequence ID 422) Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (Sequence ID 423) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 424) Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 425) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 426) Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 427) Pro-Ser-Cys-Pro (Sequence ID 428) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (Sequence ID 429) Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (Sequence ID 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 (Sequence ID 433) Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 434) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 435) Glu-Pro-Ser-Cys (SEQ ID NO: 436) Ser-Cys-Pro (SEQ ID NO: 437)

[0144] More specifically, the hinge sequence includes, but is not limited to, the amino acid sequence of sequence number 428 (Pro-Ser-Cys-Pro) or sequence number 437 (Ser-Cys-Pro).

[0145] In a more specific embodiment of the persistent conjugate of the present invention, the N-terminus of the immunoglobulin Fc region in the conjugate is proline, and the conjugate has the Fc region linked to a linker via the nitrogen atom of the proline.

[0146] In one embodiment of the persistent conjugate of the present invention, the immunoglobulin Fc region may be in the form of a dimer in which the two chains of the immunoglobulin Fc region form a homodimer or heterodimer due to the presence of a hinge sequence. The conjugate of chemical formula (1) of the present invention is in a form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0147] In this invention, "N-terminus" means the amino terminus of a protein or polypeptide, and includes the very end of the amino terminus, or one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids from the very end. The immunoglobulin Fc region of this invention includes a hinge sequence at the N-terminus, but is not limited to this.

[0148] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes some or all of the heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), excluding the variable regions of the heavy and light chains of the immunoglobulin, provided that it has substantially equivalent or improved effects to those of the natural immunoglobulin. In addition, it may be a region in which a very long partial amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0149] For example, the immunoglobulin Fc region of the present invention is a combination of 1) the CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) the CH1 domain and CH2 domain, 3) the CH1 domain and CH3 domain, 4) the CH2 domain and CH3 domain, 5) at least one of the CH1 domain, CH2 domain, CH3 domain and CH4 domain and an immunoglobulin hinge region (or a part of a hinge region), or 6) a dimer of each domain of the heavy chain constant region and the light chain constant region, but is not limited to these.

[0150] In the present invention, the immunoglobulin Fc region is in the form of a dimer or polymer composed of single-chain immunoglobulins consisting of domains of the same origin, but is not limited to these forms.

[0151] Furthermore, as one embodiment of the persistent conjugate of the present invention, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, where the Fc region dimer F and X are covalently linked via a single linker L containing an ethylene glycol repeating unit. In a specific example of this embodiment, X is covalently linked via linker L to only one of the two polypeptide chains of the Fc region dimer F. In a more specific example of this embodiment, of the two polypeptide chains of the Fc region dimer F to which X is linked, only one molecule of X is covalently linked via L. In the most specific example of this embodiment, F is a homodimer.

[0152] In other embodiments of the persistent conjugate of the present invention, two molecules of X may be symmetrically bound to one Fc region in a dimeric form, where the immunoglobulin Fc region and X may be linked to each other by L. However, the present invention is not limited to the above examples.

[0153] Furthermore, the immunoglobulin Fc region of the present invention includes not only natural amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means one in which at least one amino acid residue of a natural amino acid sequence is deleted, inserted, non-conservative or conservatively substituted, or has a different sequence due to a combination thereof.

[0154] For example, in the case of IgG Fc, amino acid residues 214-238, 297-299, 318-322, or 327-331, which are known to be important for binding, are used as suitable modification sites.

[0155] Furthermore, various derivatives are used, such as derivatives in which the disulfide bond-forming site has been removed, derivatives in which several amino acids at the N-terminus of the natural Fc are deleted, and derivatives in which a methionine residue has been added to the N-terminus of the natural Fc. In addition, to eliminate the effector function, the complement binding site, such as the C1q binding site, may be removed, and the ADCC (antibody-dependent cell-mediated cytotoxicity) site may also be removed. Techniques for producing such immunoglobulin Fc region sequence derivatives are disclosed in Patent Documents 2 and 3, among others.

[0156] Amino acid substitutions in proteins and peptides that do not alter the overall activity of the molecule are well known in the art (Non-Patent Literature 13). The most common substitutions are those of 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. Modifications may also be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0157] The aforementioned Fc derivative may exhibit biological activity equivalent to that of the Fc region of the present invention, and may also have improved structural stability against heat, pH, etc., of the Fc region.

[0158] Furthermore, such Fc regions may be obtained from natural sources isolated from the living bodies of humans or animals such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, or they may be recombinants or derivatives obtained from transformed animal cells or microorganisms. Here, the method of obtaining from natural sources may involve isolating total immunoglobulins from the living bodies of humans or animals and then treating them with proteolytic enzymes. Treatment with papain cleaves them into Fab and Fc, and treatment with pepsin cleaves them into pF'c and F(ab)2. These can then be separated into Fc or pF'c using methods such as size exclusion chromatography. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0159] Furthermore, the immunoglobulin Fc region may be a natural glycan, an increased glycan compared to the natural one, a decreased glycan compared to the natural one, or a form from which the glycans have been removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering techniques using microorganisms are used to increase, decrease, or remove the immunoglobulin Fc glycans. Here, the immunoglobulin Fc region from which the glycans have been removed has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cell-mediated cytotoxicity or complement-dependent cell-mediated cytotoxicity is reduced or eliminated, thus not inducing unwanted immune responses in the body. For these reasons, an immunoglobulin Fc region from which the glycans have been removed or which has been deglycosylated is a form suitable for its original purpose as a drug carrier.

[0160] In this invention, "deglycosylation" refers to the Fc region from which sugar has been removed by an enzyme, and "aglycosylation" refers to the Fc region that is produced in prokaryotes, and more specifically in Escherichia coli, and has not been glycosylated.

[0161] On the other hand, the immunoglobulin Fc region is of human origin, or of animal origin such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and in more specific embodiments, it is of human origin.

[0162] Furthermore, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, or an Fc region resulting from a combination thereof or a hybrid thereof. In a more specific embodiment, it may be derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, it may be derived from IgG, which is known to extend the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited to these.

[0163] As a specific example, the immunoglobulin Fc region may be a fragment of human IgG4 Fc, and may be a homodimer in which two monomers are linked by a disulfide bond (inter-chain form) between the third amino acid cysteine ​​of each monomer. Here, the homodimer has / may have two disulfide bonds (intra-chain form) in each monomer, namely between the 35th and 95th cysteine ​​and between the 141st and 199th cysteine.

[0164] Each monomer consists of 221 amino acids, and the amino acids forming the homodimer consist of a total of 442 amino acids, but are not limited to these. Specifically, an immunoglobulin Fc fragment is formed when two monomers having the amino acid sequence of SEQ ID NO: 438 (consisting of 221 amino acids) form a homodimer through a disulfide bond between the third amino acid, cysteine, of each monomer. The monomers of the homodimer independently form an internal disulfide bond between the 35th and 95th cysteine ​​positions, and an internal disulfide bond between the 141st and 199th cysteine ​​positions, but are not limited to these.

[0165] In chemical formula (1), F contains a monomer having the amino acid sequence of SEQ ID NO: 438, and F is a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 438, but is not limited to these.

[0166] For example, the immunoglobulin Fc region is a homodimer containing the amino acid sequence of SEQ ID NO: 439 (consisting of 442 amino acids), but is not limited to this.

[0167] As a specific example, the immunoglobulin Fc region and X mentioned above are not glycosylated, but this is not the only example.

[0168] On the other hand, "combination" in the present invention means that when forming a dimer or polymer, polypeptides encoding single-chain immunoglobulin Fc regions of the same origin bind to single-chain polypeptides of different origins. That is, a dimer or polymer can be produced from at least two fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0169] In this invention, "hybrid" means that within the constant region of a single-chain immunoglobulin, there are sequences corresponding to immunoglobulin Fc fragments of at least two different origins. Various forms of hybrids are possible in this invention. That is, hybrids of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include a hinge.

[0170] On the other hand, IgG is also divided into subclasses IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations thereof or hybridization thereof are also possible. Specifically, these are the IgG2 and IgG4 subclasses, and more specifically, the Fc region of IgG4, which has almost no effector function such as complement-dependent cytotoxicity (CDC).

[0171] Furthermore, the aforementioned conjugates exhibit improved efficacy and duration compared to natural interleukin-2 or aldesleukin, or to X without F modification. Such conjugates include, but are not limited to, the forms described above, as well as forms encapsulated in biodegradable nanoparticles.

[0172] The "polyethylene glycol linker" in the present invention includes a biocompatible polymer in which at least two ethylene glycol repeating units are bonded. The repeating units are linked to each other by any covalent bond other than peptide bonds. The polyethylene glycol linker is one component that forms part of the compound of the present invention, and in this specification, the linker is used interchangeably with a "non-peptide linker" or a "non-peptide polymer".

[0173] In one specific embodiment, the conjugate may be formed in which F (specifically, an immunoglobulin Fc region) and X (specifically, an interleukin-2 analog) are covalently linked to each other via a non-peptide linker containing reactive groups bound to both ends of F (specifically, an immunoglobulin Fc region) and X (specifically, an interleukin-2 analog).

[0174] Specifically, the non-peptide linker in the present invention contains a reactive group at its terminus and can form a conjugate by reacting with other components constituting the conjugate. When a non-peptide linker having reactive functional groups at both ends forms a conjugate by bonding to X and F of chemical formula (1) via the respective reactive groups, the non-peptide linker or non-peptide polymer is also called a non-peptide polymer linker moiety or non-peptide linker linker moiety.

[0175] In one specific embodiment, L (polyethylene glycol linker) is a linker containing ethylene glycol repeating units, such as polyethylene glycol, but is not limited thereto. The polyethylene glycol used herein encompasses, but is not limited to, ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEGs). Furthermore, derivatives thereof known in the art and derivatives readily available in the art are also included in the present invention.

[0176] The polyethylene glycol linker contains ethylene glycol repeating units and may also contain functional groups used in the formation of the conjugate at its terminals until it is configured as a conjugate. The persistent conjugate according to the present invention is in a form in which X and F are linked via the functional groups, but is not limited thereto. In the present invention, the non-peptide linker contains two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0177] Specifically, the linker may include repeating units represented by chemical formula (2). For example, polyethylene glycol (PEG) is one such example, but it is not limited to this.

[0178] [ka]

[0179] Here, n is 10 to 2400, n is 10 to 480, or n is 50 to 250, but is not limited to these ranges.

[0180] The PEG portion of the aforementioned persistent conjugate is -(CH2CH2O) n -Not just the structure, but also the connecting elements and their (CH2CH2O) n -This includes, but is not limited to, oxygen atoms interposed between them.

[0181] As a specific example, the ethylene glycol repeating unit is represented as [OCH2CH2]n, and the n value is a natural number, determined such that the average molecular weight of the [OCH2CH2]n portion in the interleukin-2 analog conjugate, for example, the number-average molecular weight, is greater than 0 to about 100 kDa, but is not limited to these examples. As another specific example, the n value is a natural number, and the average molecular weight of the [OCH2CH2]n portion in the interleukin-2 analog conjugate, for example, the number-average molecular weight is approximately 1 to approximately 100 kDa, approximately 1 to approximately 80 kDa, approximately 1 to approximately 50 kDa, approximately 1 to approximately 30 kDa, approximately 1 to approximately 25 kDa, approximately 1 to approximately 20 kDa, approximately 1 to approximately 15 kDa, approximately 1 to approximately 13 kDa, approximately 1 to approximately 11 kDa, approximately 1 to approximately 10 kDa, approximately 1 to approximately 8 kDa, approximately 1 to approximately 5 kDa, approximately 1 to approximately 3.4 kDa, approximately 2 to approximately 30 kDa, approximately 3 to approximately 30 kDa, approximately 3 to approximately 27 kDa, approximately 3 to approximately 25 kDa, approximately 3 to approximately 22 kDa, approximately 3 to approximately 20 kDa, approximately 3 to approximately 18 kDa, approximately 3 to approximately 1 6kDa, approx. 3-15kDa, approx. 3-13kDa, approx. 3-11kDa, approx. 3-10kDa, approx. 3-8kDa, approx. 3-5kDa, approx. 3-3.4kDa, approx. 8-30kDa, approx. 8-27kDa, approx. 8-25kDa, approx. 8-22kDa, approx. 8-20kDa, approx. 8-18kDa, approx. 8- The ranges are 16kDa, approximately 8-15kDa, approximately 8-13kDa, approximately 8-11kDa, approximately 8-10kDa, approximately 9-15kDa, approximately 9-14kDa, approximately 9-13kDa, approximately 9-12kDa, approximately 9-11kDa, approximately 9.5-10.5kDa, or approximately 10kDa, but are not limited to these ranges.

[0182] Furthermore, while the aforementioned conjugate in one specific embodiment has a structure in which an interleukin-2 analog and an immunoglobulin Fc region (F) are covalently linked via a linker (L) containing ethylene glycol repeating units, it is not limited to this structure.

[0183] In other specific embodiments of the persistent conjugate, L may be a linker containing ethylene glycol repeating units, and F may be a dimeric immunoglobulin Fc region. More specifically, but not limited to, one molecule of X is covalently linked to any Fc region of the dimeric immunoglobulin Fc region via the linker containing the ethylene glycol repeating units. Furthermore, in yet another specific embodiment, but not limited to, one end of the linker containing the ethylene glycol repeating units is linked to only one of the two Fc region chains of the dimeric immunoglobulin Fc region.

[0184] The molecular weight of the polyethylene glycol linker used in the present invention is in the range of greater than 0 to 200 kDa, specifically in the range of about 1 to 100 kDa, about 1 to 50 kDa, about 1 to 30 kDa, about 2 to 30 kDa, about 1 to 20 kDa, more specifically in the range of about 3.4 kDa to 10 kDa, and even more specifically about 3.4 kDa, but is not limited to these ranges. Furthermore, the non-peptide linker of the present invention that is bonded to the polypeptide corresponding to F may be a combination of different types of polymers, not just one type of polymer.

[0185] In this invention, "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc. Any numerical value within a range equivalent to or similar to the numerical value following the term "approximately" is acceptable, but is not limited to these.

[0186] Specifically, the non-peptide linker may have reactive groups at both ends in a state where it is not bound to F and X, and may be bound to F and X via the reactive groups.

[0187] As a specific example, both ends of the linker are bonded to the thiol group, amino group, or hydroxyl group of the immunoglobulin Fc region, and to the thiol group, amino group, azide group, or hydroxyl group of the interleukin-2 analog (X), but are not limited to these.

[0188] Specifically, the linker is a reactive group whose two ends are bound to the immunoglobulin Fc region and the interleukin-2 analog (X), respectively. More specifically, it is a reactive group that is bound to the thiol group of cysteine ​​in the immunoglobulin Fc region and at least one selected from the group consisting of an amino group located at the N-terminus, lysine, arginine, glutamine, and / or histidine, and a hydroxyl group located at the C-terminus, and to the thiol group of cysteine ​​in the interleukin-2 analog (X) and at least one selected from the group consisting of an amino group of lysine, arginine, glutamine, and / or histidine, an azide group of azidrisine, and a hydroxyl group, but is not limited to these.

[0189] More specifically, the linker's reactive group is at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited to these.

[0190] In the above, examples of aldehyde groups include, but are not limited to, propionaldehyde groups and butyraldehyde groups.

[0191] In the above, succinimide derivatives include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate.

[0192] The linker may be linked to an immunoglobulin Fc region, F, and an interleukin-2 analog, X, via the reactive group, and converted into a linker linkage.

[0193] Furthermore, the final product generated by reductive alkylation via aldehyde bonds is far more stable than that linked by amide bonds. The aldehyde reactive group selectively reacts with the N-terminus at low pH, and can form covalent bonds with lysine residues at high pH, ​​such as pH 9.0.

[0194] Furthermore, the reactive groups at both ends of the linker may be the same or different. For example, it may have aldehyde groups at both ends, or one end may have a maleimide group and the other end may have an aldehyde group, propionaldehyde group, or butyraldehyde group. However, it is not limited to this, as long as F, specifically the immunoglobulin Fc region and X, are bound to each end of the linker.

[0195] For example, one end of the linker may contain a maleimide group as a reactive group, while the other end may contain an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like.

[0196] When polyethylene glycol having hydroxyl reactive groups at both ends is used as a linker, the interleukin-2 analog sustained-type conjugate of the present invention can be prepared by activating the hydroxyl groups as various reactive groups as described above through known chemical reactions, or by using commercially available polyethylene glycol having modified reactive groups.

[0197] In one specific embodiment, the linker is linked to a cysteine ​​residue of X, more specifically to the -SH group of cysteine, but is not limited to this.

[0198] Specifically, the linker's reactive group may be linked to the -SH group of the cysteine ​​residue. The reactive group is as described above. When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X by a thioether bond, and the aldehyde group can be linked to F, specifically the -NH2 group of immunoglobulin Fc, by a reductive amination reaction, but is not limited to this.

[0199] In other specific embodiments, the linker is linked to a lysine residue of X, more specifically to the amino group of lysine, but is not limited to these embodiments.

[0200] Furthermore, in the aforementioned conjugate, the linker's reactive group may be linked to the -NH2 located at the N-terminus of the immunoglobulin Fc region, but is not limited thereto.

[0201] Furthermore, in the aforementioned compound, the interleukin-2 analog according to the present invention may be linked to a linker having a reactive group via its C-terminus, but this is merely one example.

[0202] In this invention, "C-terminus" refers to the carboxyl end of a peptide, and for the purposes of this invention, it refers to the position where the linker is attached. For example, although not limited to these, it includes not only the very last amino acid residue of the C-terminus, but also all amino acid residues surrounding the C-terminus, specifically the 1st to 20th amino acid residues from the very end.

[0203] Furthermore, the aforementioned conjugates exhibit improved efficacy persistence compared to X without F modification, and such conjugates include not only the aforementioned forms but also forms encapsulated in biodegradable nanoparticles, among others.

[0204] The interleukin-2 analog sustained-release conjugates of the present invention either have completely lost their binding affinity compared to natural interleukin-2, aldesleukin, or sustained-release conjugates containing the same, or have an interleukin-2α receptor binding affinity of approximately 0.001 times or more, approximately 0.005 times or more, approximately 0.01 times or more, approximately 0.05 times or more, approximately 0.1 times or more, approximately 0.3 times or more, approximately 0.5 times or more, approximately 0.6 times or more, approximately 0.7 times or more, approximately 0.8 times or more, approximately 0.9 times or more, approximately 1.1 times or more, approximately 1.3 times or more, approximately 1.5 times or more, or approximately 1.7 times or more. However, these values ​​are not limited, and any conjugates in which the binding affinity has changed compared to natural interleukin-2 or aldesleukin are included in the present invention.

[0205] Specifically, the interleukin-2 analog sustained-release conjugates of the present invention have an interleukin-2β receptor binding affinity of approximately 0.1 times, 0.3 times, 0.5 times, 0.7 times, 1.0 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 130 times, 150 times, or 200 times or more compared to the interleukin-2β receptor binding affinity of natural interleukin-2, aldesleukin, or sustained-release conjugates containing the same. However, these values ​​are not limited, and any substance with a binding affinity that is different from or increased compared to natural interleukin-2 or aldesleukin is included in the present invention.

[0206] In this specification, unless otherwise specified, the detailed descriptions and claims relating to the “interleukin-2 analog” or “conjugate” in which an interleukin-2 analog is covalently linked to a biocompatible substance according to the present invention apply to all categories that include not only the interleukin-2 analog or conjugate, but also salts of the interleukin-2 analog or conjugate (e.g., pharmaceutically acceptable salts of the interleukin-2 analog) or solvates thereof. Therefore, even if the specification only refers to “interleukin-2 analog” or “conjugate,” the description applies equally to specific salts, specific solvates, and specific solvates of specific salts. These salt forms may be, for example, any pharmaceutically acceptable salt.

[0207] The type of salt is not particularly limited. However, it is preferable that it be in a form that is safe and effective for individuals, such as mammals, but is not particularly limited thereto.

[0208] The term "pharmaceutically acceptable" means a substance that can be used effectively for its desired purpose without inducing excessive toxicity, irritation, or allergic reactions, within the bounds of pharmaceutical judgment.

[0209] In this invention, "pharmaceutically acceptable salts" include salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromate, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0210] Furthermore, in this invention, "solvate" means a product in which the interleukin-2 analog or a salt thereof according to the present invention forms a complex with a solvent molecule.

[0211] The composition according to the present invention may be a composition comprising the interleukin-2 analog or its sustained-release conjugate, and more specifically, it may be a pharmaceutical composition having cancer prevention or therapeutic use, and may also be administered in combination with an immune checkpoint inhibitor or a composition containing the same. The interleukin-2 analog and its sustained-release conjugate are as described above. More specifically, it may be a composition comprising a pharmacologically effective amount of the interleukin-2 analog or its sustained-release conjugate, further comprising a pharmaceutically acceptable carrier, and may also be administered in combination with a pharmacologically effective amount of an immune checkpoint inhibitor or a composition containing the same.

[0212] Alternatively, the composition of the present invention may further contain an immune checkpoint inhibitor, but is not limited thereto.

[0213] Specific examples of compositions according to the present invention include interleukin-2 analogs having any amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 106, or sustained-release conjugates containing the same. More specifically, examples include, but are not limited to, compositions containing an interleukin-2 analog having any amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 42, 53, 87, 105, and 106, or the said interleukin-2 analog or sustained-release conjugate containing the same, which are administered in combination with an immune checkpoint inhibitor or a composition containing the same.

[0214] The present invention provides for a composition comprising an interleukin-2 analog or a sustained-release interleukin-2 analog conjugate, which is administered concurrently, sequentially, or in reverse order with a composition comprising an immune checkpoint inhibitor, but is not limited thereto.

[0215] In this invention, "pharmacologically effective dose" means a safe dose in which the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor exert a preventive or therapeutic effect against cancer while being free from toxicity or side effects in the patient. Specifically, it means, but is not limited to, a dose that shows significant activity on interleukin-2 receptors (e.g., β and / or α receptors) or a dose that activates T cells.

[0216] The composition according to the present invention may exhibit at least one of the following properties, but any composition that exhibits an improved immune response, an anti-cancer effect, or the like is acceptable. (i) Higher blood exposure compared to aldesleukin monotherapy or aldesleukin combined with an immune checkpoint inhibitor. (ii) Superior tumor growth suppression compared to aldesleukin monotherapy or aldesleukin combined with an immune checkpoint inhibitor. (iii) Superior memory T cell generation response compared to aldesleukin monotherapy or aldesleukin combined with an immune checkpoint inhibitor. (iv) Superior suppression of immune checkpoint protein activation compared to aldesleukin monotherapy or aldesleukin combined with an immune checkpoint inhibitor. (v) Superior T cell activation compared to aldesleukin monotherapy or aldesleukin combined with an immune checkpoint inhibitor.

[0217] Interleukin-2, known as a T cell growth factor, is a protein involved in immunomodulation. It has the activity to proliferate T cells, stimulate B cells, and act on T cells to secrete gamma interferon. Based on this immunomodulatory activity of interleukin-2, cancer prevention and treatment effects can be obtained by eliminating cancer cells using the body's immune system.

[0218] In particular, the interleukin-2 analog of the present invention has increased binding affinity to the interleukin-2β receptor, which plays a major role in signal transduction, thus providing a more effective anticancer effect in the individual's immune system. Furthermore, the sustained-release conjugate containing the interleukin-2 analog exhibits increased binding affinity to the interleukin-2β receptor and, as its persistence increases, the amount of blood exposure increases, resulting in excellent bioavailability. Ultimately, it possesses excellent tumor growth inhibition and memory T cell generation ability, thus exhibiting effective cancer prevention or therapeutic effects. Moreover, because it possesses excellent memory T cell generation ability and can exert an effect of suppressing cancer recurrence through the individual's immune memory response, it can be used as a safe and effective cancer treatment agent.

[0219] 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, which in turn utilize the immune checkpoints of T cells to attack cancer cells that evade the immune system, thus providing an excellent combination therapy effect.

[0220] In this invention, "immune checkpoint inhibitor" refers to a therapeutic agent that activates T cells and attacks cancer cells by binding to immune checkpoint proteins involved in T cell suppression and blocking their activity.

[0221] The aforementioned immune checkpoint inhibitors are therapeutic agents that maintain the original function of immune cells (e.g., T cells) by interfering with the binding of immune checkpoint proteins (e.g., PD-1) to ligands on cancer cells (e.g., PD-L1).

[0222] The aforementioned immune checkpoint inhibitors include peptides, antibodies or their antigen-binding fragments, nucleic acid molecules, and small molecules, but any substance that has antagonistic activity is acceptable.

[0223] Examples of immune checkpoint inhibitors of the present invention include anti-PD-1 antibodies or their antigen-binding fragments.

[0224] PD-1 (programmed cell death-1) is the most well-known immune checkpoint protein. It resides on the outside of immune cells and can specifically bind to the PD-L1 (programmed death-ligand 1) protein on various cells in the human body. When PD-L1 and PD-1 bind, immune cell function is inactivated, suppressing the immune response and preventing unwanted autoimmune reactions. However, cancer cells express large amounts of PD-L1 protein on their surface, and by binding to PD-1 on immune cells, they can disable T cells and prevent the body's immune response. In this case, cancer cells can evade the immune response and survive.

[0225] The immune checkpoint inhibitor of the present invention has an antagonistic effect against immune checkpoint proteins that cancer cells use to evade the immune system, thereby ensuring that an immune response that neutralizes cancer cells occurs appropriately, and thus providing an anticancer effect using the body's own immune response.

[0226] In the present invention, the anti-PD-1 (programmed cell death-1) antibody is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514, but is not limited to these.

[0227] The pharmaceutical composition for cancer prevention or treatment of the present invention, which contains an interleukin-2 analog or a sustained-release conjugate thereof, is administered in combination with an immune checkpoint inhibitor or a composition containing the same. Therefore, due to the synergistic effect of the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor, it may exhibit excellent anticancer efficacy while minimizing side effects.

[0228] While immune checkpoint inhibitors are known to exhibit different responsiveness depending on the phenotype of the tumor microenvironment (TME), their responsiveness is extremely low in immune nonpenetrating tumors (cold tumors), making their use as cancer treatment difficult.

[0229] The interleukin-2 analog or its sustained-release conjugate of the present invention can induce the transformation of immune-impermeable tumors into invasive tumors (hot tumors) by altering the tumor microenvironment, which is less responsive to immune checkpoint inhibitors. Therefore, administering an immune checkpoint inhibitor together with the interleukin-2 analog or its sustained-release conjugate yields excellent therapeutic effects.

[0230] PD-1 (programmed cell death-1), one of the immune checkpoint proteins, is located on the outside of immune cells and can specifically bind to the PD-L1 (programmed death-ligand 1) protein on various cells in the human body.

[0231] The present invention provides a pharmaceutical composition for cancer prevention or treatment comprising an interleukin-2 analog or a sustained-release conjugate thereof, which can more effectively treat cancers that are less responsive to PD-1 antagonists, but is not limited thereto.

[0232] In the present invention, the cancers 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, gastric cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, perianal gland cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin lymphoma, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, This includes, but is not limited to, lymphocytic lymphoma, renal and pelvic cancer, CNS tumors, primary CNS lymphoma, spinal cord tumors, brain tumors, gliomas (astrocytoma, glioblastoma, oligodendroglioma, ependymoma), germ cell tumors, meningiomas, brainstem gliomas, pituitary adenomas, schwannomas, congenital tumors, craniopharyngiomas, or brain tumors.

[0233] Specifically, the cancer is selected from the group consisting of colorectal cancer, liver cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, kidney cancer, lung cancer, skin cancer, melanoma, breast cancer, bladder cancer, and gastric cancer. Alternatively, the cancer according to the present invention is a primary cancer, a recurrent cancer, or a metastatic cancer, but is not limited to these. It may also include metastatic renal cell carcinoma or metastatic melanoma. More specifically, the cancer is a carcinoma that is less responsive to a PD-1 antagonist.

[0234] The pharmaceutical composition of the present invention contains, but is not limited to, an interleukin-2 analog or its sustained-release conjugate in an amount of 0.01 to 99% by weight / volume.

[0235] Specifically, the compositions of the present invention include, but are not limited to, an interleukin-2 analog or its sustained-release conjugate and an immune checkpoint inhibitor, each administered at a dose of 0.00001 mg / mL to 100 mg / mL.

[0236] More specifically, the composition of the present invention contains a sustained-release conjugate of an interleukin-2 analog in an amount of 0.0001 to 2120 mg / kg, and an immune checkpoint inhibitor is administered in combination with the sustained-release conjugate of the interleukin-2 analog in an amount of 0.0001 to 1000 mg / kg, but is not limited thereto. Alternatively, the composition of the present invention contains an interleukin-2 analog in an amount of 0.00005 to 500 mg / kg, and an immune checkpoint inhibitor is administered in combination with an amount of 0.0001 to 1000 mg / kg, but is not limited thereto.

[0237] However, the dosage of the immune checkpoint inhibitor may be modified to obtain the superior effects of co-administration with the interleukin-2 analog or its sustained-release conjugate of the present invention.

[0238] For example, the dose containing the immune checkpoint inhibitor in the composition of the present invention is indicated based on the PD-1 inhibitor, and if other immune checkpoint inhibitors are included, the dose of the immune checkpoint inhibitor is equivalent to the dose of the PD-1 inhibitor, but is not limited thereto.

[0239] Furthermore, the interleukin-2 analog or its sustained-release conjugate contained in the composition of the present invention and the immune checkpoint inhibitor may be administered in appropriate divided doses, and the number of doses may be the same or different. For example, if the interleukin-2 analog or its sustained-release conjugate of the composition is administered once a week, the immune checkpoint inhibitor may be administered once, twice, or more times a week, but is not limited to these doses.

[0240] In the present invention, "prevention" means any action that suppresses or delays cancer or tumors by administering the interleukin-2 analog (for example, in the form of the interleukin-2 analog itself or a sustained-release conjugate to which a biocompatible substance is bound) or a composition containing the same, and an immune checkpoint inhibitor or a composition containing the same.

[0241] In the present invention, "treatment" means any action that improves or favorably alters the symptoms of cancer by administering the interleukin-2 analog (for example, in the form of the interleukin-2 analog itself or a sustained-release conjugate to which a biocompatible substance is bound) or a composition containing the same, and an immune checkpoint inhibitor or a composition containing the same.

[0242] The use of the interleukin-2 analog or its sustained-release conjugate of the present invention in conjunction with immune checkpoint inhibitors offers significant advantages, including improved quality of life for patients with chronic conditions that require daily administration, due to groundbreaking improvements in blood exposure, blood half-life, and sustained in vivo efficacy.

[0243] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier or diluent. Such a pharmaceutically acceptable carrier or diluent may be non-spontaneously occurring.

[0244] In this invention, "pharmaceutically acceptable" means a sufficient amount to produce a therapeutic effect and without causing side effects, which can be easily determined by a person skilled in the art based on known factors in the medical field such as the type of cancer, the patient's age, weight, health condition, sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of administrations, the duration of treatment, the formulation, and drugs used simultaneously.

[0245] The pharmaceutical composition comprising the interleukin-2 analog or its sustained-release conjugate, or an immune checkpoint inhibitor, of the present invention may contain pharmaceutically acceptable excipients. For oral administration, the excipients may include binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc. For injectable preparations, a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., may be used. For topical administration, bases, excipients, lubricants, preservatives, etc., may be used, but are not limited to these.

[0246] The composition of the present invention can be manufactured in various forms by mixing it with pharmaceutically acceptable excipients as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it can be manufactured in single-use ampoules or multi-dose formulations. In addition, it can be manufactured in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0247] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The formulation may also further contain fillers, anti-agglomerates, lubricants, wetting agents, fragrances, preservatives, and the like.

[0248] Furthermore, the pharmaceutical composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0249] Furthermore, the composition may be formulated into a unit-dose type formulation suitable for intravenous administration to a patient by conventional methods in the field of pharmacy, specifically into a formulation useful for the administration of protein pharmaceuticals, and may be administered orally, or by parenteral administration routes including, but not limited to, skin, intravenous, intramuscular, intraarterial, intramedullary, intrameningeal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, local, sublingual, vaginal, or rectal routes using administration methods commonly used in the art.

[0250] Furthermore, the conjugate can be used by mixing it with various carriers acceptable for drugs, such as physiological saline and organic solvents. In order to improve stability and absorbability, carbohydrates such as glucose, sucrose, and dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, and other stabilizers can be used as drugs.

[0251] Another aspect of the present invention provides a method for preventing or treating cancer, which includes administering a pharmaceutical composition containing an interleukin-2 analog or a sustained conjugate containing the same in combination with an immune checkpoint inhibitor or a composition containing the same to an individual who needs it. In the above method, the pharmaceutical composition containing an interleukin-2 analog or a sustained conjugate containing the same and the immune checkpoint inhibitor or the composition containing the same are administered simultaneously, sequentially, or in reverse order, but are not limited thereto.

[0252] The interleukin-2 analog and / or the sustained conjugate of the interleukin-2 analog, the immune checkpoint inhibitor, the compositions containing them, cancer, prevention, and treatment are as described above.

[0253] Yet another aspect for realizing the present invention is a method for preventing or treating cancer, which includes co-administering an interleukin-2 analog or a sustained conjugate containing the same and an immune checkpoint inhibitor to an individual.

[0254] The above method may be to administer a composition containing an interleukin-2 analog or a sustained conjugate containing the same and an immune checkpoint inhibitor to an individual, or may be to co-administer an interleukin-2 analog or a sustained conjugate containing the same and an immune checkpoint inhibitor in separate formulations, but is not limited thereto.

[0255] The method of the present invention may administer (i) an interleukin-2 analog or a sustained conjugate containing the same, and (ii) an immune checkpoint inhibitor as one formulation, or may administer individual formulations simultaneously, individually, sequentially or in reverse order, but is not limited thereto.

[0256] The interleukin-2 analog or the sustained conjugate containing the same, the immune checkpoint inhibitor, the composition, cancer, prevention and treatment are as described above.

[0257] The individual in the present invention means an individual who has developed cancer or an individual suspected of having cancer, and includes mammals such as humans, mice, and livestock. Any individual that can be treated with the interleukin-2 analog and / or conjugate, immune checkpoint inhibitor, or the composition containing them of the present invention may be used.

[0258] "Administration" in the present invention means introducing a predetermined substance (for example, an interleukin-2 analog or its sustained conjugate, or an immune checkpoint inhibitor) into a patient by any appropriate method. The administration route can be any general route as long as it can deliver to the target in vivo, and examples include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, etc., but is not particularly limited thereto.

[0259] The method of the present invention may include administering a pharmaceutical composition containing the interleukin-2 analog or its sustained-release conjugate in a pharmaceutically effective amount, and administering an immune checkpoint inhibitor or a pharmaceutical composition containing the same in a pharmaceutically effective amount. The preferred total daily dose is determined by the attending physician within the bounds of sound medical judgment and can be administered in one dose or in several divided doses. However, for the purposes of the present invention, the specific therapeutically effective dose for a particular patient is preferably different depending on the type and degree of response to be achieved, whether other formulations are used in some cases, the specific composition, the patient's age, weight, general health condition, sex, diet, administration time, route of administration, secretion rate of the composition, duration of treatment, drugs administered together with or simultaneously with the specific composition, and other factors, as well as similar factors well known in the pharmaceutical field.

[0260] In the method of the present invention, the dosage and frequency of administration are determined by the type of drug that is the active ingredient, along with various related factors such as the disease being treated, the route of administration, the patient's age, sex and weight, and the severity of the disease. Specifically, the composition of the present invention contains, but is not limited to, the interleukin-2 analog or a sustained-release conjugate thereof and an immune checkpoint inhibitor in a pharmaceutically effective amount.

[0261] The statement that the interleukin-2 analog or sustained-release conjugate and the immune checkpoint inhibitor are present in a pharmaceutically effective amount means that the interleukin-2 analog or sustained-release conjugate and the immune checkpoint inhibitor are present in an amount sufficient to achieve the desired pharmacological activity (e.g., prevention, improvement, or treatment of cancer), and that the amount is present at a pharmaceutically acceptable level, with no toxicity or side effects to the administered individual, or only minimal toxicity or side effects. Such a pharmaceutically effective amount is determined by comprehensively considering factors such as the number of administrations, the patient, and the dosage form.

[0262] The pharmaceutical composition of the present invention contains the above-mentioned components (active ingredients) in an amount of 0.01 to 99% by weight and volume, but is not particularly limited thereto.

[0263] The total effective dose of the composition of the present invention may be administered to a patient as a single dose, or as part of a fractionated treatment protocol involving multiple doses over a long period. The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the severity of the disease. Specifically, the total dose of the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor administered in combination with the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day. However, the effective dose of the interleukin-2 analog or its conjugate and the immune checkpoint inhibitor is determined by considering various factors such as the patient's age, weight, health status, sex, disease severity, diet, and excretion rate, in addition to the administration route and number of treatments of the pharmaceutical composition. Considering these factors, a person with ordinary knowledge in the field should be able to determine an appropriate effective dose of the composition of the present invention for a specific use. The pharmaceutical composition according to the present invention is not particularly limited in dosage form, administration route, and administration method, as long as it achieves the effects of the present invention.

[0264] The pharmaceutical composition of the present invention exhibits excellent in vivo persistence and potency, thereby significantly reducing the number and frequency of administrations of the pharmaceutical preparation of the present invention. The pharmaceutical composition is administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or rectal routes, but is not limited to any specific route of administration as long as the desired pharmacological effect is achieved.

[0265] For example, the pharmaceutical composition of the present invention may be administered once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, or it may be administered once or multiple times at time intervals ranging from one week to one month, but is not limited to these.

[0266] Specifically, interleukin-2 analogs or their sustained-release conjugates and immune checkpoint inhibitors may be administered at doses of approximately 0.00001 mg or more and 0.0000001 mg or more per kg of body weight of the patient per week, respectively. When the two substances are used in combination, the total dose may be approximately 0.00001 mg or more per kg of body weight of the patient per week, but is not limited to these doses.

[0267] Specifically, the method of the present invention involves administering 0.00001 to 175 mg of a sustained-release interleukin-2 analog conjugate and 0.0000001 to 100 mg of an immune checkpoint inhibitor per kg of body weight of the patient per week, but is not limited thereto. Alternatively, the method of the present invention involves administering 0.000001 to 50 mg of an interleukin-2 analog and 0.0000001 to 100 mg of an immune checkpoint inhibitor per kg of body weight of the patient per week, but is not limited thereto.

[0268] Alternatively, the method of the present invention involves administering an interleukin-2 analog or its sustained-release conjugate at a dose of 0.001 to 33,000 nmol / kg per week, and an immune checkpoint inhibitor at a dose of 0.0001 to 7,000 nmol / kg, but is not limited thereto.

[0269] Furthermore, the interleukin-2 analog or its sustained-release conjugate and the immune checkpoint inhibitor of the present invention may be administered in appropriate divided doses, and the number of doses may be the same or different. For example, if the interleukin-2 analog or its sustained-release conjugate is administered once a week, the immune checkpoint inhibitor may be administered once, twice, or more times a week, but is not limited to these doses.

[0270] However, the dosage of the immune checkpoint inhibitor may be modified to obtain the superior effects of co-administration with the interleukin-2 analog or its sustained-release conjugate of the present invention.

[0271] For example, the dosage of the administration of the immune checkpoint inhibitor is shown based on the PD-1 inhibitor. In the case of other immune checkpoint inhibitors, they are administered at a dosage corresponding to the dosage of the PD-1 inhibitor, but are not limited thereto.

[0272] Still another aspect for realizing the present invention is the use of the interleukin-2 analog or its sustained conjugate, or a composition containing the same, for the production of a medicament for preventing or treating cancer, which is a composition co-administered with an immune checkpoint inhibitor.

[0273] Still another aspect for realizing the present invention is the combined use of an interleukin-2 analog or its sustained conjugate and an immune checkpoint inhibitor for cancer prevention or treatment.

[0274] The interleukin-2 analog and / or its sustained conjugate, the immune checkpoint inhibitor, or a composition containing them, cancer, prevention, treatment, administration route, and administration frequency are as described above.

[0275] Still another aspect for realizing the present invention provides a cancer prevention or treatment use of an interleukin-2 analog or its sustained conjugate, or a composition containing the same, which is a composition co-administered with an immune checkpoint inhibitor.

[0276] The interleukin-2 analog and / or its sustained conjugate, the immune checkpoint inhibitor, or a composition containing them, cancer, prevention, treatment, administration route, and administration frequency are as described above.

[0277] In addition, in this specification, unless otherwise specified, expressions such as "comprising", "having", "containing", etc. are understood to mean including the specified integer or set of integers, but not excluding other integers or sets of integers.

[0278] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of illustrating the present invention in more detail, and the present invention is not limited to these examples. [Examples]

[0279] Preparation of expression vectors for natural interleukin-2 and interleukin-2 analogs To create a natural interleukin-2 expression vector encoding 133 amino acids, interleukin-2 synthesized based on the reported interleukin-2 sequence (NM_000586.3; SEQ ID NO: 1) was cloned into the pET-22b vector (Novagen). Furthermore, novel interleukin-2 analogs were created using the aforementioned interleukin-2 as a template, with modifications to the amino acids of interleukin-2.

[0280] The PCR conditions for amplifying the interleukin-2 analogs were 95°C for 30 seconds, 55°C for 60 seconds, and 65°C for 6.5 minutes, and this process was repeated for 16 cycles. Sequence analysis was performed on the mutagenesis products obtained under the above conditions, and it was confirmed that each interleukin-2 analog had the mutations shown in Table 1, relative to the natural product, at the target mutation site. The expression vectors thus obtained were named pET22b-interleukin-2 analogs 1 to 105.

[0281] Table 1 shows the altered sequences and analog names for each amino acid. To prepare these interleukin-2 analogs, PCR was performed using forward (F) and reverse (R) primers to amplify each analog gene.

[0282] In Table 1, Analog 1 is aldesleukin, and Primers #1 to #204 correspond to Sequence IDs 214 to 417 as specified herein. Table 1 summarizes the types of interleukin-2 analogs, as well as their mutation sites and altered sequences.

[0283] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0284] desA1 means that the first amino acid of interleukin-2, alanine, has been deleted. Table 2 shows the full-length protein sequences of the interleukin-2 analogs. Bold text in Table 2 indicates the mutation site. Table 2 is a summary of the amino acid sequences of the interleukin-2 analogs.

[0285] [Table 2-1] [Table 2-2] [Table 2-3]

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Example

[0286] Expression of interleukin-2 analog Using the expression vector prepared in Example 1, recombinant interleukin-2 analog was expressed under the regulation of the T7 promoter. For each recombinant interleukin-2 analog expression vector, the expression Escherichia coli strain, E. coli BL21DE3( E. coli B F - dcm ompT hsdS (r B - m B - ) galλ(DE3); Novagen was transformed. The transformation method used was the one recommended by Novagen. A single colony transformed by each recombinant expression vector was obtained and inoculated into 2× Luria broth containing ampicillin (50 μg / mL) and cultured at 37°C for 15 hours. The culture medium of the recombinant strain and 2× LB medium containing 30% glycerin were mixed in a 1:1 (v / v) ratio and dispensed into 1 mL each of cryo tubes, which were stored at -150°C. This was used as a cell stock for the production of recombinant proteins.

[0287] To express recombinant interleukin-2 analogs, one vial of each cell stock was lysed and inoculated into 500 mL of 2 × LB medium, and cultured with shaking at 37°C for 14-16 hours. When the absorbance value at 600 nm reached 4.0 or higher, the culture was terminated and used as the seed culture. Initial fermentation was started by inoculating the seed culture into 1.6 L of fermentation medium using a 5 L fermenter (Bioflo-320, NBS, USA). The culture conditions were 37°C, 2.0 L / min (1 vvm) of air, and 650 rpm of stirring, and the pH was maintained at 6.70 using 30% aqueous ammonia. Fermentation was carried out by fed-batch culture, with additional medium (feeding solution) added when the nutrients in the culture medium were depleted. Strain growth was monitored by OD value, and when the absorbance value reached 70 or higher, a final concentration of 500 μM IPTG was introduced. The culture was continued for approximately 23-25 ​​hours after the introduction of IPTG. After the culture was completed, recombinant strains were obtained using a centrifuge and stored at -80°C until use. [Examples]

[0288] Extraction and refolding of interleukin-2 analogs To convert the interleukin-2 analog from the interleukin-2 analog-expressing E. coli obtained in Example 2 into a soluble form, the cells were disrupted and refolded. A cell pellet equivalent to 100 mL of culture medium was suspended in 1-200 mL of disruption buffer (20 mM Tris-HCl pH 9.0, 1 mM EDTA pH 9.0, 0.2 M NaCl, 0.5% Triton X-100), and the recombinant E. coli were then disrupted using a microfluidizer at 15,000 psi. The cells were centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and the pellet was washed with 400 mL of the initial washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0). The pellet was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of a second washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 2% Triton X-100). The pellet was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of a third washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1% sodium deoxycholorate). The pellet was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of a fourth washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1 M NaCl). The washed E. coli inclusion body pellet was obtained by centrifugation under the same conditions as above. The washed inclusion pellet was resuspended in 400 mL of soluble / reducing buffer (6M Guanidine, 100 mM Tris pH 8.0, 2 mM EDTA pH 9.0, 50 mM DTT) and stirred at 50°C for 30 minutes. 100 mL of distilled water was added to the soluble / reduced interleukin-2 analog to dilute the 6M Guanidine to 4.8M Guanidine, and then the mixture was centrifuged at 13,900 g for 30 minutes, discarding the pellet and obtaining only the solution. 185.7 mL of distilled water was further added to the diluted solution to dilute the 4.8M Guanidine to 3.5M Guanidine, and then the pH was adjusted to 5.0 using 100% acetic acid.The pH-adjusted solution was stirred at room temperature for 1 hour. After impurities precipitated, the solution was centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and the pellet was washed with the final washing buffer (3.5 M Guanidine, 20 mM Sodium Acetate pH 5.0, 5 mM DTT). The pellet was obtained by centrifugation under the same conditions as above. The washed interleukin-2 analog was dissolved in 400 mL of refolding buffer (6 mM Guanidine, 100 mM Tris pH 8.0, 0.1 mM CuCl2). The refolding process was carried out by stirring the mixed solution at 4°C for 15 to 24 hours. [Examples]

[0289] Size exclusion column chromatography The interleukin-2 analog refolding solution obtained in Example 3 was concentrated to less than 1 mL for purification by applying it to a size exclusion column. The column was equilibrated with buffer (2M Guanidine, 100mM Tris pH 8.0) before introducing the refolding solution, and then eluted by running buffer after introducing the refolding solution. Since the eluted sample contained guanidine, it was changed to a stabilization solution (10mM Sodium Acetate pH 4.5, 5% Trehalose), and then its purity was measured by RP-HPLC and peptide mapping analysis. Samples with a measured purity of 80% or higher were used in experiments. [Examples]

[0290] Evaluation of receptor binding affinity of interleukin-2 analogs To measure the receptor binding affinity of the interleukin-2 analog obtained in Example 4 to the interleukin-2 α-receptor and β-receptor, respectively, surface plasmon resonance (SPR, BIACORE T200, GE Healthcare) was used. The binding affinity of the fabricated analogs to the α-receptor and β-receptor was measured and compared with that of interleukin-2 analog 1 (aldesleukin).

[0291] First, approximately 5,000 RU (resonance units) of anti-human immunoglobulin antibody (Abcam, #ab97221) were immobilized on a CM5 chip (GE Healthcare) using amine coupling. Then, interleukin 2α receptors (SYMANSIS, #4102H) or interleukin 2β receptors (SYMANSIS, #4122H), to which the human immunoglobulin Fc site had bound, were finally immobilized by binding them to the immunoglobulin antibodies via antigen-antibody binding reaction. Next, the recombinant interleukin 2 analogs prepared as described above were diluted in HBS-P+ buffer (Cytiva, BR100671) at different concentrations using a 2-fold serial dilution method. These dilutions were then passed through the CM5 chip with the finally immobilized interleukin 2 receptors, and the binding affinity of each interleukin 2 receptor was measured. Binding strength was measured using binding velocity (Ka) and dissociation velocity (Kd). The binding velocity was measured by flowing an interleukin-2 analog at a flow rate of 10 μL / min for 3 minutes, and the dissociation velocity from each interleukin-2 receptor was measured by flowing only HBS-P+ buffer at the same time and flow rate. After the measurements were completed, the receptor binding strength was evaluated using a 1:1 binding fitting model in the Biaevaluation program.

[0292]

number

[0293] In Table 3, "Undefinable" means that binding to the receptor in question was not observed in surface plasmon resonance measurements, and therefore, the physical quantity is undefined for that receptor. Table 3 summarizes the relative binding affinity of interleukin-2 analogs to interleukin-2α or β receptors compared to interleukin-2 analog 1 (aldesleukin).

[0294] [Table 3-1] [Table 3-2] [Table 3-3]

[0295] As can be seen from the test results described above (Table 3), the interleukin-2 analog of the present invention was confirmed to exhibit interleukin-2α receptor binding affinity that differs from that of natural interleukin-2 or aldesleukin, either by completely losing interleukin-2α receptor binding affinity or by decreasing or increasing it compared to interleukin-2 analog 1. In contrast, a relatively stronger binding affinity was observed for the interleukin-2β receptor compared to natural interleukin-2 or aldesleukin. Therefore, it was confirmed that the amino acid sequence of the interleukin-2 analog affects its binding to the interleukin-2α or β receptor. This suggests that the binding affinity to the interleukin-2 receptor can be altered by substituting amino acids at specific positions.

[0296] These experimental results suggest that the interleukin-2 analog according to the present invention possesses altered interleukin-2α receptor binding affinity and interleukin-2β receptor binding affinity, thereby suggesting its potential use in the development of various drugs. [Examples]

[0297] Linking reaction of interleukin-2 analog with polyethylene glycol (3.4K PEG) linker and purification of interleukin-2 analog conjugate. To prepare a persistent conjugate of the interleukin-2 analog obtained in Example 4, which is bound to the immunoglobulin Fc region, a conjugate was first prepared by linking the interleukin-2 analog to one end of a polyethylene glycol (PEG) linker. Interleukin-2 analogs 21, 41, 52, 86, 104, and 105 were used to prepare the conjugate. A polyethylene glycol with a molecular weight of 3.4 kDa and hydroxyhydrogens at both ends modified with propylaldehyde groups (ALD(2)3.4K PEG from NOF Corporation, Japan) was used as the PEG linker, and this was linked to the N-terminus of the interleukin-2 analog. The molar ratio of interleukin-2 analog to PEG linker was set to 1:15 to 1:20, and the concentration of the interleukin-2 analog was set to 1 mg / mL or less. The reaction was carried out at 2 to 10°C for 1 hour. The reaction was carried out under 100 mM potassium phosphate (pH 5.5) with the addition of 20 mM sodium cyanoborohydride (SCB) as a reducing agent. The reaction mixture was converted to 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 with a concentration gradient of triethylamine (pH 8.0) and sodium chloride to obtain the interleukin-2 analog-3.4K PEG conjugate. [Examples]

[0298] Production of interleukin-2 analog-3.4K PEG-immunoglobulin Fc region sustained-release conjugates To prepare the interleukin-2 analog-3.4K PEG-immunoglobulin Fc domain sustained-release conjugate, the molar ratio of the interleukin-2 analog-3.4K PEG conjugate obtained by the method of Example 6 to the immunoglobulin Fc domain (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 15-16 hours. The reaction solution was 100 mM potassium phosphate (pH 6.0), and 20 mM sodium cyanoborohydride was added as a reducing agent.

[0299] The immunoglobulin region used here consists of two monomers having the amino acid sequence of SEQ ID NO: 438 (consisting of 221 amino acids). These monomers form a homodimer via a disulfide bond between the third amino acid, cysteine, of each monomer. The monomers of this homodimer independently form an internal disulfide bond between the 35th and 95th cysteine ​​positions, and an internal disulfide bond between the 141st and 199th cysteine ​​positions.

[0300] Table 4 shows the amino acid sequence of immunoglobulin Fc.

[0301] [Table 4]

[0302] After the reaction was complete, the unreacted immunoglobulin Fc region was removed from the reaction solution using Butyl FF (Cytiva) with Bis-Tris (pH 6.5) and sodium chloride, and then purified using Source 15ISO (Cytiva) with sodium citrate buffer (pH 5.5) and ammonium sulfate. This yielded an interleukin-2 analog-3.4K PEG-immunoglobulin Fc region conjugate (persistent conjugate) in which the N-terminus of the interleukin-2 analog was ligated to one end of a 3.4 kDa PEG linker, and the opposite end of the 3.4 kDa PEG linker was ligated to the nitrogen of the N-terminal proline of the Fc region. This persistent conjugate was analyzed by SDS-PAGE, RP-HPLC, and SE-HPLC (Figures 1 and 2). [Examples]

[0303] Evaluation of interleukin-2 receptor binding affinity of interleukin-2 analog conjugates. To measure the receptor binding affinity of the persistent interleukin-2 analog conjugate obtained in Example 7 to the interleukin-2α and β receptors, surface plasmon resonance (SPR, BIACORE T200, GE Healthcare) was used.

[0304] Specifically, biotin-labeled human interleukin-2 receptor α and β subunits (ACROBiosystems) were immobilized in streptavidin biosensor chips (SA chip, Cytiva) at concentrations of approximately 100 RU and 500 RU, respectively. Interleukin-2 analog sustained-release conjugates or aldesleukin, diluted in HBS-EP+ buffer (Cytiva, BR100669) using a 2-fold serial dilution method, were injected at a flow rate of 20 μL / min. After a 3-minute binding process, HBS-EP+ buffer alone was injected at the same flow rate for 3 minutes to induce dissociation of the interleukin-2 analog sustained-release conjugate or aldesleukin at the interleukin-2 receptor. The binding strength was calculated from the obtained binding and dissociation constants. Binding strength evaluation was performed using a 1:1 binding fitting model in the Biaevaluation program.

[0305] The evaluation results confirmed that each interleukin-2 analog conjugate possesses its own unique interleukin-2 receptor binding affinity, and in particular, clear differences in binding affinity were observed among the candidate substances with respect to the interleukin-2 receptor α subunit. More specifically, the evaluation results showed that interleukin-2 analog conjugates 21 and 52 did not bind to the interleukin-2 receptor α subunit, while interleukin analog conjugates 41, 86, 104, and 105 showed relative binding affinities of 50.8%, 65.2%, 81.0%, and 112.9%, respectively, compared to aldesleukin. Against the interleukin-2 receptor β subunit, all interleukin-2 analog conjugates, including those 21 and 52, showed higher binding affinity compared to aldesleukin.

[0306] Table 5 summarizes the relative binding affinity (%) of interleukin-2 analog sustained-release conjugates, prepared separately for α-receptors and β-receptors, compared to the binding affinity of aldesleukin. In Table 5, each sustained-release conjugate is indicated by the number of the interleukin-2 analog that constitutes it (for example, the sustained-release conjugate of interleukin-2 analog 21 is indicated as "Interleukin-2 Analogue 21 Conjugate"). Table 5 summarizes the interleukin-2 receptor binding affinity of interleukin-2 analog conjugates.

[0307] [Table 5] [Examples]

[0308] Evaluation of the anticancer efficacy of combined administration of interleukin-2 analog sustained-release conjugates and immune checkpoint inhibitors in malignant melanoma. To confirm the anticancer effect of co-administration of the interleukin-2 analog conjugate and an immune checkpoint inhibitor according to the present invention, a malignant melanoma mouse model (B16F10 melanoma tumor syngeneic mouse model) allografted with melanoma cells (B16F10) was administered either alone with the interleukin-2 analog 86 conjugate or aldesleukin (control group), or in combination with a mouse anti-PD-1 antibody (Bio X cell, Catalog# BP0146), respectively. Subsequently, tumor size and individual survival rate were evaluated.

[0309] Specifically, B16F10 cells (ATCC) were subcutaneously injected into the thighs of C57BL / 6 mice. After several days, if tumors were observed macroscopically, eight mice were allocated to each group so that the tumor sizes were similar. Interleukin-2 analog 86 conjugates were administered at doses of 0.08 mg / kg or 6.0 mg / kg (based on the weight of the interleukin-2 analog site in the sustained-release interleukin-2 conjugate), once a week via subcutaneous route for a total of four weeks. The control group (aldesleukin administration group) received 3.0 mg / kg of proleukin injection (Novartis) via intraperitoneal route once daily for five consecutive days, followed by a two-day rest period, for a total of four weeks. The anti-PD-1 antibody monotherapy group received 10.0 mg / kg of mouse anti-PD-1 antibody (Bio X cell) via intraperitoneal route twice a week for a total of four weeks. In the anti-PD-1 antibody combination therapy groups, the interleukin-2 analog 86 conjugate 0.08 mg / kg group, the 6.0 mg / kg group, and the aldesleukin 3.0 mg / kg group each received the same dose of anti-PD-1 antibody (10.0 mg / kg) as the anti-PD-1 monotherapy group. Tumor size was 2,000 mm. 3 The point at which a humanitarian termination would occur was set.

[0310] On day 10 of administration, tumor size (Figure 3A) and the survival rate of each individual over 35 days (Figure 3B) were observed in each group. As a result, in monotherapy, the group administered 6.0 mg / kg of interleukin-2 analog 86 conjugate showed higher tumor suppression ability and individual survival rates than the control group, despite being administered only once a week. In particular, complete remission, where the tumor was completely removed, was observed in 2 out of 8 animals in the interleukin-2 analog 86 conjugate 6.0 mg / kg group, compared to no complete remission in the control group.

[0311] In combination therapy with anti-PD-1 antibodies, the group receiving interleukin-2 analog 86 conjugate in combination with an anti-PD-1 antibody showed higher tumor suppression and individual survival rates compared to the group receiving aldesleukin in combination with an anti-PD-1 antibody. In particular, in the group receiving interleukin-2 analog 86 conjugate at a very low dose compared to aldesleukin (0.08 mg / kg) in combination with an anti-PD-1 antibody, complete remission, where the tumor was completely removed, was observed in 2 out of 8 animals. Furthermore, in the group receiving interleukin-2 analog 86 conjugate at 6.0 mg / kg in combination with an anti-PD-1 antibody, a 100% individual survival rate and complete remission in 7 out of 8 animals were observed, demonstrating outstanding anticancer efficacy. In contrast, complete remission was not observed in the group receiving aldesleukin in combination with an anti-PD-1 antibody. [Examples]

[0312] Evaluation of the anticancer efficacy of combined administration of an interleukin-2 analog sustained-release conjugate and an immune checkpoint inhibitor in a Lewis lung cancer mouse model. To evaluate the anticancer efficacy in a mouse model of lung cancer, Lewis lung carcinoma (LL / 2) cells were allogeneically transplanted into Lewis lung cancer mouse models (LL / 2 tumor syngeneic mouse models). Interleukin-2 analog 86 conjugate or aldesleukin was administered alone, or in combination with a mouse anti-PD-1 antibody (Bio X cell, Catalog# BP0146), and the subsequent antitumor efficacy was evaluated.

[0313] LL / 2 cells (ATCC) were subcutaneously injected into the thighs of C57BL / 6 mice. After several days, if tumors were observed macroscopically, 10 mice were allocated to each group so that the tumor sizes were similar. Each group was divided into a monotherapy group and a combination therapy group with an immune checkpoint inhibitor. The monotherapy group was assigned to receive either interleukin-2 analog 86 conjugate, aldesleukin, or an anti-PD-1 antibody. Specifically, the interleukin-2 analog 86 conjugate group received a dose of 6.0 mg / kg of the conjugate subcutaneously once a week for a total of two doses, based on IL-2 levels. The control group received 3.0 mg / kg of aldesleukin once daily for five consecutive days via the intraperitoneal route, with a two-day rest period, for a total of two doses. Finally, the group receiving immune checkpoint inhibitor monotherapy was given anti-PD-1 antibody via the intraperitoneal route once every 3-4 days for a total of two doses per week for two weeks.

[0314] In addition to the monotherapy group, the immune checkpoint inhibitor combination therapy group was further administered each interleukin-2 analog 86 conjugate and an anti-PD-1 antibody to the control group, with the administration concentrations and intervals being the same as those of the monotherapy group.

[0315] On day 12 of administration, tumor size was observed in each group and analyzed using the Bliss independence model to evaluate drug interactions when administered in combination with immune checkpoint inhibitors.

[0316] As a result, the interleukin-2 analog 86 conjugate not only demonstrated excellent antitumor efficacy when administered alone in a Lewis lung cancer mouse model (Figure 4), but also showed a significant synergistic effect when used in combination with immune checkpoint inhibitors that differentiate it from aldezleukins (Figures 5A and 5B).

[0317] The Lewis lung cancer mouse model is a representative cold tumor model, and it has been reported that treatment with immune checkpoint inhibitors, including anti-PD-1 antibodies, alone is insufficient (Non-Patent Literature 14). In this example, no significant anticancer efficacy was observed with the administration of immune checkpoint inhibitors alone (Figure 4), but a synergistic effect was confirmed when the interleukin-2 analog 86 conjugate according to the present invention was administered in combination with an immune checkpoint inhibitor. Therefore, it is judged that the interleukin-2 analog conjugate of the present invention induced the tumor microenvironment (TME) of the Lewis lung model to be converted from an immune-impermeable tumor to an invasive tumor (hot tumor), and thus it is understood to show superior efficacy when administered in combination with an immune checkpoint inhibitor. [Examples]

[0318] Evaluation of the anticancer efficacy of co-administration of a sustained-release interleukin-2 analog conjugate and an anti-PD-1 antibody in a mouse model of pancreatic ductal adenocarcinoma (PDAC), and confirmation of memory responses in a cancer cure model. To evaluate the anticancer efficacy of interleukin-2 analog sustained-release conjugates alone and in combination with anti-PD-1 antibodies against pancreatic cancer (pancreatic ductal adenocarcinoma), one of the most aggressive malignancies, an allogeneic transplant tumor model was created by subcutaneously injecting mouse-derived pancreatic ductal adenocarcinoma cells (panc02) into the lateral region of 6-week-old female C57BL / 6 mice. After 14 days, tumor volume (~80 mm) was measured. 3 Based on this criterion, 7 individuals were randomly distributed to each group.

[0319] The study divided participants into three groups: one receiving interleukin-2 analog 86 conjugate alone, one receiving anti-PD-1 antibody alone, and one receiving both drugs in combination. Each group received a total of six weeks of treatment according to the administration method. The antitumor efficacy in each experimental group was evaluated by tumor growth inhibition (TGI) using the following formula. Detailed drug dosages, administration routes, frequency, and antitumor efficacy for each group are shown in Table 6.

[0320]

number

[0321] [Table 6]

[0322] The interleukin-2 analog 86 conjugate showed antitumor efficacy from a dose of 1.7 mg / kg and increased in a dose-dependent manner up to a maximum of 25 mg / kg. While no tumor suppression efficacy was observed in the 0.3 mg / kg dose group of the interleukin-2 analog 86 conjugate or the anti-PD-1 antibody monotherapy group compared to the negative control group, a significant level of tumor suppression was observed when both drugs were administered in combination at the same doses. In particular, complete remission was observed in the group administered interleukin-2 analog 86 conjugate monotherapy at doses of 8.5 mg / kg or higher, and complete remission was observed in all individuals in the group administered monotherapy at 25 mg / kg and in combination with the anti-PD-1 antibody.

[0323] In individuals who achieved complete remission, panc02 tumor cells identical to those of the primary cancer were subcutaneously injected laterally 89 days after the initial drug administration to investigate the effect of preventing recurrence through memory responses. As a result, recurrence prevention through memory responses was confirmed in more than 70% of the group that received interleukin-2 analog 86 conjugate (25 mg / kg) alone and the group that received it in combination with an anti-PD-1 antibody (Figure 6). These results suggest that interleukin-2 analog 86 conjugate may be used not only for the treatment of primary pancreatic cancer but also for preventing recurrence of the same tumor.

[0324] These experimental results suggest that the persistent-release conjugate of the interleukin-2 analog according to the present invention exhibits potent anticancer efficacy and a low risk of side effects by showing binding affinity to modified interleukin-2α and β receptors. The structure of the persistent-release conjugate, with its linked human immunoglobulin Fc domain, increases blood exposure, suggesting that it can increase the in vivo immune response with high activity even at lower doses, allowing for longer administration intervals compared to conventional interleukin-2 therapy. Furthermore, it exhibits excellent anticancer efficacy through synergistic action with immune checkpoint inhibitors. Therefore, when the interleukin-2 analog conjugate according to the present invention is administered in combination with an immune checkpoint inhibitor, it can be used for safe and effective anticancer treatment. This suggests that the interleukin-2 analog or the persistent-release conjugate containing it according to the present invention induces an immune memory response in the individual, thereby suppressing cancer recurrence.

[0325] From the above description, those skilled in the art in the field to which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. The present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0326] [Explanation of government-supported research and development] This research was funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare, and supported by the National New Drug Development Project of the National New Drug Development Corporation (RS-2022-00165557).

Claims

1. A pharmaceutical composition for cancer prevention or treatment containing an interleukin-2 analog (analog) comprising any sequence selected from the amino acid sequences of SEQ ID NOs: 3 to 106, The pharmaceutical composition is characterized by being administered in combination with an immune checkpoint inhibitor.

2. The interleukin-2 analog is in the form of a persistent conjugate, and the persistent conjugate is represented by the following chemical formula (1): The composition according to claim 1. 【Chemistry 1】 Here, X is an interleukin-2 analog containing any sequence selected from the amino acid sequences of SEQ ID NOs: 3 to 106. L is polyethylene glycol linker, F is the immunoglobulin Fc region in dimeric form. The dash indicates a covalent bond between X and L, and between L and F. The persistent conjugate is formed in which one end of L is covalently bonded to only one polypeptide chain within the Fc region of the dimerized form, and X is covalently bonded to the opposite end of L.

3. The interleukin-2 analog contains any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 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. The composition according to claim 1 or 2.

4. The composition according to claim 1 or 2, wherein the interleukin-2 analog contains any sequence selected from the group consisting of the amino acid sequences of 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 interleukin-2 analog contains any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105, and 106. The composition according to claim 1 or 2.

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

7. The aforementioned interleukin-2 analog has increased binding affinity to the interleukin-2β receptor compared to aldesleukin. The composition according to claim 1 or 2.

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

9. The immunoglobulin Fc region is the IgG4 Fc region. The composition according to claim 2.

10. The immunoglobulin Fc region is non-glycosylated. The composition according to claim 2.

11. The persistent conjugate is formed in which X is covalently linked to any of the Fc regions of the immunoglobulin Fc region of the dimeric form via the polyethylene glycol linker. The composition according to claim 2.

12. The polyethylene glycol linker is a linker with a molecular weight of 1 kDa to 100 kDa. The composition according to claim 2.

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

14. The aforementioned cancers 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 gland cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, and lymphoma. It is one of the following selected conditions: bulbar lymphoma, renal and pelvic cancer, CNS tumors, primary CNS lymphoma, spinal cord tumors, brain tumors, gliomas (astrocytoma, glioblastoma, oligodendroglioma, ependymoma), germ cell tumors, meningiomas, brainstem gliomas, pituitary adenomas, schwannomas, congenital tumors, craniopharyngiomas, and brain tumors. The composition according to claim 1 or 2.

15. The aforementioned cancer is a cancer that has low responsiveness to PD-1 antagonists. The composition according to claim 1 or 2.

16. The aforementioned composition is administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or rectal routes. The composition according to claim 1 or 2.

17. The aforementioned composition is administered at time intervals ranging from one week to one month. The composition according to claim 1 or 2.

18. The aforementioned composition is characterized by being administered in combination with a composition containing an immune checkpoint inhibitor, either simultaneously, individually, sequentially, or in reverse order. The composition according to claim 1 or 2.

19. The aforementioned immune checkpoint inhibitor is a PD-1 antagonist. The composition according to claim 1 or 2.

20. The immune checkpoint inhibitor is at least one selected from an anti-PD-1 antibody or its antigen-binding fragment. The composition according to claim 19.

21. The anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, cemiprimab, spartalizumab, camrelizumab, sintilimab, tislerizumab, tripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514. The composition according to claim 20.

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