Combination therapy of interleukin-2 analog or conjugate thereof and CTLA -4 antagonist for prevention or treatment of cancer

Abstract

The present invention relates to the combined use of an interleukin-2 analog or a long-acting conjugate thereof and a CTLA-4 antagonist, for the prevention or treatment of cancer, and a pharmaceutical composition comprising same. When administered in combination with the CTLA-4 antagonist, the interleukin-2 analog or the long-acting conjugate thereof exhibits excellent tumor growth inhibition effects and induces complete remission while also reducing side effects, and thus can be used as an excellent anti-cancer therapeutic agent.

Description

Combination therapy of interleukin 2 analogs or conjugates thereof and CTLA-4 antagonists for the prevention or treatment of cancer

[0001] The present invention relates to a combination therapy for the prevention or treatment of cancer of an interleukin 2 analog or a conjugate thereof; and a CTLA-4 antagonist.

[0002]

[0003] Interleukin 2 is an important immunostimulant with a molecular weight of approximately 15 kDa, composed of a total 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 allows it to be used to treat various immune-related diseases, including cancer and AIDS (Korean Patent Publication No. 10-2017-0070091). Currently, Interleukin 2 (trademark Proleukin) is an FDA-approved drug for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, due to severe toxicity associated with high-dose Interleukin 2 therapy, the applicable patient population is limited, and in practice, this therapy is administered to only a small number of eligible patients. Toxicity associated with Interleukin 2 includes severe fever, nausea, vomiting, vascular leak, severe hypotension, pulmonary edema, and vascular leak syndrome, which causes liver damage.

[0004]

[0005] The Interleukin 2 receptor consists of three subunit receptors. These subunits are composed of the alpha chain (IL-2Rα, CD25), the beta chain (IL-2Rβ or CD122), and the gamma chain (IL-2Rγ or CD132), and Interleukin 2 can exert various functions by binding to various combinations of these receptor subunits. A single Interleukin 2 alpha receptor is referred to as a low-affinity Interleukin 2 receptor and does not participate in signal transduction. The complex of Interleukin 2 beta and gamma receptors binds to Interleukin 2 with moderate affinity. The complex of Interleukin 2 alpha, beta, and gamma receptors binds to Interleukin 2 with high affinity. The complex of Interleukin 2 beta and gamma receptors is required for effective signal transduction through the kinase activation of multiple signaling pathways. In particular, the Interleukin 2 beta and gamma coupled receptor is prominent in CD8+ cells and natural killer (NK) cells. In addition, the high-affinity complex of interleukin 2 alpha, beta, and gamma receptors is usually CD4 + It is found not only in T regulatory cells (Tregs) but also in recently activated T cells. The interleukin 2 beta receptor is CD8 + Since it is distributed in T cells or natural killer (NK) cells and participates in the body's immune response, research is being conducted to develop therapeutic agents by enhancing the activity of beta receptors to activate immunity. As an example of such research, interleukin 2 analogs and their sustained-release conjugates have been studied to enhance their utility as therapeutic agents by regulating their binding affinity to interleukin 2 alpha, beta, and gamma receptors (WO2021-201615 A1 and WO2022-211537 A1).

[0006]

[0007] Meanwhile, immune cells generally possess proteins called immune checkpoints on their cell membranes, which suppress unnecessary autoimmune responses and enable them to eliminate cancer cells by detecting tumor-specific antigens expressed due to changes such as mutations occurring in the cells. However, to evade such immune mechanisms, cancer cells alter the function of these immune checkpoints, thereby suppressing T cell function and preventing the proper occurrence of an immune response.

[0008]

[0009] CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) is a representative immune checkpoint that induces immune cells to suppress the immune system. CTLA-4 is a protein expressed on T cells that functions to reduce the immune response by binding to CD80 (B7-1) and CD86 (B7-2) on the surface of antigen-presenting cells. Due to this action of CTLA-4 as an immune checkpoint, T cell function is blocked, ultimately preventing T cells from recognizing cancer cells and inducing their death. The possibility has been suggested that inhibiting CTLA-4 could treat cancer by preventing T cell inactivation and increasing T cell proliferation (e.g., ipilimumab or tremelimumab).

[0010]

[0011] As such, treatment methods that activate the body's immune system to enable immune cells to act on cancer cells and obtain an anticancer effect are being actively researched. However, when drugs that stimulate this immune system are used for anticancer treatment, side effects caused by the overactivation of immune cells become a problem. Therefore, it is necessary to discover safe and effective anticancer agents and treatment regimens.

[0012]

[0013] There is a need to develop safe therapeutic agents and treatment regimens utilizing them that can activate the immune system to exhibit anticancer effects.

[0014]

[0015] One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising an interleukin 2 analog or a sustained-release conjugate thereof, characterized in that it is administered in combination with a CTLA-4 antagonist.

[0016] Another objective of the present invention is to provide a regimen for the prevention or treatment of cancer using an interleukin 2 analog or a sustained-release conjugate thereof; and a CTLA-4 antagonist.

[0017] Another objective of the present invention is to provide a combination comprising an interleukin 2 analog or a sustained-release conjugate thereof; and a CTLA-4 antagonist.

[0018] Another objective of the present invention is to provide a pharmaceutical composition for the prevention, improvement, or treatment of cancer comprising the above combination.

[0019] Another objective of the present invention is 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 a CTLA-4 antagonist.

[0020] Another objective of the present invention is to provide a method for the prevention or treatment of cancer comprising the step of administering and / or using the combination, pharmaceutical composition, or pharmaceutical kit to an individual in need thereof.

[0021] Another object of the present invention is to provide a method for preventing, improving, or treating cancer, comprising the steps 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 in need of a composition containing a pharmaceutically effective amount of a CTLA-4 antagonist.

[0022] Another object of the present invention is to provide the use of the above combination, pharmaceutical composition, or pharmaceutical kit for the prevention, improvement, or treatment of cancer; and / or use for the manufacture of a drug for the prevention, improvement, or treatment of cancer.

[0023]

[0024] A pharmaceutical composition for the prevention or treatment of cancer comprising an interleukin 2 analog according to the present invention or a sustained-release conjugate containing the same can be used as an excellent anticancer treatment by administering it in combination with a CTLA-4 antagonist to exhibit excellent tumor growth inhibition and complete tumor remission while reducing side effects.

[0025]

[0026] Figure 1 is the SDS-PAGE result of the interleukin 2 analog persistent couple (analogs 21, 41, and 52).

[0027] Figures 2a to 2c show the results of purity analysis of interleukin 2 analog persistent conjugates (analogs 21, 41, and 52).

[0028] Figure 3 shows the results of evaluating the antitumor efficacy of combination therapy in an animal model of triple-negative breast cancer (TNBC) using an interleukin 2 analog long-acting conjugate and an anti-CTLA-4 antibody. It also shows the tumor size in mouse models administered either with interleukin 2 analog conjugate 86 alone or in combination with an anti-CTLA-4 antibody. In Figure 3, the dosage of the long-acting conjugate is based solely on the weight of the interleukin 2 analog portion of the entire conjugate.

[0029] Figure 4 shows the results of evaluating the antitumor efficacy of combination therapy with an interleukin 2 analog sustained-release conjugate and an anti-CTLA-4 antibody in a melanoma allograft mouse model. It confirms the tumor size and survival rate in mouse models administered either with interleukin 2 analog conjugate 86 alone or with interleukin 2 analog conjugate 86 in combination with an anti-CTLA-4 antibody. In Figure 4, the dosage of the sustained-release conjugate is based solely on the weight of the interleukin 2 analog portion within the entire conjugate.

[0030]

[0031] 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 a CTLA-4 antagonist. The interleukin 2 (IL-2) analog may comprise a sequence in which one or more amino acids are mutated from natural interleukin 2.

[0032] As one specific example, the interleukin 2 analog is characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 3 to 106.

[0033] In another specific embodiment, the sustained conjugate is characterized as being a sustained conjugate represented by the following chemical formula 1:

[0034] [Chemical Formula 1]

[0035] X - L - F

[0036] In this case, X is an interleukin 2 analog comprising any one sequence selected from the amino acid sequences of SEQ ID NOs 3 to 106;

[0037] L is a polyethylene glycol linker;

[0038] F is the dimeric form of the immunoglobulin Fc region;

[0039] - represents the covalent bond connection between X and L, and between L and F, and

[0040] In the above sustained conjugate, one end of L is covalently connected to only one polypeptide chain of the dimeric Fc region, and X is covalently connected to the opposite end of this L.

[0041] A composition according to any one of the preceding embodiments is characterized in that the sustained-release conjugate includes, as part of the conjugate, an interleukin 2 analog in which the binding affinity to the interleukin 2 alpha receptor is altered and the binding affinity to the interleukin 2 beta receptor is increased compared to natural interleukin 2 or aldesleukin.

[0042] A composition according to any one of the aforementioned embodiments, wherein the interleukin 2 analog is characterized by having an increased binding affinity to the interleukin 2 beta receptor compared to aldesleukin.

[0043] A composition according to any one of the prior embodiments, wherein the interleukin 2 analog comprises any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs 10, 13 to 15, 17, 20 to 22, 32, 35, 36, 42, 53, 54, 56, 58 to 60, 62, 71, 72, 74 to 78, 85, 87, 89, 91 to 94, and 97 to 106.

[0044] A composition according to any one of the prior embodiments, wherein the interleukin 2 analog comprises any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs 17, 22, 42, 53, 56, 58 to 60, 62, 71, 72, 74 to 77, 87, 89, 91 to 93, 98 to 101, and 103 to 106.

[0045] A composition according to any one of the aforementioned embodiments is characterized in that the interleukin 2 analog comprises any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 22, 42, 53, 87, 105, and 106.

[0046] A composition according to any one of the aforementioned embodiments is characterized in that the interleukin 2 analog further comprises one or more amino acids at the C-terminus.

[0047] A composition according to any one of the preceding embodiments is characterized in that the CTLA-4 antagonist is one or more selected from an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

[0048] A composition according to any one of the aforementioned embodiments is characterized in that the anti-CTLA-4 antibody is ipilimumab or tremelimumab.

[0049] A composition according to any one of the preceding embodiments is characterized in that the sustained conjugate comprises an IgG4 Fc region.

[0050] A composition according to any one of the preceding embodiments is characterized in that the sustained conjugate comprises a non-glycosylated immunoglobulin Fc region.

[0051] A composition according to any one of the aforementioned embodiments is characterized in that the immunoglobulin Fc region is derived from a non-glycosylated Fc region derived from human IgG4.

[0052] A composition according to any one of the aforementioned embodiments, wherein the immunoglobulin Fc region has a structure in which two polypeptide chains are connected by disulfide bonds, and is characterized by being connected only through the nitrogen atom of one of the two chains.

[0053] A composition according to any one of the aforementioned embodiments is characterized in that the immunoglobulin Fc region comprises a monomer having the amino acid sequence of SEQ ID NO. 438.

[0054] A composition according to any one of the preceding embodiments is characterized in that the immunoglobulin Fc region is a homomer of the monomer of the amino acid sequence of SEQ ID NO. 438.

[0055] A composition according to any one of the aforementioned embodiments is characterized in that the immunoglobulin Fc region is connected through the nitrogen atom of the N-terminal proline.

[0056] A composition according to any one of the preceding embodiments, wherein the sustained conjugate is characterized in that X is covalently connected to one of the Fc regions of the dimeric immunoglobulin through the polyethylene glycol linker.

[0057] A composition according to any one of the preceding embodiments is characterized in that one end of the linker is connected to only one of the two Fc region chains of the dimeric immunoglobulin Fc region.

[0058] A composition according to any one of the preceding embodiments is characterized in that the polyethylene glycol linker is a linker with a molecular weight of 1 kDa to 100 kDa.

[0059] A composition according to any one of the preceding embodiments is characterized in that the composition further comprises a pharmaceutically acceptable excipient.

[0060] A composition according to any one of the preceding embodiments, wherein 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, stomach cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, pro-anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, 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, lymphocytic lymphoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain tumor, glioma (astrocytoma, glioblastoma, oligodendroglioma) It is characterized by being any one selected from the group consisting of ependymoma, germ cell tumor, meningioma, brainstem glioma, pituitary adenoma, schwannoma, congenital tumor, craniopharyngioma, and brain tumor.

[0061] A composition according to any one of the aforementioned embodiments is characterized in that the breast cancer is triple-negative breast cancer.

[0062] A composition according to any one of the aforementioned embodiments is characterized in that the cancer is a cancer type that is low in responsiveness to a CTLA-4 antagonist.

[0063] A composition according to any one of the aforementioned embodiments is characterized in that the cancer having low responsiveness to the CTLA-4 antagonist is an immune-non-invasive tumor (cold tumor).

[0064] A composition according to any one of the preceding embodiments is characterized by being administered via a route of intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, nasal administration, pulmonary administration, or rectal administration.

[0065] A composition according to any one of the preceding embodiments is characterized by being administered at time intervals ranging from one week to one month.

[0066] A composition according to any one of the preceding embodiments is characterized by being administered concurrently, sequentially, or in reverse order with a CTLA-4 antagonist or a composition containing the same.

[0067] A composition according to any one of the preceding embodiments is characterized in that the composition further comprises a CTLA-4 antagonist.

[0068] Another aspect of the present invention is a method for preventing or treating cancer comprising the step of administering to an individual the interleukin 2 analog or sustained-release conjugate thereof, or a composition containing the same, and a CTLA-4 antagonist or a composition containing the same.

[0069] Another aspect of the present invention is the combined use of the interleukin 2 analog or sustained-release conjugate thereof, or a composition containing the same, and a CTLA-4 antagonist or a composition containing the same for the prevention or treatment of cancer.

[0070] Another aspect of the present invention is to provide a drug for the prevention or treatment of cancer, wherein the composition is administered in combination with a CTLA-4 antagonist, and the interleukin 2 analog or sustained-release conjugate thereof, or a composition containing the same.

[0071] Another aspect of the present invention is a pharmaceutical kit comprising the interleukin 2 analog or its sustained-release conjugate, or the same, for administering the interleukin 2 analog or its sustained-release conjugate in combination with a CTLA-4 antagonist.

[0072]

[0073] The specific details for implementing the present invention are described as follows. Meanwhile, each description and embodiment disclosed herein may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not to be limited by the specific descriptions provided below. Additionally, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0074]

[0075] Throughout this specification, standard one- and three-character codes for amino acids are used. Additionally, amino acids referred to by abbreviations in this specification are described according to the IUPAC-IUB nomenclature.

[0076] Alanine A Arginine R

[0077] Asparagine N Aspartic acid D

[0078] Cysteine ​​C, Glutamic Acid E

[0079] Glutamine Q Glycine G

[0080] Histidine H, Isoleucine I

[0081] Leucine L-Lysine K

[0082] Methionine M Phenylalanine F

[0083] Proline P Serine S

[0084] Threonine T Tryptophan W

[0085] Tyrosine Y Valine V

[0086]

[0087] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising an interleukin 2 analog or a sustained-release conjugate thereof, wherein the composition is administered in combination with a CTLA-4 antagonist. For the interleukin 2 analog and the sustained-release conjugate thereof of the present invention, reference may be made to WO2021-201615 A1 and WO2022-211537 A1, and said two documents are incorporated herein by reference.

[0088] The interleukin 2 analog of the present invention is characterized by having a modified binding affinity to the interleukin 2 receptor, particularly an increased binding affinity to the interleukin 2 beta receptor. Specifically, the interleukin 2 analog of the present invention may have an increased binding affinity to the interleukin 2 beta receptor compared to natural interleukin 2 or known aldesleukin, and more specifically, may have a modified (increased or decreased) binding affinity to the interleukin 2 alpha receptor, and may include a sequence in which one or more amino acids are mutated from natural interleukin 2.

[0089]

[0090] One embodiment of the composition of the present invention may include, but is not limited to, an interleukin 2 analog comprising any one of the amino acid sequences selected from SEQ ID NOs 3 to 106, essentially composed of, or composed of.

[0091]

[0092] Specifically, the composition of the present invention may be a pharmaceutical composition for the prevention or treatment of cancer comprising an interleukin 2 analog or a sustained-release conjugate thereof, and more specifically, may be a pharmaceutical composition for the prevention or treatment of cancer administered in combination with a CTLA-4 antagonist while comprising an interleukin 2 analog or a sustained-release conjugate thereof and a pharmaceutically acceptable excipient, but is not limited thereto.

[0093]

[0094] The pharmaceutical composition of the present invention is

[0095] a) administered as a mixture comprising (i) an interleukin 2 analog or a sustained-release conjugate thereof; and (ii) a CTLA-4 antagonist; or

[0096] b) (i) an interleukin 2 analog or a sustained-release conjugate thereof; and (ii) a CTLA-4 antagonist may be administered in a separate form, but is not limited thereto.

[0097] For example, an interleukin 2 analog or its sustained-release conjugate; and a CTLA-4 antagonist may be formulated into a single preparation or formulated separately. If the interleukin 2 analog or its sustained-release conjugate; and a CTLA-4 antagonist are in a separate form, the interleukin 2 analog or its sustained-release conjugate; and a CTLA-4 antagonist may be formulated into separate preparations that can be administered simultaneously, individually, sequentially, or in reverse order.

[0098] In the present invention, concomitant administration should be understood not only as simultaneous administration, but also as a form of administration in which an interleukin 2 analog or its sustained-release conjugate; and a CTLA-4 antagonist act together on an individual so that each substance can perform a function equivalent to or greater than its inherent function. Accordingly, the term "concomitant" herein should be understood to include simultaneous, individual, sequential, or reverse administration of an interleukin 2 analog or its sustained-release conjugate; and a CTLA-4 antagonist. Where said administration is sequential, reverse, or individual, the order of administration is not particularly limited, provided that the interval between the administration of the secondary component is such that the beneficial effects of said concomitant administration are not lost.

[0099]

[0100] The interleukin 2 analog or its sustained-release conjugate and CTLA-4 antagonist of the present invention; or a composition comprising these may be provided in the form of a kit, but is not limited thereto. In the present invention, the term “kit” may refer to a composition according to the present invention for co-administering an interleukin 2 analog or its sustained-release conjugate and a CTLA-4 antagonist. Specifically, the kit according to the present invention may include an interleukin 2 analog or its sustained-release conjugate and a CTLA-4 antagonist formulated as a single preparation, or may include individual preparations of an interleukin 2 analog or its sustained-release conjugate and a CTLA-4 antagonist, and may additionally include a substance necessary for co-administration of the two substances, but is not limited thereto.

[0101]

[0102] In the present invention, the term "interleukin 2 (IL-2)" refers to an immunomodulator, which is a type of cytokine that transmits signals in the immune system. Interleukin 2 is generally known as an important immune stimulant of about 15 kDa.

[0103] In the present invention, the term "interleukin 2 analog" means that one or more amino acids in the natural sequence have been mutated, and in particular, in the present invention, it may be an interleukin 2 analog in which the amino acids of natural interleukin 2 have been mutated, such that the binding affinity to the interleukin 2 receptor is reduced or increased compared to the natural form. Specifically, the interleukin 2 analog of the present invention may be non-naturally occurring.

[0104] The above-mentioned natural interleukin 2 may be human interleukin 2, and its sequence may be obtained from known databases, etc. Specifically, it may be the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0105] In the present invention, the meaning that natural interleukin 2 may be the amino acid sequence of SEQ ID NO. 1 means that not only the sequence identical to SEQ ID NO. 1, but also sequences with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO. 1 also fall within the category of natural interleukin 2 of the present invention, and the amino acid mutation position means that when sequences with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO. 1 are aligned, the corresponding position on the amino acid sequence of SEQ ID NO. 1 is mutated.

[0106]

[0107] In the present invention, the term “aldesleukin” refers to a commercially available interleukin 2 analog, which may have the trademark Proleukin®, and specifically may have the amino acid sequence of SEQ ID NO. 2. In the present invention, it is used interchangeably with “interleukin 2 analog 1.” The interleukin 2 analog according to the present invention may have an interleukin 2 alpha receptor binding affinity and / or an increased interleukin 2 beta receptor binding affinity compared to the interleukin 2 analog 1.

[0108] Although the interleukin 2 alpha receptor is known not to be involved in the interleukin 2 signaling pathway, it increases the binding affinity of interleukin 2 with other interleukin 2 receptors (beta or gamma) by 10 to 100 times, and CD4 + It is expressed in regulatory T cells, etc.

[0109] The interleukin 2 beta receptor is CD8 +Since it is mainly distributed in T cells or natural killer (NK) cells and performs a major function in activating immune responses and macrophage activity, tumor cell death and activation of the body's immune response can be expected through the activation of interleukin 2 beta receptors.

[0110] Therefore, the interleukin 2 analog of the present invention, which has increased binding affinity to the interleukin 2 beta receptor, can have therapeutic effects such as tumor suppression and death, and reduced side effects.

[0111]

[0112] As one example, the above interleukin 2 analog may include, essentially consist of, or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs 3 to 106, but is not limited thereto.

[0113] In one specific embodiment, the interleukin 2 analog may comprise, essentially constitute, or be composed of any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs 10, 13 to 15, 17, 20 to 22, 32, 35, 36, 42, 53, 54, 56, 58 to 60, 62, 71, 72, 74 to 78, 85, 87, 89, 91 to 94, and 97 to 106, but is not limited thereto.

[0114] In other specific embodiments, the interleukin 2 analog may include, essentially constitute, or be composed of any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs 17, 22, 42, 53, 56, 58 to 60, 62, 71, 72, 74 to 77, 87, 89, 91 to 93, 98 to 101, and 103 to 106, but is not limited thereto.

[0115] In other specific embodiments, the interleukin 2 analog may include, essentially constitute, or be composed of any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 10, 13, 14, 15, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 56, 58, 59, 60, 62, 71, 72, 74, 75, 76, 77, 78, 85, 87, 89, 91, 92, 93, 94, 95, 98, 99, 100, 101, 103, 104, 105, and 106, but is not limited thereto.

[0116] In other specific embodiments, the interleukin 2 analog may include, essentially constitute, or be composed of any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 10, 13, 14, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 87, 89, 91, 92, 93, 94, 98, 99, 100, 101, 103, 104, and 105, but is not limited thereto.

[0117] In other specific embodiments, the interleukin 2 analog may include, essentially constitute, or be composed of any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 22, 42, 53, 87, 105 and 106, but is not limited thereto.

[0118] In addition, the interleukin 2 analog may additionally include one or more amino acids at the C-terminus, but is not limited thereto.

[0119]

[0120] In addition, even if the present specification describes an "interleukin 2 analog consisting of a specific sequence number," if it has the same or corresponding activity as an interleukin 2 analog consisting of the amino acid sequence of the said 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, and it is obvious that even in cases having such sequence additions or mutations, it falls within the scope of the present invention.

[0121] The interleukin 2 analog of the present invention may include, but is not limited to, an amino acid sequence 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 3 to 106.

[0122] In the present invention, the terms 'homology' or 'identity' refer to the degree of mutual relationship between two given amino acid sequences or base sequences and may be expressed as a percentage.

[0123] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Practically, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderate or high stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0124] The terms homology and identity can often be used interchangeably.

[0125] Whether any two base sequences or peptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later). (Includes the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Center for Biotechnology Information Database, or ClustalW.

[0126] Homology, similarity, or identity of base sequences or peptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48: 443. In summary, the GAP program is defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, as used in the present invention, the terms “homology” or “identity” indicate relevance between sequences.

[0127]

[0128] The interleukin 2 analog of the present invention can be used as a substitute for a novel interleukin 2 that alters in vitro activity by weakening or increasing the binding affinity to interleukin 2 alpha and / or beta receptors. In particular, it can be used as an effective therapeutic agent due to activity to both receptors, as it not only increases the binding affinity to beta receptors but also alters (increases or decreases) the binding affinity to alpha receptors.

[0129]

[0130] In the present invention, such modifications for the preparation of an analog of interleukin 2 include all modifications using L-type or D-type amino acids and / or non-natural type amino acids; and / or modifications by modifying the natural type sequence, for example, by modifying side chain functional groups, intramolecular covalent bonds, for example, forming rings between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexalation, biotinylation, etc.

[0131] In addition, it includes all of the addition of one or more amino acids to the amino and / or carboxyl terminus of natural interleukin 2.

[0132] The aforementioned substituted or added amino acids may include the 20 amino acids typically observed in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased through commercial peptide synthesis companies, for example, American Peptide Company or Bachem in the United States, or Anygen in Korea.

[0133] Amino acid derivatives can also be obtained in the same way, and to give just a few examples, 4-imidazoacetic acid can be used.

[0134]

[0135] In addition, the interleukin 2 analog according to the present invention may be in a modified form in which its N-terminus and / or C-terminus, etc. are chemically modified or protected by an organic group, or amino acids are added to the peptide terminals, etc., in order to protect against protein-cleaving enzymes in vivo and increase stability.

[0136] In particular, in the case of chemically synthesized peptides, since the N- and C-terminals are charged, the N-terminal may be acetylated and / or the C-terminal may be amidated to remove these charges, but is not specifically limited thereto.

[0137]

[0138] The interleukin 2 analog of the present invention can be synthesized through a solid-phase synthesis method, can be produced by a recombination method, and can be manufactured by commissioning commercially, but is not limited thereto.

[0139] In addition, the interleukin 2 analog of the present invention can be synthesized by methods well known in the field, for example, by an automated peptide synthesizer, depending on its length, and can also be produced by genetic engineering technology.

[0140] Specifically, the interleukin 2 analog of the present invention may be prepared by a standard synthesis method, a recombinant expression system, or any other method in the art. Accordingly, the interleukin 2 analog according to the present invention may be synthesized by a number of methods, including, for example, a method comprising the following:

[0141] (a) a method for synthesizing a peptide stepwise or by fragment assembly by means of a solid-phase or liquid-phase method, and isolating and purifying the final peptide product; or

[0142] (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from a host cell culture; or

[0143] (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

[0144] A method for obtaining a peptide fragment by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0145]

[0146] In the present invention, the binding affinity of any interleukin 2 analog (or sustained conjugate containing it) to a natural interleukin 2 receptor can be measured using a method of measuring affinity for the receptor, such as using surface plasmon resonance (SPR).

[0147] Specifically, there may be a method of measuring binding affinity by immobilizing an interleukin 2 receptor on a sensor chip using the protein-ligand binding principle during SPR analysis, inducing binding with the immobilized receptor by flowing an interleukin 2 analog diluted in an experimental buffer using a serial dilution method, and then inducing dissociation of the receptor and the interleukin 2 analog by flowing only the experimental buffer at the same flow rate, or by first immobilizing an antibody against the human immunoglobulin Fc region on the sensor chip, immobilizing an interleukin 2 receptor bound to the Fc region, and then flowing an interleukin 2 analog to measure binding affinity, but is not limited thereto.

[0148] More specifically, biotin-labeled human interleukin 2 receptors are immobilized on a streptavidin biosensor chip, and an interleukin 2 analog-sustained conjugate diluted in HBS-EP+ buffer by a 2x serial dilution method is flowed at a flow rate of 20 μL / min for 3 minutes, followed by flowing only HBS-EP+ buffer at the same flow rate for 3 minutes to induce dissociation of the interleukin 2 receptors and the interleukin 2 analog-sustained conjugate, and then the binding constants and dissociation constants obtained can be measured according to a 1:1 binding fitting model using a Bioaevaluation program, but are not limited thereto.

[0149]

[0150] The interleukin 2 analog of the present invention may have reduced or increased interleukin 2 alpha receptor binding affinity compared to natural interleukin 2 or aldesleukin (or interleukin 2 analog 1).

[0151] Specifically, the interleukin 2 analog of the present invention may have an interleukin 2 alpha receptor binding affinity of about 0.001 times or more, about 0.005 times or more, about 0.01 times or more, about 0.05 times or more, about 0.1 times or more, about 0.3 times or more, about 0.5 times or more, about 0.7 times or more, about 0.9 times or more, about 1.1 times or more, about 1.3 times or more, about 1.5 times or more, or about 1.7 times or more compared to the interleukin 2 alpha receptor binding affinity of natural interleukin 2 or aldesleukin, but the numerical value is not limited, and if the binding affinity is changed compared to natural interleukin 2 or aldesleukin, it falls within the scope of the present invention.

[0152] Alternatively, based on the binding affinity of aldesleukin to the interleukin 2 alpha receptor (100%), the interleukin 2 analog of the present invention may lose its binding affinity at all, or have a binding affinity of about 1% or more, about 5% or more, about 7% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 70% or more, about 90% or more, about 100% or more, about 150% or more, or about 200% or more, but the values ​​are not limited, and if the binding affinity is altered compared to natural interleukin 2 or aldesleukin, it falls within the scope of the present invention.

[0153]

[0154] In addition, the interleukin 2 analog of the present invention may have an interleukin 2 beta receptor binding affinity of about 0.1 times or more, about 0.3 times or more, about 0.5 times or more, about 0.7 times or more, about 1.0 times or more, about 10 times or more, about 20 times or more, about 30 times or more, about 40 times or more, about 50 times or more, about 60 times or more, about 70 times or more, about 80 times or more, about 90 times or more, or about 100 times or more compared to the interleukin 2 beta receptor binding affinity of natural interleukin 2 or aldesleukin, but the numerical value is not limited, and if the binding affinity is changed or increased compared to natural interleukin 2 or aldesleukin, it falls within the scope of the present invention.

[0155] Alternatively, based on the binding affinity of aldesleukin to the interleukin 2 beta receptor (100%), the interleukin 2 analog of the present invention may have a binding affinity of about 5% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 50% or more, about 100% or more, about 200% or more, about 500% or more, about 700% or more, about 1000% or more, about 1500% or more, about 3000% or more, about 5000% or more, about 7000% or more, about 10000% or more, about 12000% or more, about 15000% or more, about 20000% or more, or about 25000% or more, but the values ​​are not limited, and if the binding affinity is increased compared to natural interleukin 2 or aldesleukin, it falls within the scope of the present invention.

[0156]

[0157] In the present invention, the term "approximately" includes a range encompassing ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​within a range equivalent to or similar to the numerical value following the term "approximately," but is not limited thereto.

[0158] The interleukin 2 analog of the present invention is characterized by having a modified binding affinity to interleukin 2 alpha receptors and an increased binding affinity to interleukin 2 beta receptors compared to natural interleukin 2 or aldesleukin.

[0159]

[0160] In a specific embodiment of the present invention, in order to produce the interleukin 2 analog of the present invention, an interleukin 2 analog was produced by introducing a mutation based on natural interleukin 2 (Sequence No. 1). The interleukin 2 analog produced in the present invention may include any one of the amino acid sequences of Sequence Nos. 3 to 106, or may be encoded by any one of the nucleotide sequences of Sequence Nos. 108 to 211.

[0161]

[0162] The nucleic acid encoding the interleukin 2 analog of the present invention may be modified to introduce a mutation (amino acid deletion, substitution, and / or addition) to an amino acid at a specific position in the base sequence encoding the natural form of interleukin 2 of SEQ ID NO. 1, and specifically may include a base sequence encoding any one of the amino acid sequences of SEQ ID NOs. 3 to 106. As an example, the nucleic acid of the present invention may have or include any one of the base sequences of SEQ ID NOs. 108 to 211.

[0163] The base sequence of the present invention may have various modifications made to the coding region within a range that does not change the amino acid sequence of the interleukin 2 analog of the present invention, taking into account the degeneracy of codons or codons preferred by the organism to express the nucleic acid of the present invention. Specifically, the nucleic acid of the present invention may have or include a nucleotide sequence having homology or identity of 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% with any one of the sequences of SEQ ID NOs 108 to 211, or may be composed of or essentially composed of a nucleotide sequence having homology or identity of 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% with any one of the sequences of SEQ ID NOs 108 to 211, but is not limited thereto.

[0164] In addition, the nucleic acid of the present invention may include, without limitation, probes that can be prepared from known gene sequences, for example, sequences that can be hybridized under stringent conditions with a sequence complementary to all or part of the nucleic acid sequence of the present invention. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8).

[0165] Hybridization requires that two nucleic acids have complementary sequences, even though a mismatch between bases may be possible depending on the degree of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, regarding DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the nucleic acids of the present invention may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

[0166] The appropriate strictness for hybridizing the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the art (e.g., J. Sambrook et al., i.e.).

[0167] As for homology or identity, it is as described above.

[0168]

[0169] In addition, the interleukin 2 analog of the present invention may have an increased half-life in the body compared to natural interleukin or aldesleukin, but is not particularly limited thereto. For example, a biocompatible material for increasing the half-life (e.g., immunoglobulin Fc region) may be bound to the interleukin 2 analog directly or through a linker to form a sustained-release conjugate with an increased half-life, but is not limited thereto.

[0170] The sustained-release conjugate according to the present invention not only includes an interleukin 2 analog that has increased binding affinity to an interleukin 2 beta receptor, but also combines it with an immunoglobulin Fc region as a representative carrier to increase its half-life, thereby increasing the half-life of the interleukin 2 analog, increasing blood exposure, and increasing the in vivo immune response, so that the inhibition and reduction of cancer cell growth can be effectively achieved.

[0171]

[0172] The above description may be applied to other embodiments or other aspects of the present invention, but is not limited thereto.

[0173]

[0174] Other embodiments of the composition of the present invention may include, but are not limited to, a sustained combination of the interleukin 2 analog.

[0175]

[0176] In the present invention, the interleukin 2 analog sustained-release conjugate may be in the form in which a biocompatible material is conjugated to the interleukin 2 analog to increase its in vivo half-life. In this specification, the biocompatible material may be mixed with a carrier.

[0177] In the present invention, the sustained conjugate may exhibit increased efficacy duration compared to an interleukin 2 analog without a carrier, and in the present invention, such a conjugate is referred to as a "sustained conjugate" or "conjugate."

[0178] Meanwhile, such combinations may be non-naturally occurring.

[0179]

[0180] In one embodiment of the present invention, the sustained conjugate is a sustained conjugate represented by the following chemical formula 1:

[0181] [Chemical Formula 1]

[0182] X - L - F

[0183] At this time, X is the above-mentioned Interleukin 2 analog;

[0184] L is a polyethylene glycol linker;

[0185] F is the dimeric form of the immunoglobulin Fc region;

[0186] - represents the covalent bond connection between X and L, and between L and F, and

[0187] In the above sustained conjugate, one end of L is covalently connected to only one polypeptide chain of the dimeric Fc region, and X is covalently connected to the opposite end of this L.

[0188] More specifically, the sustained conjugate may be one molecule of X connected to one polypeptide chain in the Fc region through L. Additionally, in the sustained conjugate of the present invention, X and L, and L and F may be connected to each other by covalent bonds, and the conjugate may be a conjugate in which X, L, and F are each connected through covalent bonds in the order of Chemical Formula 1.

[0189]

[0190] The interleukin 2 analog of the sustained-release conjugate of the present invention is characterized by having a modified binding affinity to the interleukin 2 receptor, particularly an increased binding affinity to the interleukin 2 beta receptor, when it exists alone without forming part of the conjugate. Specifically, the interleukin 2 analog of the present invention may have an increased binding affinity to the interleukin 2 beta receptor compared to natural interleukin 2 or known aldesleukin when it exists alone without forming part of the conjugate, and more specifically, the binding affinity to the interleukin 2 alpha receptor may also be modified (increased or decreased).

[0191]

[0192] In the present invention, the interleukin 2 analog may correspond to the composition of one moiety constituting the complex. Specifically, it corresponds to X in Chemical Formula 1, and the interleukin 2 analog is as described above.

[0193] In the above conjugate, F is a substance capable of increasing the half-life of X, that is, an interleukin 2 analog, and corresponds to one component of the moiety constituting the conjugate of the present invention.

[0194] The above F may be bonded to X by a covalent chemical bond, and F and X may be bonded to each other through L by a covalent chemical bond.

[0195] Specifically, the above F is an immunoglobulin Fc region, and the immunoglobulin Fc region may be an IgG Fc region or a deglycosylated IgG4 Fc region, but is not particularly limited thereto.

[0196] In a specific example of the present invention, the F (immunoglobulin Fc region) may be a dimer composed of two polypeptide chains, and may have a structure in which one end of L is connected to only one of the two polypeptide chains, but is not limited thereto.

[0197]

[0198] One or more amino acid side chains within the peptide of the present invention may be conjugated to such biocompatible materials to increase solubility and / or half-life in vivo and / or increase bioavailability. Such modification may also reduce the clearance of therapeutic proteins and peptides.

[0199] The biocompatible material described above may be water-soluble (amphiphilic or hydrophilic) and / or non-toxic and / or pharmaceutically acceptable.

[0200]

[0201] In one specific embodiment, the sustained conjugate of the present invention may be an interleukin 2 analog linked to an immunoglobulin Fc region, but is not limited thereto.

[0202]

[0203] In the present invention, "immunoglobulin Fc region" refers to a region comprising a heavy chain constant region 2 (CH2) and / or a heavy chain constant region 3 (CH3), excluding the heavy chain and light chain variable regions of the immunoglobulin. The immunoglobulin Fc region may be a component forming a moiety of the conjugate of the present invention. Specifically, it corresponds to F in Chemical Formula 1.

[0204] In this specification, the term Fc region encompasses not only the natural sequence obtained from the papain digestion of immunoglobulin but also derivatives thereof, such as modified sequences in which one or more amino acid residues of the natural sequence are modified by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, thereby differing from the natural form. It is presupposed that the derivatives, substituents, and modified sequences possess the ability to bind to FcRn. In the present invention, F may be a human immunoglobulin region, but is not limited thereto. In this specification, “biocompatible material” or “carrier” may refer to the Fc region.

[0205] The above F (immunoglobulin Fc region) is a structure in which two polypeptide chains are connected by disulfide bonds, and may be a structure in which the two chains are connected only through nitrogen atoms of one of the chains, but is not limited thereto. The connection through nitrogen atoms may be made through reductive amination to the epsilon-amino atom of lysine or the N-terminal amino group.

[0206] A reductive amination reaction refers to a reaction in which an amine group or an amino group of a reactant reacts with an aldehyde of another reactant (i.e., a functional group capable of reductive amination) to produce an amine, and then forms an amine bond through a reduction reaction; it is an organic synthesis reaction that is widely known in the relevant technical field.

[0207] As one embodiment of the sustained conjugate of the present invention, the sustained conjugate may be such that the immunoglobulin Fc region is connected to a linker through its N-terminal nitrogen atom.

[0208]

[0209] These immunoglobulin Fc regions may include a hinge portion in the heavy chain constant region, but are not limited thereto.

[0210] In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.

[0211] The term "hinge sequence" in this invention refers to a site located in the heavy chain that forms a dimer of the immunoglobulin Fc region through an inter disulfide bond.

[0212] In the present invention, the hinge sequence may be a hinge sequence having the following amino acid sequence that has been modified by deleting a portion thereof to have only one cysteine ​​residue, but is not limited thereto:

[0213] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro(Sequence No. 418).

[0214] The hinge sequence above may be one in which the 8th or 11th cysteine ​​residue of the hinge sequence of SEQ ID NO. 418 is deleted to contain only one cysteine ​​residue. The hinge sequence of the present invention may be composed 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 sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (Sequence No. 419), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (Sequence No. 420), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (Sequence No. 421), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (Sequence No. 422), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (Sequence No. 423), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (Sequence No. 424), Glu-Lys-Tyr-Gly-Pro-Pro-Cys(Sequence No. 425), Glu-Ser-Pro-Ser-Cys-Pro(Sequence No. 426), Glu-Pro-Ser-Cys-Pro(Sequence No. 427), Pro-Ser-Cys-Pro(Sequence No. 428), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro(Sequence No. 429), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro(Sequence No. 430), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro(Sequence No. 431), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys(Sequence No. 432), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (Sequence No. 433), Glu-Ser-Lys-Pro-Ser-Cys-Pro (Sequence No. 434), Glu-Ser-Pro-Ser-Cys-Pro (Sequence No. 435), Glu-Pro-Ser-Cys (Sequence No. 436), Ser-Cys-Pro (Sequence No. 437).

[0215] More specifically, the hinge sequence may include the amino acid sequence of SEQ ID NO. 428 (Pro-Ser-Cys-Pro) or SEQ ID NO. 437 (Ser-Cys-Pro), but is not limited thereto.

[0216] In one more specific form of the sustained conjugate of the present invention, the N-terminus of the immunoglobulin Fc region within the conjugate is proline, and the conjugate is such that the Fc region is connected to a linker through the nitrogen atom of the proline.

[0217] In one embodiment of the sustained-release conjugate of the present invention, the immunoglobulin Fc region may be in a dimeric form in which two chains of the immunoglobulin Fc region form a homodimer or a heterodimer by the presence of a hinge sequence. The conjugate of Formula 1 of the present invention may be in a form in which one end of the linker is connected to one chain of the immunoglobulin Fc region of the dimeric form, but is not limited thereto.

[0218] The term "N-terminus" of the present invention refers to the amino terminus of a protein or polypeptide, and may include the outermost amino terminus, or one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids from the outermost terminus. The immunoglobulin Fc region of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.

[0219]

[0220] In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region comprising some or all of the heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), excluding only the heavy chain and light chain variable regions of the immunoglobulin, insofar as it has substantially equivalent or enhanced effects to the natural form. In addition, it may be a region in which some significantly long amino acid sequences corresponding to CH2 and / or CH3 have been removed.

[0221] For example, the immunoglobulin Fc region of the present invention may be a combination of 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains among the CH1 domain, a CH2 domain, a CH3 domain and a 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 thereto.

[0222] In the present invention, the immunoglobulin Fc region may be in a dimeric or polymeric form composed of short-chain immunoglobulins having domains of the same origin, but is not limited thereto.

[0223]

[0224] In addition, as one embodiment of the sustained conjugate of the present invention, the immunoglobulin Fc region F is a dimer composed of two polypeptide chains, wherein the Fc region dimer F and X are covalently connected through a linker L containing an ethylene glycol repeating unit. In one specific embodiment of this embodiment, X is covalently connected through linker L to only one of the two polypeptide chains of the Fc region dimer F. In a more specific example of this embodiment, only one molecule of X is covalently connected through L to the polypeptide chain to which X is connected among the two polypeptide chains of the Fc region dimer F. In the most specific example of this embodiment, F is a homodimer.

[0225] In another embodiment, the immunoglobulin Fc region F is a dimer composed of two polypeptide chains, and one end of L may be connected to only one of the two polypeptide chains, but is not limited thereto.

[0226] In another embodiment of the sustained-release conjugate of the present invention, it is also possible for two molecules of X to be symmetrically bound to a single Fc region in a dimeric form. In this case, the immunoglobulin Fc region and X may be connected to each other by L. However, the invention is not limited to the examples described above.

[0227] In addition, 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 having a different sequence in which one or more amino acid residues of the natural amino acid sequence are deleted, inserted, non-conservative or conservatively substituted, or a combination thereof.

[0228] For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, can be used as suitable sites for modification.

[0229] In addition, various types of derivatives are possible, such as by removing a site capable of forming disulfide bonds, removing some amino acids from the N-terminus of the natural form Fc, or adding a methionine residue to the N-terminus of the natural form Fc. Furthermore, to eliminate effector function, complement binding sites, such as the C1q binding site, may be removed, or the ADCC (antibody dependent cell mediated cytotoxicity) site may be removed. Techniques for manufacturing sequence derivatives of such immunoglobulin Fc regions are disclosed in International Patent Publication No. WO 97 / 34631, International Patent Publication No. 96 / 32478, etc.

[0230] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are known in the art (H. Neuras, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications may be made through phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0231] The Fc derivative described above may exhibit biological activity equivalent to the Fc region of the present invention and may have increased structural stability of the Fc region against heat, pH, etc.

[0232] In addition, this Fc region may be obtained from a natural form isolated in vivo from animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or it may be a recombinant form obtained from transformed animal cells or microorganisms, or a derivative thereof. Here, the method of obtaining from the natural form may be a method of obtaining the total immunoglobulin by isolating it from the body of a human or animal and then treating it with a protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography, etc. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from microorganisms.

[0233] Furthermore, the immunoglobulin Fc region may be in the form of a natural glycosylation, an increased glycosylation compared to the natural form, a decreased glycosylation compared to the natural form, or a form with the glycosylation removed. Conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms, can be utilized for increasing, decreasing, or removing these immunoglobulin Fc glycosylations. Here, the immunoglobulin Fc region with the glycosylation removed exhibits significantly reduced binding affinity to complement (c1q), and since antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, it does not induce unnecessary immune responses in vivo. In this regard, the form with the glycosylation removed or deglycosylated immunoglobulin Fc region is considered to be more suitable for its original purpose as a drug carrier.

[0234] In the present invention, "deglycosylation" refers to the Fc region from which sugar has been removed by an enzyme, and "aglycosylation" refers to the Fc region that has not been glycosylated, produced in prokaryotes, or in a more specific embodiment, in E. coli.

[0235] Meanwhile, the immunoglobulin Fc region may be of human or animal origin such as cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and in a more specific embodiment, it is of human origin.

[0236] Additionally, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, it is derived from IgG known to enhance the half-life of ligand-binding proteins. In a 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 thereto.

[0237] In addition, as one specific example, the immunoglobulin Fc domain may be a fragment of human IgG4 Fc, and may be a homomer in which two monomers are connected through a disulfide bond (inter-chain form) between the cysteine, which is the 3rd amino acid of each monomer, wherein the homomer has or may have two disulfide bonds (intra-chain form), i.e., between the cysteine ​​at positions 35 and 95 and between the cysteine ​​at positions 141 and 199 of each monomer.

[0238] The number of amino acids in each monomer may consist of 221 amino acids, and the amino acids forming the homomer may consist of a total of 442 amino acids, but are not limited thereto. Specifically, the immunoglobulin Fc region may form a homomer through a disulfide bond between two monomers having the amino acid sequence of SEQ ID NO. 438 (composed of 221 amino acids), and the monomers of the homomer may each independently form an internal disulfide bond between cysteine ​​at positions 35 and 95 and an internal disulfide bond between cysteine ​​at positions 141 and 199, but are not limited thereto.

[0239] F of the above chemical formula 1 may include a monomer having the amino acid sequence of SEQ ID NO. 438, and F may be a homomer of the monomer having the amino acid sequence of SEQ ID NO. 438, but is not limited thereto.

[0240] As one example, the immunoglobulin Fc region may be a homomer containing the amino acid sequence of SEQ ID NO. 439 (composed of 442 amino acids), but is not limited thereto.

[0241] In one specific example, the immunoglobulin Fc region and X may not be glycosylated, but are not limited thereto.

[0242]

[0243] Meanwhile, in the present invention, "combination" means that when forming a dimer or a multimer, a polypeptide encoding a short-chain immunoglobulin Fc region of the same origin forms a combination with a short-chain polypeptide of a different origin. That is, it is possible to prepare a dimer or a multimer from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0244] In the present invention, "hybrid" is a term meaning that a sequence corresponding to two or more immunoglobulin Fc fragments of different origins exists within a single-chain immunoglobulin constant region. In the present invention, various forms of hybrids are possible. That is, a hybrid of a domain consisting of one to four domains from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc is possible and may include a hinge.

[0245] Meanwhile, IgG can also be divided into subclasses of IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations or hybridizations thereof are also possible. Specifically, it is the IgG2 and IgG4 subclasses, and most specifically, it is the Fc region of IgG4, which has almost no effector function such as complement-dependent cytotoxicity (CDC).

[0246] In addition, the above-described conjugate may have increased efficacy compared to natural interleukin 2 or aldesleukin, or compared to X without F modification, and such conjugate includes, but is not limited to, the above-described form as well as a form encapsulated in biodegradable nanoparticles.

[0247]

[0248] In the present invention, the "polyethylene glycol linker" comprises a biocompatible polymer in which two or more ethylene glycol repeating units are bonded. The repeating units are connected to each other through any covalent bond other than a peptide bond. The polyethylene glycol linker may be a component forming the moiety of the conjugate of the present invention, and in this specification, the linker may be used interchangeably with "non-peptide linker" or "non-peptide polymer."

[0249] In one specific embodiment, the conjugate may be such that F and X are covalently connected to each other through a non-peptide linker comprising reactive groups at both ends that can be combined with F (specifically an immunoglobulin Fc region) and X (specifically an interleukin 2 analog).

[0250] Specifically, in the present invention, the nonpeptide linker includes a reactive group at its end and can form a complex through a reaction with other components constituting the complex. When a nonpeptide linker having reactive functional groups at both ends forms a complex by combining with X and F of Formula 1 through each reactive group, the nonpeptide linker or nonpeptide polymer may be named a nonpeptide polymer linker moiety or a nonpeptide linker moiety.

[0251]

[0252] In one specific embodiment, L (polyethylene glycol linker) may be a linker containing ethylene glycol repeating units, e.g., polyethylene glycol, but is not limited thereto. In this specification, the term polyethylene glycol encompasses all forms of ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG), but is not specifically limited thereto. Furthermore, derivatives thereof already known in the art and derivatives that can be easily prepared at the level of the art are also included within the scope of the invention.

[0253] The above-described polyethylene glycol linker may include an ethylene glycol repeating unit and, prior to being formed into a conjugate, may include a functional group at the terminal that is used in the manufacture of the conjugate. The sustained conjugate according to the present invention may be in the form where X and F are connected through the functional group, but is not limited thereto. In the present invention, the above-described non-peptide linker may include two or three or more functional groups, and each functional group may be the same or different from one another, but is not limited thereto.

[0254] Specifically, the linker may comprise a repeating unit represented by the following chemical formula 2. Examples include, but are not limited to, polyethylene glycol (PEG):

[0255] [Chemical Formula 2]

[0256]

[0257] Here, n = 10 to 2400, n = 10 to 480, or n = 50 to 250, but is not limited thereto.

[0258] In the above sustained-release conjugate, the PEG moiety is -(CH2CH2O) n - Not only the structure, but also the connecting elements and this -(CH2CH2O) n Oxygen atoms interposed between them may also be included, but are not limited thereto.

[0259]

[0260] In one specific example, the ethylene glycol repeating unit may be represented as, for example, [OCH2CH2]n, and the value of n may be a natural number such that the average molecular weight of the [OCH2CH2]n site in the interleukin 2 analog conjugate, e.g., the number average molecular weight, is greater than 0 and about 100 kDa, but is not limited thereto. As another example, the above n value is a natural number representing the average molecular weight of the [OCH2CH2]n site within the interleukin 2 analog conjugate, e.g., the number average molecular weight, 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, and approximately 3 Up to about 22 kDa, about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, about 3 to about 15 kDa, about 3 to about 13 kDa, about 3 to about 11 kDa, about 3 to about 10 kDa, about 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.It may be 4 kDa, about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa, about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9.5 to about 10.5 kDa, or about 10 kDa, but is not limited thereto.

[0261]

[0262] In addition, in one specific embodiment, the conjugate may be a structure in which an interleukin 2 analog and an immunoglobulin Fc region (F) are covalently connected through a linker (L) containing an ethylene glycol repeating unit, but is not limited thereto.

[0263] In another specific embodiment, in the sustained conjugate, L is a linker containing an ethylene glycol repeating unit, and F may be an immunoglobulin Fc region in a dimeric form. More specifically, one molecule of X may be covalently connected to one of the Fc regions of the dimeric immunoglobulin Fc regions through the linker containing the ethylene glycol repeating unit, but is not limited thereto. Additionally, in another specific embodiment, one end of the linker containing the ethylene glycol repeating unit may be connected to only one of the two Fc region chains of the dimeric immunoglobulin Fc regions, but is not limited thereto.

[0264] The molecular weight of the polyethylene glycol linker that can be used in the present invention may be in the range of greater than 0 and 200 kDa, specifically, in the range of about 1 to 100 kDa, in the range of about 1 to 50 kDa, in the range of about 1 to 30 kDa, in the range of about 2 to 30 kDa, in the range of about 1 to 20 kDa, more specifically in the range of about 3.4 kDa to 10 kDa, more specifically in the range of about 3.4 kDa, but is not limited thereto. In addition, the non-peptide linker of the present invention that is combined with the polypeptide corresponding to F may be a combination of different types of polymers as well as a single type of polymer.

[0265] In the present invention, the term "approximately" includes a range encompassing ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​within a range equivalent to or similar to the numerical value following the term "approximately," but is not limited thereto.

[0266]

[0267] Specifically, the non-peptide linker may have reactive groups at both ends in a state not bound to F and X, and may bind to F and X through said reactive groups.

[0268] In one specific example, both ends of the linker may be bound to the thiol group, amino group, hydroxyl group of the immunoglobulin Fc region and the thiol group, amino group, azide group, hydroxyl group of the interleukin 2 analog (X), but are not limited thereto.

[0269] Specifically, the linker may include, but is not limited to, a reactive group that can be coupled to an immunoglobulin Fc region and an interleukin 2 analog (X) at each of its ends, specifically a thiol group of cysteine ​​of the immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine and / or histidine; and / or a hydroxyl group located at the C-terminus, and a reactive group that can be coupled to a thiol group of cysteine ​​of the interleukin 2 analog (X); an amino group of lysine, arginine, glutamine and / or histidine; an azide group of azidolysine; and / or a hydroxyl group.

[0270] More specifically, the reactive group of the linker may be one or more selected from the group consisting of aldehyde groups, maleimide groups, and succinimide derivatives, but is not limited thereto.

[0271] In the above, propionaldehyde or butylaldehyde groups may be given as examples of aldehyde groups, but are not limited thereto.

[0272] In the above, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimidyl, hydroxysuccinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate may be used as succinimidyl derivatives, but are not limited thereto.

[0273] The above linker can be connected to the immunoglobulin Fc region F and the interleukin 2 analog X through the above-mentioned reactor, and can be converted into a linker connection.

[0274] In addition, the final product produced by reductive alkylation via an aldehyde bond is much more stable than that linked by an amide bond. The aldehyde reactive group selectively reacts at the N-terminus at low pH and can form a covalent bond with a lysine residue at high pH, ​​for example, pH 9.0.

[0275] Additionally, the reactive groups at both ends of the linker may be identical or different from each other; for example, both ends may have aldehyde groups, or one end may have a maleimide group and the other end may have an aldehyde group, a propionaldehyde group, or a butylaldehyde group. However, it is not particularly limited thereto if F, specifically the immunoglobulin Fc region and X, can be bound to each end of the linker.

[0276] For example, one end of the linker may include a maleimide group as a reactive group, and the other end may include an aldehyde group, a propionaldehyde group, or a butylaldehyde group, etc.

[0277] When polyethylene glycol having hydroxyl reactive groups at both ends is used as a linker, the hydroxyl groups can be activated into the various reactive groups by known chemical reactions, or the interleukin 2 analog persistence conjugate of the present invention can be prepared by using commercially available polyethylene glycol having modified reactive groups.

[0278] In one specific embodiment, the linker may be connected to a cysteine ​​residue of X, more specifically to the -SH group of cysteine, but is not limited thereto.

[0279] Specifically, the reactive group of the linker may be connected to the -SH group of the cysteine ​​residue, and all previously described provisions apply to the reactive group. If maleimide-PEG-aldehyde is used, the maleimide group may be connected to the -SH group of X via a thioether bond, and the aldehyde group may be connected to F, specifically the -NH2 group of immunoglobulin Fc, via a reductive amination reaction, but is not limited thereto.

[0280] In other specific embodiments, the linker may be connected to a lysine residue of X, more specifically, to an amino group of lysine, but is not limited thereto.

[0281] Additionally, in the above conjugate, the reactive group of the linker may be connected to -NH2 located at the N-terminus of the immunoglobulin Fc region, but is not limited thereto.

[0282] In addition, in the above combination, the interleukin 2 analog according to the present invention may be connected through a linker having a reactor and a C-terminus, but this is one example.

[0283] In the present invention, "C-terminus" refers to the carboxyl terminus of a peptide and refers to a position that can bind to a linker for the purposes of the present invention. Examples include, but are not limited to, all amino acid residues surrounding the C-terminus as well as the terminal amino acid residue of the C-terminus, and specifically, may include the first to 20th amino acid residue from the terminal end, but are not limited thereto.

[0284]

[0285] In addition, the aforementioned conjugate may have increased efficacy duration compared to X without modification of F, and such conjugate includes not only the aforementioned form but also forms encapsulated in biodegradable nanoparticles.

[0286]

[0287] The interleukin 2 analog sustained-release conjugate of the present invention may have, when compared to the binding affinity of natural interleukin 2, aldesleukin, or a sustained-release conjugate containing the same, either lose binding affinity at all or have an interleukin 2 alpha-receptor binding affinity of about 0.001 times or more, about 0.005 times or more, about 0.01 times or more, about 0.05 times or more, about 0.1 times or more, about 0.3 times or more, about 0.5 times or more, about 0.6 times or more, about 0.7 times or more, about 0.8 times or more, about 0.9 times or more, about 1.1 times or more, about 1.3 times or more, about 1.5 times or more, or about 1.7 times or more, but the numerical value is not limited, and if the binding affinity is altered compared to natural interleukin 2 or aldesleukin, it falls within the scope of the present invention.

[0288] Additionally, specifically, the interleukin 2 analog sustained-release conjugate of the present invention may have an interleukin 2 beta-receptor binding affinity of about 0.1 times or more, about 0.3 times or more, about 0.5 times or more, about 0.7 times or more, about 1.0 times or more, about 10 times or more, about 20 times or more, about 30 times or more, about 40 times or more, about 50 times or more, about 60 times or more, about 70 times or more, about 80 times or more, about 90 times or more, about 100 times, about 130 times, about 150 times, or about 200 times or more compared to the interleukin 2, aldesleukin, or a sustained-release conjugate containing the same, but the numerical value is not limited, and if the binding affinity is changed or increased compared to the natural interleukin 2 or aldesleukin, it falls within the scope of the present invention.

[0289]

[0290] Unless otherwise indicated in this specification, the description in the detailed specification or claims regarding the "interleukin 2 analog" or the "conjugate" in which an interleukin 2 analog is covalently linked to a biocompatible material according to the present invention applies to the category including not only the said interleukin 2 analog or conjugate, but also the salt of said interleukin 2 analog or conjugate (e.g., a pharmaceutically acceptable salt of said interleukin 2 analog), or the solvate form thereof. Therefore, even if the specification only states "interleukin 2 analog" or "conjugate," such description applies likewise to said specific salt, said specific solvate, and said specific solvate of said specific salt. Such salt forms may be, for example, any pharmaceutically acceptable salt. The type of said salt is not particularly limited. However, it is preferable that it be a form that is safe and effective for individuals, e.g., mammals, but is not specifically limited thereto.

[0291] The type of the above salt is not particularly limited. However, it is desirable that it be in a form that is safe and effective for individuals, such as mammals, but is not specifically limited thereto.

[0292] The above term, "pharmaceuticalally acceptable," means a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reactions, within the scope of pharmaceutical judgment.

[0293] In the present invention, the term “pharmaceuticalally acceptable salt” includes a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium, etc.

[0294] In addition, the term "solvent" used in the present invention refers to an interleukin 2 analog or a salt thereof according to the present invention forming a complex with a solvent molecule.

[0295]

[0296] The composition according to the present invention may be a composition comprising the interleukin 2 analog or a sustained-release conjugate thereof, and specifically, may be a pharmaceutical composition having use for cancer prevention or treatment and administered in combination with a CTLA-4 antagonist or a composition comprising the same. The interleukin 2 analog and the sustained-release conjugate thereof are as described above. More specifically, it may be administered in combination with a pharmacologically effective amount of the interleukin 2 analog or the sustained-release conjugate thereof, additionally comprising a pharmaceutically acceptable carrier, and a pharmacologically effective amount of a CTLA-4 antagonist or a composition comprising the same.

[0297] Alternatively, the composition of the present invention may further comprise a CTLA-4 antagonist, but is not limited thereto.

[0298]

[0299] Specific examples of the composition according to the present invention include, but are not limited to, a composition comprising an interleukin 2 analog comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 3 to 106 or a sustained-release conjugate comprising the same, more specifically, an interleukin 2 analog comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 53, 87, 105, and 106 or said interleukin 2 analog or a sustained-release conjugate comprising the same, which may be administered in combination with a CTLA-4 antagonist or a composition comprising the same.

[0300]

[0301] The composition comprising the interleukin 2 analog or the interleukin 2 analog sustained-release conjugate of the present invention may be administered concurrently, sequentially, or in reverse order with a composition comprising a CTLA-4 antagonist, but is not limited thereto.

[0302] In the present invention, “pharmacologically effective dose” means a safe dosage of the interleukin 2 analog or sustained-release conjugate thereof and the CTLA-4 antagonist that exhibits a preventive or therapeutic effect against cancer without causing toxicity or side effects to the patient. Specifically, it may mean a dosage capable of exhibiting significant activity on interleukin 2 receptors (e.g., beta and / or alpha receptors) or a dosage capable of activating T cells, but is not limited thereto.

[0303]

[0304] The composition according to the present invention may exhibit one or more of the following characteristics, but is not limited to exhibiting an increase in immune response and anticancer effects, etc.:

[0305] (i) Higher blood exposure compared to administration of aldesleukin alone or combination of aldesleukin and a CTLA-4 antagonist;

[0306] (ii) Superior tumor growth inhibition and induction of complete remission compared to administration of aldesleukin alone or combination of aldesleukin and CTLA-4 antagonists;

[0307] (iii) Superior memory T cell generation response compared to administration of aldesleukin alone or administration of aldesleukin and CTLA-4 antagonist in combination.

[0308] (iv) Superior inhibition of immune checkpoint protein activation compared to administration of aldesleukin alone or administration of aldesleukin in combination with a CTLA-4 antagonist; and

[0309] (v) Superior T cell activation compared to administration of aldesleukin alone or combination of aldesleukin and a CTLA-4 antagonist.

[0310]

[0311] Interleukin 2, known as a T cell growth factor, is a protein involved in immune regulation that proliferates T cells, stimulates B cells, and acts on T cells to secrete γ-interferon. Based on this immune-regulating activity of Interleukin 2, it is possible to achieve preventive and therapeutic effects against cancer by utilizing the body's immune system to eliminate cancer cells.

[0312] In particular, the interleukin 2 analog of the present invention has an increased binding affinity to the interleukin 2 beta receptor, which plays a major role in signal transduction, and this leads to a more effective anticancer effect in the individual's immune system. In addition, the sustained-release conjugate containing the interleukin 2 analog exhibits excellent bioavailability by having increased binding affinity to the interleukin 2 beta receptor while also having high persistence and high blood exposure, and ultimately possesses excellent tumor growth inhibitory ability, thereby demonstrating an effective cancer prevention or treatment effect.

[0313]

[0314] When a CTLA-4 antagonist is used in combination with an interleukin 2 analog or a sustained-release conjugate thereof according to the present invention, excellent anticancer effects can be obtained by utilizing the immune checkpoints of T cells to prevent cancer cells from evading immunity while enabling T cells to attack cancer cells.

[0315]

[0316] In the present invention, the “CTLA-4 antagonist” is a substance that inhibits the action of CTLA-4 (Cytotoxic T-lymphocyte associated protein 4), an immune checkpoint protein used by cancer cells to evade immunity, and plays a role in allowing immune cells (e.g., T cells) to maintain their original functions by specifically binding to human CTLA-4 and blocking the interaction between CTLA-4 and CD80 (B7.1) and CD86 (B7.2), which are ligands to which CTLA-4 binds.

[0317]

[0318] The above CTLA-4 antagonist may include peptides, antibodies or antigen-binding fragments thereof, nucleic acid molecules, and small molecules capable of inhibiting the action of CTLA-4, but is not limited thereto as long as they are capable of antagonizing action.

[0319] Examples of the CTLA-4 antagonists of the present invention include anti-CTLA-4 antibodies, fragments thereof, or antigen-binding fragments thereof.

[0320] The anti-CTLA-4 antibody of the present invention refers to any form of immunoglobulin molecule capable of binding to CTLA-4, and may include, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, and chimeric antibodies.

[0321] An example of an anti-CTLA-4 antibody according to the present invention may be ipilimumab or tremelimumab, but is not limited thereto.

[0322]

[0323] The anti-CTLA-4 antibody fragment or the antigen-binding fragment thereof of the present invention may refer to a fragment of an antibody having the ability to specifically bind to CTLA-4, for example, a fragment having one or more CDR regions. Examples include fragments including Fab, Fab', F(ab')2, Fd, Fv, and CDR, a single-strand variable fragment antibody (scFv), and polypeptides capable of binding to CTLA-4, but are not limited thereto.

[0324]

[0325] A pharmaceutical composition for the prevention or treatment of cancer comprising an interleukin 2 analog or a sustained-release conjugate thereof according to the present invention may be administered in combination with a CTLA-4 antagonist or a composition containing the same to exhibit excellent anticancer efficacy through the synergistic action of the interleukin 2 analog or the sustained-release conjugate thereof and the CTLA-4 antagonist, while reducing side effects.

[0326]

[0327] The cancer of the present invention may be a cancer that is low responsiveness to CTLA-4 antagonists or an immune-non-invasive tumor (cold tumor), but is not limited thereto.

[0328]

[0329] It is known that immune checkpoint inhibitors, including CTLA-4 antagonists, exhibit different responsiveness depending on the phenotype of the tumor microenvironment (TME). However, because the responsiveness of immune checkpoint inhibitors is very low in immune-non-invasive tumors (cold tumors), it is known that there are limitations in using them as cancer treatments.

[0330] The interleukin 2 analog or sustained-release conjugate thereof of the present invention can induce a transition from an immune-non-invasive tumor to an invasive tumor (hot tumor) by altering the tumor microenvironment that is low in responsiveness to CTLA-4 antagonists. Therefore, when a CTLA-4 antagonist and an interleukin 2 analog or sustained-release conjugate thereof are administered together, cancers with low responsiveness to CTLA-4 antagonists can be treated more effectively, but are not limited thereto.

[0331] More specifically, in the present invention, 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, stomach cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, pro-anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, 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, lymphocytic lymphoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain tumor, glioma (astrocytoma, glioblastoma, oligodendroglioma) It may include, but is not limited to, ependymoma, germ cell tumor, meningioma, brainstem glioma, pituitary adenoma, schwannoma, congenital tumor, craniopharyngioma, or brain tumor.

[0332] Specifically, the cancer may be any one 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 stomach cancer. More specifically, the breast cancer may be triple-negative breast cancer, but is not limited thereto.

[0333]

[0334] Alternatively, the cancer according to the present invention may be primary cancer, recurrent cancer, or metastatic cancer, but is not limited thereto. Additionally, it may include metastatic renal cell carcinoma or metastatic melanoma.

[0335] Although not specifically limited thereto, the pharmaceutical composition of the present invention may contain 0.01 to 99% by weight to volume of an interleukin 2 analog or a sustained-release conjugate thereof.

[0336] Specifically, the composition of the present invention may comprise an interleukin 2 analog or a sustained-release conjugate thereof; and a CTLA-4 antagonist administered at a dose of 0.00001 mg / mL to 1,000 mg / mL, but is not limited thereto.

[0337] More specifically, the composition of the present invention may contain a sustained-release conjugate of an interleukin 2 analog at an amount of 0.0001 to 2120 mg / kg and a CTLA-4 antagonist at an amount of 0.0001 to 1000 mg / kg, administered in combination with the sustained-release conjugate of the interleukin 2 analog, but is not limited thereto. Alternatively, the composition of the present invention may contain an interleukin 2 analog at an amount of 0.00005 to 500 mg / kg and a CTLA-4 antagonist at an amount of 0.0001 to 1000 mg / kg, administered in combination thereto, but is not limited thereto.

[0338] However, the dosage of the CTLA-4 antagonist may be changed to exhibit an excellent co-administration effect with the interleukin 2 analog of the present invention or its sustained-release conjugate.

[0339] For example, the dosage of the CTLA-4 antagonist in the composition of the present invention may be expressed based on the anti-CTLA-4 antibody, and if other CTLA-4 antagonists are included, the CTLA-4 antagonist may be included in a dosage corresponding to the dosage of the anti-CTLA-4 antibody, but is not limited thereto.

[0340] In addition, the interleukin 2 analog or its sustained-release conjugate and the CTLA-4 antagonist included in the composition of the present invention may be administered in appropriately divided doses, and the doses may be the same or different. For example, when the interleukin 2 analog or its sustained-release conjugate of the composition is administered once a week, the CTLA-4 antagonist may be administered once, twice, or more times a week, but is not limited thereto.

[0341]

[0342] In the present invention, the term "prevention" means any act of suppressing or delaying cancer or tumor by administering the interleukin 2 analog (e.g., the interleukin 2 analog itself or a sustained-release conjugate form in which a biocompatible material is bound thereto) or a composition containing the same; and a CTLA-4 antagonist or a composition containing the same.

[0343] In the present invention, the term "treatment" means any act in which the symptoms of cancer are improved or benefited by administering the interleukin 2 analog (e.g., the interleukin 2 analog itself or a sustained-release conjugate form in which a biocompatible material is bound thereto) or a composition containing the same; and a CTLA-4 antagonist or a composition containing the same.

[0344] The use of the interleukin 2 analog or its sustained-release conjugate and CTLA-4 antagonist of the present invention has the great advantage of improving the quality of life of patients by reducing the frequency of administration for chronic patients who need to be administered daily, due to a dramatic increase in blood exposure, blood half-life, and sustained efficacy in vivo.

[0345]

[0346] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier or diluent. Such pharmaceutically acceptable carrier or diluent may be of non-natural origin.

[0347]

[0348] In the present invention, the term "pharmaceuticalally acceptable" means a sufficient amount to produce a therapeutic effect and not causing side effects, and can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the type of cancer, the patient's age, weight, health, gender, the patient's sensitivity to drugs, route of administration, method of administration, frequency of administration, duration of treatment, and drugs used in combination or concurrently.

[0349] A pharmaceutical composition comprising an interleukin 2 analog or a sustained-release conjugate thereof of the present invention, or a CTLA-4 antagonist, may include pharmaceutically acceptable excipients. The excipients are not particularly limited thereto, but for oral administration, binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavors, etc. may be used; for injectables, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used; and for topical administration, bases, excipients, lubricants, preservatives, etc. may be used.

[0350] The formulations of the composition of the present invention can be prepared in various ways by mixing with pharmaceutically acceptable excipients as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be prepared in the form of a single-dose ampoule or a multi-dose formulation. Additionally, it can be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0351] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, preservatives, etc. may be additionally included.

[0352] In addition, the pharmaceutical composition of the present invention may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid formulations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations, and suppositories.

[0353] In addition, the above composition may be formulated into a unit dosage form suitable for administration into a patient's body according to conventional methods in the pharmaceutical field, specifically in a form useful for administering protein drugs, and administered orally or by a parenteral administration route including the skin, intravenously, intramuscularly, intra-arterially, intramedullaryly, intrathecally, intraventricularly, pulmonaryly, transdermally, subcutaneously, intraabdominally, intranasally, gastrointestinally, topically, sublingually, vaginally, or rectal route using administration methods conventionally used in the industry, but is not limited thereto.

[0354] In addition, the above conjugate may be used in combination with various carriers permitted as pharmaceuticals, such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used as pharmaceuticals to increase stability or absorption.

[0355]

[0356] Another aspect of the present invention provides a method for the prevention or treatment of cancer comprising the step of administering a pharmaceutical composition comprising an interleukin 2 analog or a sustained-release conjugate containing the same to an individual in need, in combination with a CTLA-4 antagonist or a composition containing the same. In the method, the pharmaceutical composition comprising the interleukin 2 analog or the sustained-release conjugate containing the same and the CTLA-4 antagonist or the composition containing the same may be administered simultaneously, sequentially, or in reverse order, but are not limited thereto.

[0357] The above interleukin 2 analogs and / or sustained-release conjugates of interleukin 2 analogs, CTLA-4 antagonists, compositions containing these, cancer, prevention and treatment are as described above.

[0358] Another embodiment of the present invention is a method for preventing or treating cancer comprising the step of co-administering an interleukin 2 analog or a sustained-release conjugate containing the same, and a CTLA-4 antagonist to an individual.

[0359] The above method may involve administering a preparation comprising an interleukin 2 analog or a sustained-release conjugate containing the same, and a CTLA-4 antagonist to an individual, or administering an interleukin 2 analog or a sustained-release conjugate containing the same, and a CTLA-4 antagonist as separate preparations in combination, but is not limited thereto.

[0360] The method of the present invention may involve administering (i) an interleukin 2 analog or a sustained-release conjugate containing the same and (ii) a CTLA-4 antagonist as a single agent, or administering individual agents simultaneously, individually, sequentially, or in reverse order, but is not limited thereto.

[0361] The above-mentioned interleukin 2 analog or sustained-release conjugate containing the same, CTLA-4 antagonist, composition, cancer, prevention, and treatment are as described above.

[0362]

[0363] In the present invention, the individual refers to an individual that has developed or is suspected of having cancer, and includes mammals such as humans, mice, and livestock, but any individual that can be treated with the interleukin 2 analog and / or conjugate, CTLA-4 antagonist, or the composition containing these of the present invention is included without limitation.

[0364] In the present invention, the term “administration” means introducing a specific substance (e.g., an interleukin 2 analog or a sustained-release conjugate thereof, and / or a CTLA-4 antagonist) to a patient by any appropriate method, and the route of administration is not particularly limited thereto but may be administered through any general route capable of reaching a target in vivo, e.g., intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, nasal administration, pulmonary administration, or rectal administration.

[0365]

[0366] The method of the present invention may include administering a pharmaceutical composition comprising the interleukin 2 analog or a sustained-release conjugate thereof in a pharmaceutically effective amount, and administering a CTLA-4 antagonist or a pharmaceutical composition comprising the same in a pharmaceutically effective amount. A suitable total daily dose may be determined by the treating physician within the scope of proper medical judgment and may be administered in a single dose or divided into several doses. However, for the purposes of the present invention, it is preferable to apply a specific therapeutically effective dose for a specific patient differently depending on various factors and similar factors well known in the pharmaceutical field, including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the patient's age, weight, general health status, gender and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used together or concurrently with the specific composition.

[0367]

[0368] In the method of the present invention, the dosage and frequency of administration are determined according to the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease. Specifically, the composition of the present invention may contain, but is not limited to, a pharmaceutically effective amount of the interleukin 2 analog or a sustained-release conjugate containing it and a CTLA-4 antagonist.

[0369] Including the above-mentioned interleukin 2 analog or sustained-release conjugate and CTLA-4 antagonist in a pharmaceutically effective amount means an extent to which the desired pharmacological activity (e.g., prevention, improvement, or treatment of cancer) resulting from the interleukin 2 analog or sustained-release conjugate and CTLA-4 antagonist can be obtained, and may also mean a pharmaceutically acceptable level where no toxicity or side effects occur or are minimal in the administered subject, but is not limited thereto. Such a pharmaceutically effective amount may be determined by comprehensively considering the frequency of administration, the patient, the formulation, etc.

[0370] Although not specifically limited thereto, the pharmaceutical composition of the present invention may contain the above component (active ingredient) in an amount of 0.01 to 99% by weight to volume.

[0371]

[0372] The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or administered via a fractionated treatment protocol involving multiple doses administered over a long period. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Specifically, the preferred total dose of the interleukin 2 analog or its sustained-release conjugate and CTLA-4 antagonist administered in combination according to the present invention may be about 0.0001 mg to 500 mg per kg of the patient's body weight per day. However, since the effective dose for the patient is determined by considering various factors such as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate, as well as the route of administration and frequency of treatment of the pharmaceutical composition, a person of ordinary knowledge in the art would be able to determine an appropriate effective dose for a specific use of the composition of the present invention in light of these factors. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

[0373] The pharmaceutical composition of the present invention exhibits excellent in vivo persistence and potency, thereby significantly reducing the number and frequency of administration of the pharmaceutical formulation of the present invention. The pharmaceutical composition may be administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, nasal, pulmonary, or rectal administration routes, but is not limited to a specific administration route as long as the desired pharmacological effect can be obtained.

[0374] For example, the pharmaceutical composition of the present invention may be administered once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, or may be administered once or multiple times at time intervals ranging from one week to one month, but is not limited thereto.

[0375] Specifically, interleukin 2 analogs or sustained-release conjugates thereof and CTLA-4 antagonists may be administered at a dose of at least 0.00001 mg and 0.0000001 mg per kg of patient body weight per week, respectively, and when the two substances are used in combination, the total dose may be at least 0.00001 mg per kg of patient body weight per week, but is not limited thereto.

[0376] Specifically, the method of the present invention may involve administering 0.00001 to 175 mg of an interleukin 2 analog sustained-release conjugate and 0.0000001 to 100 mg of a CTLA-4 antagonist per 1 kg of patient body weight per week, but is not limited thereto. Alternatively, the method of the present invention may involve administering 0.000001 to 50 mg of an interleukin 2 analog and 0.0000001 to 100 mg of a CTLA-4 antagonist per 1 kg of patient body weight per week, but is not limited thereto.

[0377] Alternatively, the method of the present invention may involve administering an interleukin 2 analog or a sustained-release conjugate thereof at a dose of 0.001 to 33,000 nmol / kg and a CTLA-4 antagonist at a dose of 0.0001 to 7,000 nmol / kg for one week, but is not limited thereto.

[0378] Additionally, the interleukin 2 analog or its sustained-release conjugate and the CTLA-4 antagonist of the present invention may be administered in appropriately divided doses, and the doses may be the same or different. For example, when the interleukin 2 analog or its sustained-release conjugate is administered once a week, the CTLA-4 antagonist may be administered once, twice, or more times a week, but is not limited thereto.

[0379]

[0380] However, the dosage of the CTLA-4 antagonist may be modified to exhibit an excellent co-administration effect with the interleukin 2 analog of the present invention or its sustained-release conjugate. In the present invention, the dosage of the CTLA-4 antagonist may be expressed based on the anti-CTLA-4 antibody, and in the case of other CTLA-4 antagonists, the dosage may be administered at a dose corresponding to the dosage of the anti-CTLA-4 antibody, but is not limited thereto.

[0381]

[0382] Another embodiment of the present invention is the use of the interleukin 2 analog or sustained-release conjugate thereof for the manufacture of a drug for the prevention or treatment of cancer; or a composition comprising the same, which is administered in combination with a CTLA-4 antagonist.

[0383] Another embodiment of the present invention is the combined use of an interleukin 2 analog or a sustained-release conjugate thereof and a CTLA-4 antagonist for the prevention or treatment of cancer.

[0384] The above-mentioned interleukin 2 analogs and / or sustained-release conjugates thereof, CTLA-4 antagonists, or compositions containing these, cancer, prevention, treatment, administration routes, and number of administrations are as described above.

[0385]

[0386] Another embodiment of the present invention is to provide a cancer prevention or treatment use of an interleukin 2 analog or a sustained-release conjugate thereof; or a composition comprising the same, administered in combination with a CTLA-4 antagonist.

[0387] The above-mentioned interleukin 2 analogs and / or sustained-release conjugates thereof, CTLA-4 antagonists, or compositions containing these, cancer, prevention, treatment, administration routes, and number of administrations are as described above.

[0388]

[0389] Meanwhile, unless the context otherwise requires in this specification, expressions such as “includes,” “includes,” “containing,” etc., should be understood to mean the inclusion of a specified integer or group of integers, but not to exclude other integers or sets of integers.

[0390]

[0391] The present invention will be explained in more detail below through examples. These examples are solely for the purpose of explaining the invention more specifically, and the scope of the invention is not limited by these examples.

[0392]

[0393] Example 1: Preparation of natural interleukin 2 and interleukin 2 analog expression vectors

[0394]

[0395] To construct 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). In addition, a novel interleukin 2 analog was constructed by modifying the amino acids of interleukin 2 using the above interleukin 2 as a template.

[0396] PCR conditions for amplifying interleukin 2 analogs were 95°C for 30 seconds, 55°C for 60 seconds, and 65°C for 6.5 minutes, and this process was repeated 16 times. Sequence analysis was performed on the mutation products obtained under the above conditions, and it was confirmed that the mutations indicated in Table 1 below based on the natural form were present at the target mutation sites of each interleukin 2 analog. The expression vectors obtained in this way were named pET22b-interleukin 2 analogs 1 to 105.

[0397] The amino acid change sequences and analog names for each are shown in Table 1 below. To construct these interleukin 2 analogs, PCR was performed using forward (F) and reverse (R) primers to amplify each analog gene.

[0398] In Table 1 below, Analog 1 is aldesleukin, and Primers #1 to #204 correspond to sequence numbers 214 to 417, respectively. Table 1 summarizes the types of interleukin 2 analogs, mutation sites, and their modified sequences.

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411] desA1 means the deletion of alanine, the first amino acid of interleukin 2. Table 2 below shows the full-length protein sequences of interleukin 2 analogs. Bold text in Table 2 below indicates mutation locations. Table 2 summarizes the amino acid sequences of interleukin 2 analogs.

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423] Example 2: Expression of Interleukin 2 Analog

[0424]

[0425] Recombinant interleukin 2 analogs were expressed under T7 promoter regulation using the expression vector prepared in Example 1 above. Each recombinant interleukin 2 analog expression vector was used to express the E. coli strain, E. coli BL21DE3 (E. coli BL21DE3). - dcmompThsdS(r B - m B -)galλ(DE3); Novagen) was transformed. The transformation method used was the method recommended by Novagen. Each single colony transformed with each recombinant expression vector was taken, inoculated into 2X Luria Broth medium containing ampicillin (50 μg / mL), and incubated at 37°C for 15 hours. The recombinant strain culture and 2X LB medium containing 30% glycerol were mixed in a 1:1 (v / v) ratio, 1 mL of each was dispensed into cryo-tubes, and stored at -150°C. This was used as a cell stock for the production of recombinant proteins.

[0426] For the expression of recombinant interleukin 2 analogs, one vial of each cell stock was thawed, inoculated into 500 mL of 2X LB, and cultured with shaking at 37°C for 14 to 16 hours. Culture was terminated when the absorbance value at 600 nm reached 4.0 or higher, and the resulting culture was used as the seed culture. Using a 5 L fermenter (Bioflo-320, NBS, USA), the seed culture was inoculated into 1.6 L of fermentation medium, and initial fermentation was initiated. Culture conditions were maintained at a temperature of 37°C, an air flow rate of 2.0 L / min (1 vvm), a stirring speed of 650 rpm, and 30% ammonia water, with the pH maintained at 6.7. Fermentation proceeded as fed-batch culture by adding a feeding solution when nutrients in the culture medium became limited. The growth of the strain was monitored by the OD value, and IPTG at a final concentration of 500 μM was introduced when the absorbance value was 70 or higher. Culture was continued for approximately 23 to 25 hours after the introduction of IPTG, and after the culture was finished, the recombinant strain was harvested using a centrifuge and stored at -80℃ until use.

[0427]

[0428]

[0429] Example 3: Extraction and refolding of interleukin 2 analogs

[0430]

[0431] To convert the interleukin 2 analog from the interleukin 2 analog-expressing E. coli obtained in Example 2 above into a soluble form, cells were lysed and refolded. A cell pellet corresponding to 100 mL of culture medium was suspended in 1-200 mL of lysis buffer (20 mM Tris-HCl pH 9.0, 1 mM EDTA pH 9.0, 0.2 M NaCl, 0.5% Triton X-100), and then the recombinant E. coli was lysed at 15,000 psi using a microfluidizer. The pellet was centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and the pellet was washed with 400 mL of first wash buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0). Centrifuged under the same conditions as above, discarded the supernatant, and washed the pellet with 400 mL of a second wash buffer solution (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 2% Triton X-100). Centrifuged under the same conditions as above, discarded the supernatant, and washed the pellet with 400 mL of a third wash buffer solution (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1% sodium deoxycholorate). Centrifuged under the same conditions as above, discarded the supernatant, and washed the pellet with 400 mL of a fourth wash buffer solution (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1 M NaCl). Centrifuged under the same conditions as above to obtain a washed E. coli inclusion body pellet. The washed inclusion pellet was resuspended in 400 mL of soluble / reduction buffer (6 M Guanidine, 100 mM Tris pH 8.0, 2 mM EDTA pH 9.0, 50 mM DTT) and stirred at 50°C for 30 minutes. 100 mL of distilled water was added to the soluble / reduced interleukin 2 analog to 6 M Guanidine 4.After diluting with 8 M Guanidine, the solution was centrifuged at 13,900 g for 30 minutes, the pellet was discarded, and only the solution was obtained. 185.7 mL of distilled water was added to the diluted solution to dilute 4.8 M Guanidine to 3.5 M Guanidine, and the pH was adjusted to 5.0 using 100% acetic acid. The pH-adjusted solution was stirred at room temperature for 1 hour. The solution from which impurities had precipitated was centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and the pellet was washed with a final wash buffer solution (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. Washed interleukin 2 analogs were dissolved in 400 mL of refolding buffer solution (6 mM Guanidine, 100 mM Tris pH 8.0, 0.1 mM CuCl2). The refolding process was performed by stirring the mixed solution at 4°C for 15–24 hours.

[0432]

[0433] Example 4: Size Exclusion Column Chromatography

[0434]

[0435] The interleukin 2 analog refolding solution obtained in Example 3 above was concentrated to less than 1 mL for purification using a size exclusion column. The column was equilibrated with a buffer solution (2 M Guanidine, 100 mM Tris pH 8.0) before the introduction of the refolding solution, and elution was performed by flowing the buffer solution after the introduction of the refolding solution. Since the eluted sample contained Guanidine, it was replaced with a stabilization solution (10 mM Sodium Acetate pH 4.5, 5% Trehalose), and its purity was measured using RP-HPLC and peptide mapping analysis. If the measured purity was 80% or higher, the sample was used for the experiment.

[0436]

[0437] Example 5: Evaluation of Receptor Binding Affinity of Interleukin 2 Analogue

[0438]

[0439] Surface plasmon resonance (SPR, BIACORE T200, GE healthcare) was used to measure the receptor binding strength of the interleukin 2 analog obtained in Example 4 above to the interleukin 2 alpha receptor and beta receptor, respectively. The binding strength of the prepared analog to the alpha receptor and beta receptor was measured, and each binding strength was compared with that of interleukin 2 analog 1 (aldesleukin).

[0440]

[0441] First, approximately 5,000 RU (resonance units) of an anti-human immunoglobulin antibody (Abcam, #ab97221) was immobilized on a CM5 chip (GE Healthcare) via amine coupling. Subsequently, interleukin 2 alpha receptors (SYMANSIS, #4102H) or interleukin 2 beta receptors (SYMANSIS, #4122H) bound to the human immunoglobulin Fc region were bound to the immunoglobulin antibodies, respectively, for final immobilization. Next, the recombinant interleukin 2 analogs prepared above were diluted to various concentrations in HBS-P+ buffer (Cytiva, BR100671) using a two-fold serial dilution method. These dilutions were then flowed onto the CM5 chip finally immobilized with interleukin 2 receptors to measure the binding affinity of each receptor. Binding affinity is [calculated using] binding velocity (K a ) and nautical mileage (K dMeasurements were taken using [a specific method], and the binding rate was measured by flowing the interleukin 2 analog at a flow rate of 10 uL / min for 3 minutes, and the dissociation rate from each interleukin 2 receptor was measured by flowing only HBS-P+ buffer at the same time and flow rate. Once the measurements were completed, the binding affinity of the receptors was evaluated according to a 1:1 binding fitting model in the Biaevaluation program.

[0442]

[0443] Relative bonding strength K D (%) = Analog 1(aldesleukin) Dissociation constant(K d ) / Analog dissociation constant (K d ) × 100

[0444]

[0445] In Table 3 below, “undefinable” indicates that the corresponding physical quantity cannot be defined for that receptor because binding to that receptor was not observed in surface plasmon resonance measurements. Table 3 summarizes the relative binding affinities of interleukin 2 analogs to interleukin 2 alpha or beta receptors relative to interleukin 2 analog 1 (aldesleukin).

[0446]

[0447]

[0448]

[0449]

[0450]

[0451] As specified in the test results (Table 3) above, it was confirmed that the interleukin 2 analogs of the present invention exhibit interleukin 2 alpha receptor binding affinities different from natural interleukin 2 or aldesleukin, such as completely losing interleukin 2 alpha receptor binding affinity or showing a decrease or increase compared to interleukin 2 analog 1. On the other hand, for interleukin 2 beta receptors, a stronger binding affinity was confirmed compared to natural interleukin 2 or aldesleukin. Through this, it was confirmed that the amino acid sequence of the interleukin 2 analogs has an influence on the binding of interleukin 2 alpha or beta receptors. This suggests that the binding affinity of interleukin 2 receptors can be changed by substituting amino acids at specific positions.

[0452] The experimental results described above suggest that the interleukin 2 analog according to the present invention has altered interleukin 2 alpha receptor binding affinity and interleukin 2 beta receptor binding affinity, and can be utilized in the development of various drugs using this.

[0453]

[0454] Example 6: Linkage reaction of interleukin 2 analog and polyethylene glycol (3.4K PEG) linker and purification of interleukin 2 analog linker

[0455]

[0456] To prepare a sustained-release conjugate in which the interleukin 2 analog obtained in Example 4 is bound to the immunoglobulin Fc region, a linker was first prepared by connecting the interleukin 2 analog to one end of a polyethylene glycol (PEG) linker. Interleukin 2 analogs No. 21, 41, 52, 86, 104, and 105 were used for the preparation of the linker, and polyethylene glycol (ALD(2)3.4K PEG from NOF, Japan) with a molecular weight of 3.4 kDa and hydroxyl hydrogens at both ends modified with propylaldehyde groups was used as the PEG linker and reacted to link to the N-terminus of the interleukin 2 analog. The molar ratio of the interleukin 2 analog to the PEG linker was 1:15 to 1:20, and the concentration of the interleukin 2 analog was 1 mg / mL or less, and 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), and 20 mM sodium cyanoborohydride (SCB) was added 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 triethylamine (pH 8.0) and sodium chloride concentration gradient to obtain the interleukin 2 analog-3.4K PEG conjugate.

[0457]

[0458] Example 7: Preparation of Interleukin 2 Analog-3.4K PEG-Immunoglobulin Fc Region Sustained Conjugate

[0459]

[0460] To prepare an interleukin 2 analog-3.4K PEG-immunoglobulin Fc region sustained-release conjugate, the molar ratio of the interleukin 2 analog-3.4K PEG conjugate obtained using the method of Example 6 to the immunoglobulin Fc region (SEQ ID NO. 438) was set to 1:10, and the reaction was carried out at 2 to 10°C for 15 to 16 hours with a total protein concentration of 30 mg / mL. At this time, the reaction mixture was 100 mM potassium phosphate (pH 6.0), and 20 mM sodium cyanoborohydride was added as a reducing agent.

[0461] The immunoglobulin domain used at this time is formed by two monomers having the amino acid sequence of SEQ ID NO. 438 (composed of 221 amino acids) forming a homomer through a disulfide bond between the cysteine, which is the 3rd amino acid of each monomer, and the monomers of the homomer each independently form an internal disulfide bond between the cysteine ​​at numbers 35 and 95 and an internal disulfide bond between the cysteine ​​at numbers 141 and 199.

[0462]

[0463] Table 4 summarizes the amino acid sequence of immunoglobulin Fc.

[0464]

[0465]

[0466]

[0467] After the reaction was completed, the reaction solution was purified by removing the unreacted immunoglobulin Fc region from Butyl FF (Cytiva) using Bis-Tris (pH 6.5) and sodium chloride, and by purifying it with Source 15ISO (Cytiva) using sodium citrate buffer (pH 5.5) and ammonium sulfate, thereby obtaining an interleukin 2 analog-3.4K PEG-immunoglobulin Fc region conjugate (sustained conjugate) in which the N-terminus of the interleukin 2 analog is connected to one end of a 3.4 kDa PEG linker and the opposite end of the 3.4 kDa PEG linker is connected to the nitrogen of the N-terminal proline of the Fc region. This sustained conjugate was analyzed using SDS-PAGE, RP-HPLC, and SE-HPLC (Figs. 1 and 2).

[0468]

[0469] Example 8: Evaluation of Interleukin 2 Receptor Binding Affinity of Interleukin 2 Analog Conjugate

[0470]

[0471] Surface plasma resonance (SPR, BIACORE T200, GE healthcare) was used to measure the receptor binding affinity of the interleukin 2 analog sustained conjugate obtained in Example 7 above and the interleukin 2 alpha and beta receptors, respectively.

[0472]

[0473] Specifically, approximately 100 RU and 500 RU of biotin-labeled human interleukin 2 receptor alpha and beta subunits (ACROBiosystems), respectively, were immobilized on a streptavidin biosensor chip (SA chip, Cytiva). Interleukin 2 analog sustained-release conjugates or aldesleukins diluted in HBS-EP+ buffer (Cytiva, BR100669) using a 2x serial dilution method were flowed at a flow rate of 20 μL / min. After a 3-minute binding process, HBS-EP+ buffer alone was flowed at the same flow rate for 3 minutes to induce dissociation of the interleukin 2 analog sustained-release conjugates or aldesleukins from the interleukin 2 receptor. The binding affinity was calculated using the obtained binding and dissociation constants. Binding affinity evaluation was performed using a 1:1 binding fitting model in the Biaevaluation program.

[0474] The evaluated interleukin 2 analog conjugates were confirmed to have unique binding affinities to the interleukin 2 receptor, and in particular, clear differences in binding affinity were observed among the candidate substances at the interleukin 2 receptor alpha subunit. A closer look at the results reveals that interleukin 2 analog conjugates 21 and 52 did not bind to the interleukin 2 receptor alpha 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. In the case of the interleukin 2 receptor beta subunit, high binding affinity compared to aldesleukin was observed in all interleukin 2 analog conjugates, including interleukin 2 analog conjugates 21 and 52.

[0475] Table 5 below summarizes the relative binding strength (%) of interleukin 2 analog sustained-release conjugates prepared for alpha and beta receptors, compared based on the binding strength of aldesleukin. In Table 5, the corresponding sustained-release conjugate is indicated by the number of the interleukin 2 analog constituting it (e.g., the sustained-release conjugate of interleukin 2 analog 21 is indicated as “Interleukin 2 analog 21 conjugate”). Table 5 summarizes the binding strength of interleukin 2 analog conjugates to interleukin 2 receptors.

[0476]

[0477]

[0478]

[0479] Example 9: Evaluation of anticancer efficacy by co-administration of an interleukin 2 analog conjugate and an anti-CTLA-4 antibody in a triple-negative breast cancer (TNBC) mouse model

[0480]

[0481] In order to confirm the anticancer effect when an interleukin 2 analog conjugate according to the present invention and a CTLA-4 antagonist are administered in combination, the anticancer efficacy was evaluated when an interleukin 2 analog conjugate of the present invention No. 86 (SEQ No. 87) and an anti-CTLA-4 antibody (#BP0164, BioXcell) were administered alone and in combination to a triple-negative breast cancer animal model.

[0482]

[0483] To this end, an orthotopic tumor model was constructed by injecting 4T1 cells, mouse-derived triple-negative mammary cancer cells, into the mammary fat pads of 6-week-old female Balb / C mice. After 15 days, the tumor volume (50–60 mm²) 3Seven animals were randomly assigned to each group based on the criteria. The study was divided into a monotherapy group administered interleukin 2 analog conjugate 86 and anti-CTLA-4 antibody alone, and a combination therapy group administered both drugs in combination. The drugs were administered for a total of 4 weeks according to the dosage regimens for each drug. The anti-tumor efficacy for each experimental group was evaluated as tumor growth inhibition (TGI) using the formula below. Detailed drug dosages, administration routes, frequency, and anti-tumor efficacy for each group are shown in Table 6.

[0484]

[0485] Tumor growth inhibitory ability = (1 - Increased tumor volume of the experimental group from Day 0 to Day X / Increased tumor volume of the negative control group from Day 0 to Day X) x 100

[0486]

[0487]

[0488]

[0489] Tumor growth inhibitory ability was analyzed by measuring the tumor size of each group on day 18 based on the date of drug administration. As a result, in 4T1 tumor mouse models administered 6.0 mg / kg and 25 mg / kg doses of interleukin 2 analog conjugate 86 and 10 mg / kg dose of anti-CTLA-4 antibody, respectively, tumor growth inhibitory efficacy of approximately 53.6–61.6% was confirmed compared to the negative control group (Table 6). Their anti-tumor efficacy increased to a statistically significant level through combination administration, with 94.7% observed in the combination of 6 mg / kg of interleukin 2 analog conjugate 86 + anti-CTLA4 antibody and 100.6% in the combination of 25 mg / kg + anti-CTLA4 antibody, showing a significant level of tumor growth inhibitory efficacy compared to the administration of each drug alone (Figure 3). In addition, regarding the complete tumor remission rate, complete remission was observed in only 1 out of 7 mice administered 25 mg / kg of interleukin 2 analog conjugate 86 alone, whereas complete tumor remission was observed in 42.9% of individuals (3 out of 7) in the groups administered 6.0 mg / kg and 25 mg / kg of interleukin 2 analog conjugate and anti-CTLA-4 antibody in combination (Table 6). From these results, it was confirmed that the combined administration of the interleukin 2 analog conjugate and anti-CTLA-4 antibody according to the present invention exhibits superior tumor suppression efficacy and a complete tumor remission rate compared to single administration.

[0490]

[0491] Example 10: Evaluation of anticancer efficacy by co-administration of an interleukin 2 analog conjugate and an anti-CTLA4 antibody in a melanoma (B16F10) mouse model

[0492]

[0493] To evaluate the anticancer effect when an interleukin 2 analog conjugate according to the present invention is administered in combination with a CTLA-4 antagonist, an efficacy experiment was performed on a melanoma allogeneic mouse model (B16F10 tumor syngeneic mouse model). The melanoma cell line (B16F10, ATCC) was used as the melanoma (B16F10) mouse model, and the antitumor efficacy between the single and combined administration groups of the interleukin 2 analog conjugate No. 86 (SEQ No. 87) according to the present invention and the anti-CTLA-4 antibody (#BP0164, BioXcell) was compared and analyzed.

[0494]

[0495] Specifically, B16F10 cells were injected subcutaneously into the thighs of 6-week-old female C57BL / 6 mice. Approximately 5 days later, when the tumors had grown large enough to be visually observed, the average tumor size of each group was approximately 63 mm. 3 Seven animals were assigned to each group. Interleukin 2 analog conjugate 86 was administered subcutaneously at a dose of 0.3 mg / kg once a week for a total of 3 weeks, and the anti-CTLA4 antibody was administered intraperitoneally at a dose of 10 mg / kg twice a week for a total of 3 weeks. In the combination therapy group, each substance was administered concurrently at the same dose and schedule as in the monotherapy group. Day 10, based on the start date of administration, was the last measurement day on which more than half of the negative control group survived; on this measurement day, the tumor growth inhibition rate (TGI) was analyzed, and the median overall survival (mOS) was evaluated by tracking individual survival time. Tumor growth inhibition rate was calculated using the same method as in Example 9 above.

[0496]

[0497] As a result, the anti-CTLA4 antibody monotherapy group showed a TGI of 60.3% and an mOS of 17 days, while the interleukin 2 analog conjugate 86 monotherapy group showed a TGI of 32.4% and an mOS of 14 days. In the combination therapy groups of each drug, an synergistic effect was confirmed, with a TGI of 88.0% and an mOS of 24 days, demonstrating statistically significant anticancer efficacy compared to the monotherapy groups.

[0498]

[0499] Through the above experiment, the synergistic effect of co-administration of interleukin 2 analog conjugate 86 and anti-CTLA4 antibody in a melanoma allograft mouse model was confirmed.

[0500]

[0501]

[0502] The last measurement date when more than half of the negative control subjects survived

[0503]

[0504] The experimental results of Examples 9 and 10 as described above suggest that since the interleukin 2 analog sustained-release conjugate and the CTLA-4 antagonist according to the present invention can exhibit excellent tumor suppressive efficacy and complete tumor remission rates through synergistic action, cancer can be treated safely and effectively when the interleukin 2 analog conjugate is administered in combination with the CTLA-4 antagonist.

[0505]

[0506] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the foregoing detailed description.

Claims

1. A pharmaceutical composition for the prevention or treatment of cancer containing an interleukin 2 analog comprising any one sequence selected from the amino acid sequences of SEQ ID NOs 3 to 106, wherein the pharmaceutical composition is characterized by being administered in combination with a CTLA-4 antagonist.

2. In claim 1, the interleukin 2 analog is in the form of a sustained-release conjugate, and the sustained-release conjugate is a composition represented by the following chemical formula 1: [Chemical Formula 1] X - L - F In this case, X is an interleukin 2 analog comprising any one sequence selected from the amino acid sequences of SEQ ID NOs 3 to 106; L is a polyethylene glycol linker; F is the dimeric form of the immunoglobulin Fc region; - represents the covalent bond connection between X and L, and between L and F, and In the above sustained conjugate, one end of L is covalently connected to only one polypeptide chain of the dimeric Fc region, and X is covalently connected to the opposite end of this L.

3. A composition according to claim 1 or 2, wherein the interleukin 2 analog comprises any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs 10, 13 to 15, 17, 20 to 22, 32, 35, 36, 42, 53, 54, 56, 58 to 60, 62, 71, 72, 74 to 78, 85, 87, 89, 91 to 94, and 97 to 106.

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

5. A composition according to claim 1 or 2, wherein the interleukin 2 analog comprises any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 22, 42, 53, 87, 105 and 106.

6. A composition according to claim 1 or 2, wherein the interleukin 2 analog further comprises one or more amino acids at the C-terminus.

7. A composition according to claim 1 or 2, wherein the interleukin 2 analog has increased binding affinity to the interleukin 2 beta receptor compared to aldesleukin.

8. In paragraph 2, the immunoglobulin Fc region is a composition derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof.

9. A composition according to paragraph 2, wherein the immunoglobulin Fc region is an IgG4 Fc region.

10. A composition according to claim 2, wherein the immunoglobulin Fc region is non-glycosylated.

11. A composition according to claim 2, wherein the sustained-form conjugate is a composition in which X is covalently connected to one of the Fc regions of the dimeric immunoglobulin through the polyethylene glycol linker.

12. A composition according to paragraph 2, wherein the polyethylene glycol linker is a linker with a molecular weight of 1 kDa to 100 kDa.

13. The composition of claim 1 or 2, wherein the composition further comprises a pharmaceutically acceptable excipient.

14. In paragraph 1 or 2, 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, stomach cancer, head or neck cancer, esophageal cancer, laryngeal cancer, bone cancer, rectal cancer, pro-anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, 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, lymphocytic lymphoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain tumor, glioma (astrocytoma, glioblastoma), A composition selected from the group consisting of oligodendroglioma, ependymoma, germ cell tumor, meningioma, brainstem glioma, pituitary adenoma, schwannoma, congenital tumor, craniopharyngioma, and brain tumor.

15. A composition according to claim 1 or 2, wherein the cancer is a cancer with low responsiveness to a CTLA-4 antagonist.

16. A composition according to claim 14, wherein the breast cancer is triple-negative breast cancer.

17. The composition of claim 1 or 2, wherein the composition is administered via intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, nasal administration, pulmonary administration, or rectal administration route.

18. The composition according to claim 1 or 2, wherein the composition is administered at time intervals ranging from one week to one month.

19. A composition according to claim 1 or 2, characterized in that the composition is administered concurrently, individually, sequentially, or in reverse order with a composition containing a CTLA-4 antagonist.

20. A composition according to claim 1 or 2, wherein the CTLA-4 antagonist is one or more selected from an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

21. A composition according to claim 20, wherein the anti-CTLA-4 antibody is ipilimumab or tremelimumab.

22. A composition according to claim 1 or 2, wherein the composition further comprises a CTLA-4 antagonist.

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