Pharmaceutical composition for enhancing radiation therapy, comprising a fusion protein containing an IL-2 protein and a CD80 protein

Abstract

The present invention relates to a pharmaceutical composition for enhancing radiotherapy, which comprises a fusion protein dimer containing IL-2 protein and CD80 protein, and to cancer radiotherapy using the same. The composition for enhancing radiotherapy, which comprises the fusion protein of the present invention, can increase the effectiveness of radiotherapy in cancer treatment.

Description

[Technical Field]

[0001] Technical Field The present invention relates to a pharmaceutical composition for enhancing radiotherapy, which comprises a fusion protein containing an IL-2 protein and a CD80 protein, and to radiotherapy of cancer using the same. [Background technology]

[0002] Background technology Cancer treatment methods are broadly divided into surgery, radiation therapy, and chemotherapy. The number of cancer patients receiving radiation therapy in Korea is increasing every year, and the importance of radiation therapy in cancer treatment is also growing.

[0003] Radiation therapy is now recognized as an essential treatment for various types of cancer. However, problems such as the development of radioresistance in cancer cells and damage to normal tissues due to high-dose radiation therapy have been identified as reducing the efficacy of radiation therapy. Therefore, attempts have been made to develop radiosensitizers and radiosensitizing compounds that enhance the effectiveness of radiation therapy (Oncogene, 23 (8): 1599-1607, 2004). However, the radiosensitizers reported to date have mainly been anticancer drugs, such as taxol and cisplatin.

[0004] When anticancer drugs used to enhance the effects of radiation therapy as described above are used in combination with radiation therapy, the toxicity of the anticancer drugs may be accompanied by side effects that occur during radiation therapy, such as inflammation at the radiation therapy site, gastrointestinal disorders, nausea, vomiting, diarrhea, etc., which limits their use.

[0005] Therefore, there is an urgent need to develop radiotherapy enhancers that can optimize radiotherapy by increasing the radiosensitivity of cancer cells while minimizing side effects. Summary of the Invention [Problem to be solved by the invention]

[0006] Detailed Description of the Invention technical issues Therefore, the present inventors investigated the development of a radiotherapy potentiator that can be used in combination with radiotherapy without side effects while enhancing the effectiveness of radiotherapy against cancer. As a result, the present inventors confirmed that a fusion protein dimer containing IL-2 protein and CD80 protein in a single molecule exhibits a synergistic effect in cancer treatment when used in combination with radiation. Based on the above, the present inventors have completed the present invention. [Means for solving the problem]

[0007] Solution to the problem To achieve the above object, one aspect of the present invention provides a pharmaceutical composition for enhancing cancer radiotherapy, which comprises a fusion protein dimer comprising an IL-2 protein and a CD80 protein.

[0008] Another aspect of the present invention provides a method of radiotherapy for cancer, comprising irradiating a non-human mammal suffering from cancer at the site of the cancer and administering a pharmaceutical composition to the mammal. [Effects of the Invention]

[0009] Effect of the invention The composition of the present invention comprising the fusion protein dimer containing IL-2 protein and CD80 protein can be used in combination with radiotherapy to enhance the effect of radiotherapy, thus potentially enhancing the effect of radiotherapy on cancers that are resistant to radiation.

[0010] Furthermore, due to this enhancement of radiotherapy, it is possible to obtain the excellent anticancer effect that can be obtained by performing high-dose radiotherapy while reducing the dose of radiotherapy, which has the advantage of reducing the side effects caused by high-dose radiation exposure during radiotherapy.

[0011] Furthermore, radiation therapy can stimulate immune cells throughout the body by disseminating tumor antigens. In this regard, the fusion protein dimer containing IL-2 and CD80 further amplifies the activity of systemic immune cells, thereby enhancing the anticancer effects of radiation therapy.

[0012] Therefore, it is expected that the composition of the present invention containing the fusion protein dimer containing IL-2 protein and CD80 protein can be used as a commercialized radiotherapy adjuvant for the purpose of combination therapy. In particular, when radiation is irradiated and the pharmaceutical composition of the present invention is administered, an excellent anticancer effect is obtained even in areas not irradiated with radiation, and therefore, it is expected to be highly commercially useful. [Brief explanation of the drawings]

[0013] [Figure 1] 1a to 1c show the results of confirming the abscopal effect, which indicates the anticancer effect at the irradiated tumor site and the therapeutic effect at the non-irradiated site, when mGI-101 of the present invention was administered to mice bearing tumors formed after transplantation of the melanoma cell line B16F10 and / or when the mice were irradiated. Specifically, the graphs show (a) the tumor volume on the irradiated right side, (b) the tumor volume on the non-irradiated left side, and (c) the average volume of both tumors, which were measured using a vernier caliper when mGI-101 of the present invention was administered to tumor-bearing mice and / or when the mice were irradiated. [Figure 2] 2a to 2e show the results of confirming the anticancer effect at the irradiated tumor site when tumor-bearing mice were administered mGI-101 of the present invention and / or irradiated. Specifically, the results show tumor growth in each mouse subject when mGI-101 of the present invention was administered to the tumor on the right side of the irradiated tumor and / or irradiated. [Figure 3]Figures 3a to 3e show the results of confirming the abscopal effect, which indicates a therapeutic effect at distal sites that were not irradiated, when tumor-bearing mice were administered mGI-101 of the present invention and / or irradiated. Specifically, the results show tumor growth in each mouse subject when mGI-101 of the present invention was administered and / or irradiated to the tumor on the left side that was not irradiated. [Figure 4] Figures 4a-4c are graphs showing tumor growth inhibition by mGI-101 of the present invention and / or radiation, specifically (a) tumor growth inhibition of the irradiated right-sided tumor, (b) tumor growth inhibition of the unirradiated left-sided tumor, and (c) the average tumor growth suppression of both tumors. DETAILED DESCRIPTION OF THE INVENTION

[0014] Best Mode for Carrying Out the Invention Radiation Therapy Enhancers In one aspect of the present invention, a pharmaceutical composition for enhancing cancer radiotherapy is provided, which comprises a fusion protein dimer comprising an IL-2 protein and a CD80 protein.

[0015] In another aspect of the present invention, there is provided an anti-cancer adjuvant containing the fusion protein dimer as an active ingredient.

[0016] The term "cancer" as used herein is classified as a disease in which normal tissue cells continue to proliferate and develop without limit for some reason, regardless of the biological phenomena of the living body or the condition of the surrounding tissues. Cancers in the present invention include, but are not limited to, gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. Furthermore, the present invention targets cancers that are resistant to radiation, but is not limited to these.

[0017] The term "enhancing radiotherapy" as used herein means enhancing the sensitivity of cells to radiation in the treatment of a disease using radiation, ultimately enhancing the therapeutic effect against the disease. In particular, when administered in parallel with cancer treatment, the radiosensitivity of cancer cells may be increased, and the effects of killing cancer cells and inhibiting the proliferation of cancer cells may be exhibited.

[0018] Radiotherapy for cancer includes, but is not limited to, various known radiotherapies such as deep X-ray therapy, radium therapy, cobalt-60 mass irradiation, megavoltage radiation therapy, and radioisotope oral therapy.

[0019] In one embodiment, mice that had developed tumors after transplantation of the melanoma cell line B16F10 were treated with a combination of the fusion protein dimer of the present invention comprising the IL-2 protein and the CD80 protein and radiation therapy. As a result, it was confirmed that the fusion protein can be used as a combination or adjuvant (auxiliary agent) in anti-cancer treatment by more effectively reducing tumor growth (FIGS. 1a to 4c).

[0020] The pharmaceutical composition for enhancing radiation therapy of the present invention, which comprises a fusion protein of IL-2 protein and CD80 protein, can be applied to any cells to which radiation therapy can be applied, and is particularly preferably used to increase the radiation sensitivity of cancer cells.

[0021] The pharmaceutical composition for enhancing radiotherapy exhibits synergistic effects in cancer treatment in combination with radiotherapy, and can be used as an anti-cancer therapeutic adjuvant, a radiotherapy adjuvant, a radiotherapy enhancing agent, or a radiosensitizer.

[0022] The term "adjuvant" as used herein refers to a substance that enhances the effects of a drug, substance, method, etc. having a therapeutic effect, and when the pharmaceutical composition for enhancing radiation therapy according to the present invention is administered, it enhances the anticancer activity of the active ingredient having a therapeutic effect, thereby enhancing the anticancer activity or reducing side effects. Specifically, when used in combination with conventional cancer treatment methods such as anticancer drugs or radiation, it can exert a synergistic effect on the cancer treatment effect and increase the sensitivity of cancer cells to the anticancer drug or radiation.

[0023] In particular, it was confirmed that administration of the fusion protein dimer during radiation therapy enhances the abscopal effect, which effectively removes cancer cells in distant sites other than the irradiated site. Therefore, it was confirmed that the fusion protein dimer enhances the anti-cancer effect of radiation therapy not only in the irradiated site but also in distant sites that have not been irradiated. Therefore, it can be used as a combination therapy to enhance the effect of radiation therapy.

[0024] The fusion protein comprising the IL-2 protein and the CD80 protein contained in the pharmaceutical composition for enhancing radiation therapy is as described below.

[0025] Fusion protein containing IL-2 protein and CD80 protein As used herein, the term "IL-2" or "interleukin-2," unless otherwise specified, refers to any wild-type IL-2 obtained from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). IL-2 may be obtained from animal cells, including recombinant cells capable of producing IL-2. Furthermore, IL-2 may be wild-type IL-2 or a variant thereof.

[0026] As used herein, IL-2 or variants thereof may be collectively referred to as "IL-2 proteins" or "IL-2 polypeptides." IL-2, IL-2 proteins, IL-2 polypeptides, and IL-2 variants specifically bind to, for example, the IL-2 receptor. This specific binding can be determined by methods known to those skilled in the art.

[0027] An embodiment of IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Here, IL-2 may also be in a mature form. Specifically, mature IL-2 may not contain a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. Here, IL-2 may be used in a conceptual sense to encompass fragments of wild-type IL-2 in which a portion of the N-terminus or C-terminus of wild-type IL-2 has been truncated.

[0028] Furthermore, a fragment of IL-2 can be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids are truncated from the N-terminus of a protein having the amino acid sequence of SEQ ID NO:35 or SEQ ID NO:36. Furthermore, a fragment of IL-2 can be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids are truncated from the C-terminus of a protein having the amino acid sequence of SEQ ID NO:35 or SEQ ID NO:36.

[0029] As used herein, the term "IL-2 variant" refers to a form in which some of the amino acids of full-length IL-2 or the above-mentioned IL-2 fragments have been substituted. That is, an IL-2 variant may have an amino acid sequence that differs from that of wild-type IL-2 or a fragment thereof. However, an IL-2 variant may have activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may refer, for example, to specific binding to the IL-2 receptor, and this specific binding can be measured by methods known to those skilled in the art.

[0030] Specifically, IL-2 mutants can be obtained by substituting some of the amino acids of wild-type IL-2. In one embodiment, IL-2 mutants obtained by amino acid substitution can be obtained by substituting at least one of the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10.

[0031] Specifically, an IL-2 mutant can be obtained by substituting at least one of the amino acids at positions 38, 42, 45, 61, or 72 in the amino acid sequence of SEQ ID NO: 10 with another amino acid. Furthermore, when IL-2 is in a form in which a portion of the N-terminus is truncated in the amino acid sequence of SEQ ID NO: 35, the amino acid at the complementary position in the amino acid sequence of SEQ ID NO: 10 can be substituted with another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 mutant can be obtained by substituting at least one of the amino acids at positions 58, 62, 65, 81, or 92 in the amino acid sequence of SEQ ID NO: 35 with another amino acid. These amino acid residues correspond to the amino acid residues at positions 38, 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10, respectively. According to one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted, as long as such an IL-2 mutant maintains its IL-2 activity. According to another embodiment, 1 to 5 amino acids may be substituted.

[0032] In one embodiment, the IL-2 mutant may be in a form in which two amino acids are substituted. Specifically, the IL-2 mutant can be obtained by substituting the amino acids at positions 38 and 42 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant can be obtained by substituting the amino acids at positions 38 and 45 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant can be obtained by substituting the amino acids at positions 38 and 61 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant can be obtained by substituting the amino acids at positions 38 and 72 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant can be obtained by substituting the amino acids at positions 42 and 45 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant can be obtained by substituting the amino acids at positions 42 and 61 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant can be obtained by substituting the amino acids at positions 42 and 72 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant may be obtained by substitution of the amino acids at positions 45 and 61 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant may be obtained by substitution of the amino acids at positions 45 and 72 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant may be obtained by substitution of the amino acids at positions 61 and 72 in the amino acid sequence of SEQ ID NO: 10.

[0033] Furthermore, the IL-2 mutant may be in the form in which three amino acids are substituted. Specifically, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 42, and 45 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 42, and 61 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 42, and 72 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 45, and 61 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 45, and 72 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 61, and 72 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant can be obtained by substitution of the amino acids at positions 42, 45, and 61 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant can be obtained by substitution of the amino acids at positions 42, 45, and 72 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant can be obtained by substitution of the amino acids at positions 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10.

[0034] In addition, the IL-2 mutant may be in the form of a substitution of four amino acids. Specifically, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 42, 45, and 61 in the amino acid sequence of SEQ ID NO: 10. In one embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 42, 45, and 72 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 38, 42, 61, and 72 in the amino acid sequence of SEQ ID NO: 10. In another embodiment, the IL-2 mutant may be obtained by substituting the amino acids at positions 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10.

[0035] Furthermore, the IL-2 mutant may have five amino acid substitutions, specifically, an IL-2 mutant can be obtained by substituting the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10 with different amino acids.

[0036] Here, the "another amino acid" introduced by substitution can be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, with regard to the amino acid substitutions of the IL-2 mutant, in the amino acid sequence of SEQ ID NO: 10, the amino acid at position 38 cannot be substituted with arginine, the amino acid at position 42 cannot be substituted with phenylalanine, the amino acid at position 45 cannot be substituted with tyrosine, the amino acid at position 61 cannot be substituted with glutamic acid, and the amino acid at position 72 cannot be substituted with leucine.

[0037] The amino acid substitution in the IL-2 mutant may be such that the arginine at position 38 in the amino acid sequence of SEQ ID NO: 10 is substituted with an amino acid other than arginine. Preferably, the amino acid substitution in the IL-2 mutant may be such that the arginine at position 38 in the amino acid sequence of SEQ ID NO: 10 is substituted with alanine (R38A).

[0038] The amino acid substitution in the IL-2 mutant may be such that the 42nd amino acid, phenylalanine, in the amino acid sequence of SEQ ID NO: 10 is substituted with an amino acid other than phenylalanine. Preferably, the amino acid substitution in the IL-2 mutant may be such that the 42nd amino acid, phenylalanine, in the amino acid sequence of SEQ ID NO: 10 is substituted with alanine (F42A).

[0039] The amino acid substitution in the IL-2 mutant may be such that the tyrosine at position 45 in the amino acid sequence of SEQ ID NO: 10 is substituted with an amino acid other than tyrosine. Preferably, the amino acid substitution in the IL-2 mutant may be such that the tyrosine at position 45 in the amino acid sequence of SEQ ID NO: 10 is substituted with alanine (Y45A).

[0040] The amino acid substitution in the IL-2 mutant may be such that the glutamic acid at position 61 in the amino acid sequence of SEQ ID NO: 10 is substituted with an amino acid other than glutamic acid. Preferably, the amino acid substitution in the IL-2 mutant may be such that the glutamic acid at position 61 in the amino acid sequence of SEQ ID NO: 10 is substituted with arginine (E61R).

[0041] The amino acid substitution in the IL-2 mutant may be such that the 72nd amino acid, leucine, in the amino acid sequence of SEQ ID NO: 10 is substituted with an amino acid other than leucine. Preferably, the 72nd amino acid, leucine, in the amino acid sequence of SEQ ID NO: 10 may be substituted with glycine (L72G).

[0042] Specifically, the IL-2 mutant can be obtained by at least one substitution in the amino acid sequence of SEQ ID NO:10 selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0043] Specifically, the IL-2 mutant may be obtained by amino acid substitutions at two, three, four or five positions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0044] The IL-2 mutant may also be in a form in which two amino acids are substituted. Specifically, the IL-2 mutant may be obtained by substitution of R38A and F42A. Furthermore, in one embodiment, the IL-2 mutant may be obtained by substitution of R38A and Y45A. Furthermore, in one embodiment, the IL-2 mutant may be obtained by substitution of R38A and E61R. Furthermore, in one embodiment, the IL-2 mutant may be obtained by substitution of R38A and L72G. Furthermore, in one embodiment, the IL-2 mutant may be obtained by substitution of F42A and Y45A. Furthermore, in one embodiment, the IL-2 mutant may be obtained by substitution of F42A and E61R. Furthermore, in one embodiment, the IL-2 mutant may be obtained by substitution of F42A and L72G. Furthermore, in one embodiment, the IL-2 mutant may be obtained by substitution of E61R and L72G.

[0045] Furthermore, the IL-2 mutant may have three amino acid substitutions. Specifically, the IL-2 mutant may be obtained by substitution of R38A, F42A, and Y45A. In one embodiment, the IL-2 mutant may be obtained by substitution of R38A, F42A, and E61R. In one embodiment, the IL-2 mutant may be obtained by substitution of R38A, F42A, and L72G. In one embodiment, the IL-2 mutant may be obtained by substitution of R38A, Y45A, and E61R. In one embodiment, the IL-2 mutant may be obtained by substitution of R38A, Y45A, and L72G. In one embodiment, the IL-2 mutant may be obtained by substitution of F42A, Y45A, and E61R. In one embodiment, the IL-2 mutant may be obtained by substitution of F42A, Y45A, and L72G. Furthermore, in one embodiment, the IL-2 mutant can be obtained by the substitutions F42A, E61R and L72G. Furthermore, in one embodiment, the IL-2 mutant can be obtained by the substitutions Y45A, E61R and L72G.

[0046] The IL-2 mutant may also be in the form of a substitution of four amino acids. Specifically, the IL-2 mutant can be obtained by substitution of R38A, F42A, Y45A, and E61R. In one embodiment, the IL-2 mutant can be obtained by substitution of R38A, F42A, Y45A, and L72G. In one embodiment, the IL-2 mutant can be obtained by substitution of R38A, F42A, E61R, and L72G. In one embodiment, the IL-2 mutant can be obtained by substitution of R38A, Y45A, E61R, and L72G. In one embodiment, the IL-2 mutant can be obtained by substitution of F42A, Y45A, E61R, and L72G.

[0047] Additionally, IL-2 mutants can be obtained by the following substitutions: R38A, F42A, Y45A, E61R and L72G.

[0048] Preferably, the IL-2 variant embodiment may include any one of the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A; (b) R38A / F42A / Y45A; (c) R38A / F42A / E61R; or (d) R38A / F42A / L72G.

[0049] When IL-2 has the amino acid sequence of SEQ ID NO: 35, it may have an amino acid substitution at a position complementary to the amino acid sequence of SEQ ID NO: 10. Furthermore, even if IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, it may have an amino acid substitution at a position complementary to the amino acid sequence of SEQ ID NO: 10.

[0050] Specifically, the IL-2 variant may have the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.

[0051] The IL-2 mutants may also be characterized by low in vivo toxicity. Here, the in vivo toxicity may be a side effect caused by the binding of IL-2 to the IL-2 receptor α chain (IL-2Rα). To improve the side effects caused by the binding of IL-2 to IL-2Rα, various IL-2 mutants have been developed, and such IL-2 mutants may be those described in U.S. Pat. No. 5,229,109 and Korean Patent No. 1667096. In particular, the IL-2 mutants described herein have a reduced ability to bind to the IL-2 receptor α chain (IL-2Rα) and therefore have lower in vivo toxicity than wild-type IL-2.

[0052] As used herein, the term "CD80," also known as "B7-1," is a membrane protein present on dendritic cells, activated B cells, and monocytes. CD80 provides costimulatory signals essential for T cell activation and survival. CD80 is known to be a ligand for two distinct proteins present on the surface of T cells, CD28 and CTLA-4. CD80 consists of 288 amino acids and may specifically have the amino acid sequence of SEQ ID NO: 11. Furthermore, as used herein, the term "CD80 protein" refers to full-length CD80 or a CD80 fragment.

[0053] As used herein, the term "CD80 fragment" refers to a truncated form of CD80. Furthermore, the CD80 fragment may be the extracellular domain of CD80. One embodiment of the CD80 fragment can be obtained by removing amino acids 1 to 34 from the N-terminus, which is the CD80 signal sequence. Specifically, one embodiment of the CD80 fragment may be a protein consisting of amino acids 35 to 288 of SEQ ID NO: 11. Another embodiment of the CD80 fragment may be a protein consisting of amino acids 35 to 242 of SEQ ID NO: 11. Another embodiment of the CD80 fragment may be a protein consisting of amino acids 35 to 232 of SEQ ID NO: 11. Another embodiment of the CD80 fragment may be a protein consisting of amino acids 35 to 139 of SEQ ID NO: 11. Another embodiment of the CD80 fragment may be a protein consisting of amino acids 142 to 242 of SEQ ID NO: 11. In one embodiment, the CD80 fragment may have the amino acid sequence of SEQ ID NO: 2.

[0054] Alternatively, the IL-2 protein and the CD80 protein may be linked via a linker or a carrier. Specifically, IL-2 or a variant thereof and CD80 (B7-1) or a fragment thereof may be linked to each other via a linker or a carrier. In this specification, the terms linker and carrier may be used interchangeably.

[0055] The linker connects two proteins. Examples of linkers include 1 to 50 amino acids, albumin or a fragment thereof, and the Fc domain of an immunoglobulin. Here, the Fc domain of an immunoglobulin refers to a protein that contains the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, but does not contain the heavy and light chain variable regions and light chain constant region 1 (CH1) of an immunoglobulin. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and is preferably IgG4. Here, the Fc domain of wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 4.

[0056] Furthermore, the Fc domain of an immunoglobulin may be an Fc domain variant as well as a wild-type Fc domain. Furthermore, as used herein, the term "Fc domain variant" refers to a form that differs from the wild-type Fc domain in terms of glycosylation pattern, a form that has increased glycosylation compared to the wild-type Fc domain, or a form that has decreased glycosylation compared to the wild-type Fc domain, or a deglycosylated form. Furthermore, an unglycosylated Fc domain is also encompassed. The Fc domain or variant thereof can be adapted to have a controlled number of sialic acids, fucosylation, or glycosylation by culture conditions or genetic manipulation of the host.

[0057] The glycosylation of the immunoglobulin Fc domain may be modified by conventional methods such as chemical methods using microorganisms, enzymatic methods, or genetic engineering methods. The Fc domain variant may also be a mixture of the Fc regions of immunoglobulins, IgG, IgA, IgE, IgD, and IgM. The Fc domain variant may also be a form in which some amino acids in the Fc domain are substituted with other amino acids. An embodiment of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 12.

[0058] The fusion protein may have a structure in which the CD80 protein and the IL-2 protein, or the IL-2 protein and the CD80 protein, are linked to the N-terminus and C-terminus of the linker or carrier, respectively, using the Fc domain as a linker or carrier. The bond between the N-terminus or C-terminus of the Fc domain and CD-80 or IL-2 can be achieved by a linker peptide, if necessary.

[0059] Specifically, the fusion protein may be comprised of the following structural formula (I) or (II): N'-X-[linker (1)] n -Fc domain-[linker (2)] m -Y-C'(I) N'-Y-[linker (1)] n -Fc domain-[linker (2)] m -X-C'(II) wherein in structural formulas (I) and (II): N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is the CD80 protein, Y is the IL-2 protein, Linkers (1) and (2) are peptide linkers; n and m each independently represent 0 or 1.

[0060] Preferably, the fusion protein may have structural formula (I). The IL-2 protein is as described above. The CD80 protein is as described above. In one embodiment, the IL-2 protein may be an IL-2 variant having one to five amino acid substitutions compared to wild-type IL-2. The CD80 protein may be a fragment obtained by truncating up to about 34 consecutive amino acid residues from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having binding activity to the T cell surface receptors CTLA-4 and CD28.

[0061] Specifically, the fusion protein can have the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. According to another embodiment, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30, where identity is, for example, percent homology, and can be determined by homology comparison software such as the BlastN software from the National Center of Biotechnology Information (NCBI).

[0062] A peptide linker (1) may be included between the CD80 protein and the Fc domain. The peptide linker (1) may consist of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 consecutive amino acids. In one embodiment, the peptide linker (1) may consist of 30 amino acids. The peptide linker (1) may also contain at least one cysteine. Specifically, the peptide linker (1) may contain one, two, or three cysteines. The peptide linker (1) may also be derived from an immunoglobulin hinge. In one embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3.

[0063] The peptide linker (2) can consist of 1 to 50 consecutive amino acids, 3 to 30 consecutive amino acids, or 5 to 15 consecutive amino acids. In one embodiment, the peptide linker (2) is (G4S) n (wherein n is an integer of 1 to 10), where (G4S) n In the formula (I), n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO:5.

[0064] In another aspect of the present invention, there is provided a dimer obtained by combining two fusion proteins, each comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, wherein the fusion protein comprising IL-2 or a variant thereof and CD80 or a fragment thereof is as described above.

[0065] Here, the bond between the fusion proteins constituting the dimer can be achieved by, but is not limited to, a disulfide bond formed by cysteines present in the linker. The fusion proteins constituting the dimer may be the same fusion protein or different fusion proteins. Preferably, the dimer may be a homodimer. One embodiment of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO:9.

[0066] The pharmaceutical composition for enhancing radiation therapy of the present invention, which comprises a fusion protein of IL-2 protein and CD80 protein, may be additionally administered in combination with other anticancer drugs, thereby further enhancing the effect of radiation therapy on cancer.

[0067] The anti-cancer agent may be a chemotherapeutic anti-cancer agent, a targeted anti-cancer agent or an immunological anti-cancer agent.

[0068] Specifically, a "chemotherapeutic anti-cancer agent" can be, but is not limited to, an alkylating agent, a microtubule inhibitor, an antimetabolite, or a topoisomerase inhibitor.

[0069] The alkylating agent may be, but is not limited to, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, altretamine, procarbazine, busulfan, streptozocin, carmustine, iomustine, dacarbazine, cisplatin, carboplatin, or oxaliplatin. The microtubule inhibitor may be, but is not limited to, docetaxel, vinblastine, Oncovin, or vinorelbine. The antimetabolite may be, but is not limited to, fluorouracil, capecitabine, cytarabine, gemcitabine, fludarabine, methotrexate, pemetrexed, or mercaptopurine. The topoisomerase inhibitor may be, but is not limited to, hycamtin, camptosar, bepesid, taxol, bleomycin, adriamycin, or cerbidine.

[0070] In addition, "targeted anticancer drugs" include trastuzumab, pertuzumab, panitumumab, cetuximab, bevacizumab, ramucirumab, aflibercept, rituximab, obinutuzumab, daratumumab, denosumab, ibrutinib, dasatinib, nilotinib, imatinib, bosutinib, osimertinib, erlotinib, gefitinib, and nintedanib. These include, but are not limited to, sunitinib, sorafenib, cabozantinib, lenvatinib, regorafenib, axitinib, pazopanib, cabozantinib, trametinib, dabrafenib, abemaciclib, palbociclib, lenalidomide, ruxolitinib, alectinib, crizotinib, olaparib, or venetoclax.

[0071] Furthermore, "immune anti-cancer agents" include, but are not limited to, immune checkpoint inhibitors, immune cell therapy agents (e.g., CAR-T), antibody-drug conjugates (ADCs), bispecific antibodies, anti-cancer viruses, and anti-cancer vaccines.

[0072] The "immune checkpoint inhibitor" may be an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-TIM3 antibody, or anti-LAG3 antibody. The anti-PD-1 antibody may be pembrolizumab, nivolumab, or cemiplimab. The anti-PD-L1 antibody may be, but is not limited to, atezolizumab, avelumab, or durvalumab. The anti-CTLA-4 antibody may be ipilimumab or tremelimumab. The anti-TIM3 antibody may be MBG452. And the anti-LAG3 antibody may be, but is not limited to, BMS-986016 or LAG525. The immune cell therapy agent may be, but is not limited to, tisagenlecleucel or axicabtagene ciloleucel. The ADC can be, but is not limited to, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, or eribulin mesylate. The bispecific antibody can be, but is not limited to, blinatumomab, the anti-cancer virus can be, but is not limited to, talimogene laherparepvec, and the anti-cancer vaccine can be, but is not limited to, sipuleucel-T.

[0073] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Carriers may include distilled water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0074] Specifically, the pharmaceutical composition can be prepared as a parenteral formulation containing a pharmaceutically acceptable carrier and administered by a conventional route known in the art. Here, "pharmaceutically acceptable" means that the activity of the active ingredient is not inhibited and the toxicity is not greater than that acceptable for the subject to which it is applied (prescribed).

[0075] When the pharmaceutical composition is prepared as a parenteral preparation, it can be formulated into injections, transdermal preparations, nasal inhalants, and suppositories with a suitable carrier according to methods known in the art.When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or a mixture thereof can be used as a suitable carrier, and preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterile water for injection, isotonic solutions such as 5% dextrose, etc.The formulation of pharmaceutical compositions is known in the art, and may be specifically referred to in Remington's Pharmaceutical Sciences (19th ed., 1995), etc., which are incorporated herein by reference.

[0076] On the other hand, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "administration" means introducing a predetermined substance into a subject by an appropriate method, and the route of administration of the composition may be any common route as long as it can reach the target tissue. It may be, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0077] The term "subject" refers to all animals, including humans, rats, mice, livestock, etc. Preferably, the subject may be a mammal, including a human.

[0078] The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment and which does not cause side effects. Effective dosage levels can be readily determined by one skilled in the art depending on several factors, including the patient's sex, age, weight, and health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method and time of administration, the route of administration, the excretion rate, the duration of treatment, the combination or concomitant drug, and other factors well known in the medical field. The daily dose can range from 0.01 μg / kg to 10 g / kg, or from 0.01 mg / kg to 1 g / kg. Administration can be once a day or several times a day. Such dosages should not be construed as limiting the scope of the present invention in any respect.

[0079] Radiation therapy In another aspect of the present invention, there is provided a method for radiotherapy of cancer, comprising irradiating a cancer site in a non-human mammal suffering from cancer with radiation, and administering to the mammal a pharmaceutical composition for enhancing radiotherapy according to the present invention.

[0080] As used herein, the term "irradiation" refers to a localized treatment method that damages the DNA of malignant cells by irradiating them with radiation. Normal cells have a greater ability to repair this damage than tumor cells. Irradiation refers to a treatment that takes advantage of this difference and includes treatments that use radiation in the conventional sense.

[0081] Radiation therapy can be divided into definitive, adjuvant, and palliative radiation therapy depending on the type. Definitive radiation therapy aims for complete cure when tumors are relatively localized and have no distant metastasis. Adjuvant radiation therapy is used to prevent local recurrence after surgery. By combining radiation therapy with adjuvant radiation therapy, it can not only prevent recurrence but also narrow the surgical area, preserving tissue function. Palliative radiation therapy is used to relieve symptoms caused by cancer. Radiation is a treatment that uses high-energy rays to kill cancer cells. However, because radiation affects not only cancer cells but also surrounding normal tissue, side effects can occur. Examples include skin changes, hair loss, nausea and vomiting, diarrhea, mucositis / esophagitis, dry mouth, and changes in reproductive function.

[0082] Radiation can be administered to the whole body or part of the body at a dose of 0.1 to 100 Gy. Specifically, the dose of radiation is 0.1 to 100 Gy, 0.5 to 90 Gy, 0.7 to 80 Gy, or 0.9 to 70 Gy, preferably 1 to 60 Gy, but is not limited to these. Radiation can be administered for 1 to 26 weeks, but is not limited to these.

[0083] The pharmaceutical composition for enhancing radiotherapy of the present invention can be administered in combination with radiation during cancer treatment to obtain the effect of enhancing cancer radiotherapy, and "combined administration" means that radiation is administered simultaneously during anti-cancer therapy for treating various types of cancer cells.

[0084] As used herein, the term "treatment" can be used to encompass both therapeutic and prophylactic treatment. In this context, prevention can be used to mean alleviating or mitigating a pathological condition or disease in a subject. "Treatment" also includes any form of administration or application to treat disease in mammals, including humans. This term includes inhibiting or slowing the progression of a disease or disorder; restoring or repairing damaged or lost function, thereby partially or completely alleviating the disease; or stimulating an insufficient process; or alleviating serious disease.

[0085] In one aspect, the term "treatment" can include, but is not limited to, any action that ameliorates or benefits the symptoms of cancer by administering radiation.

[0086] In one embodiment, the term "prevention" includes, but is not limited to, any action that prevents, inhibits, or delays the symptoms of cancer using the pharmaceutical compositions of the present invention.

[0087] In one embodiment, when radiation is administered before or after the administration of the pharmaceutical composition for enhancing radiotherapy of the present invention, the effect of radiotherapy can be significantly enhanced due to a synergistic effect, and further, resistance to anticancer drugs, or metastasis or recurrence of cancer can be prevented.

[0088] The pharmaceutical composition of the present invention may be administered before or after radiation exposure, provided that the effects of the pharmaceutical composition and radiation exposure interact with each other.

[0089] The administration period of the pharmaceutical composition can be increased or decreased as appropriate depending on the type of cancer, the stage of cancer, the administration route, sex, age, body weight, etc. Also, a daily dose may be administered periodically before or after radiation exposure, or a large dose may be administered intensively over a short period of time. Specifically, the pharmaceutical composition may be administered 1 to 20 times, 1 to 18 times, 1 to 15 times, 1 to 10 times, 1 to 8 times, 1 to 5 times, or 2 to 3 times per week, but is not limited to these.

[0090] On the other hand, the pharmaceutical composition may be administered approximately 6 to 48 hours, approximately 10 to 42 hours, approximately 14 to 36 hours, or approximately 18 to 30 hours, preferably approximately 20 to 28 hours, based on the time of radiation exposure, but is not limited thereto.

[0091] The composition of the present invention may be administered by various oral or parenteral routes, as long as it can reach the target tissue. Specifically, it can be administered by conventional methods via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes.

[0092] The cancer radiotherapy of the present invention involves administering a therapeutically effective amount of the pharmaceutical composition for enhancing radiotherapy according to the present invention. A therapeutically effective amount means an amount that effectively enhances tumor sensitivity in cancer cells to radiation. It will be apparent to those skilled in the art that an appropriate total daily dose can be determined by the attending physician within the scope of sound medical judgment. It is preferable to apply a therapeutically effective amount specific to a particular patient in different ways depending on various factors, including the type and degree of response achieved, the specific composition, including whether other drugs are used if necessary, the patient's age, weight, general health, sex, and diet, the administration time, the administration route, the excretion rate of the composition, the treatment duration, and the radiation dose administered, as well as similar factors well known in the pharmaceutical arts. Therefore, it is preferable to determine an effective amount of the pharmaceutical composition for enhancing radiotherapy suitable for the purpose of the present invention, taking the above into consideration. In addition, in some cases, anti-cancer effects, including those of radiotherapy, can be enhanced by administering a known anti-cancer drug together with the pharmaceutical composition for enhancing radiotherapy according to the present invention.

[0093] In addition, the radiotherapy of the present invention can be applied to any animal capable of increasing radioresistance. Animals include humans, primates, and domestic animals such as cows, pigs, sheep, horses, dogs, and cats. Furthermore, the radiotherapy of the present invention can be used to treat all cancers with increased radioresistance. Preferred cancers include, but are not limited to, gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0094] The radiation therapy of the present invention involves administering the composition of the present invention to a subject having cancer cells or a subject suffering from cancer, where radiation irradiation refers to ionizing radiation, particularly gamma rays emitted by commonly used linear accelerators or radionuclides. When exposed to radiation, ionization occurs in vivo, chemically denaturing nucleic acids, cell membranes, etc., which are essential for cell growth and survival, and causing cancer cell death. Radiation irradiation using radionuclides can be performed externally or internally, and the dosage of anticancer drugs, radiation dose, and intermittent radiation dose can vary depending on a number of factors, such as the type and location of the tumor and the patient's response to chemotherapy or radiation therapy.

[0095] Radiation therapy of the present invention may also include brachytherapy, radionuclide therapy, external beam radiation therapy, hyperthermia (including cryoablation and hyperthermia), radiosurgery, charged particle radiation therapy, neutron therapy, photodynamic therapy, and the like.

[0096] MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention. [Example]

[0097] Preparation Example 1. Preparation of test substance Preparation Example 1.1. Preparation of hCD80-Fc-IL-2 mutant (2M): GI-101 To prepare a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant, a dimer containing the fusion protein of SEQ ID NO: 9, which contains, in this order from the N-terminus, a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) with two amino acid substitutions (R38A, F42A) (SEQ ID NO: 6), was prepared. The specific preparation method was as described in Korean Patent Application Publication No. 10-2020-0032009A. The fusion protein dimer was designated "GI-101."

[0098] Preparation Example 1.2. Preparation of mCD80-Fc-IL-2 mutant (2M): mGI-101 To prepare a fusion protein containing mouse CD80, an Fc domain, and an IL-2 mutant, a dimer containing, in order from the N-terminus, an mCD80 fragment (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 mutant (2M) with two amino acid substitutions (R38A, F42A) (SEQ ID NO: 6) was prepared. The fusion protein dimer was designated "mGI-101."

[0099] Preparation Example 1.3. Preparation of test substance: mGI-101 The frozen test substance is completely thawed at room temperature, and then prepared according to the dosage and volume using PBS as a solvent.After mixing the test substance and PBS, the mixture is gently stirred by hand without vortexing or pipetting, and then administered.The thawed test substance is kept refrigerated until administration.

[0100] Preparation example 1. Preparation of a mouse tumor model Preparation Example 1.1. Preparation of tumor cell lines B16F10 cell line, a mouse melanoma cell line, was purchased from the Korean Cell Line Bank and cultured in Dulbecco's modified MEM medium containing 10% fetal bovine serum (FBS) under conditions of 37°C and 5% CO2.

[0101] Preparation Example 1.2. Quarantine and acclimatization of mice as test subjects Six-week-old female C57BL / 6 mice were purchased from Orient Bio. Inspection and quarantine were performed by observing the animals' appearance and referring to the microbiological monitoring report of the test system provided by the supplier at the time of acquisition. Animals with normal appearance were transported to the breeding area and allowed to acclimate for 7 days in the animal room where the test was performed. During the 7-day isolation and acclimatization period, their health status was evaluated and checked for suitability for the experiment to select healthy mice.

[0102] Preparation Example 1.3. Identification of Mouse Subjects and Breeding Boxes When obtaining experimental animals, the mice were marked with a red oil-based pen on their tails (tail marking), and temporary subject identification cards (study name, subject number, breeding period) were attached to the breeding boxes during the quarantine and acclimation periods. When dividing the mice into groups, the mice were marked with a black oil-based pen on their tails, and subject identification cards (study name, group information, subject number, sex, breeding time, and administration period) were attached to each cage.

[0103] Preparation Example 1.4. Transplantation of tumor cell lines After the quarantine and acclimation period, selected healthy C57BL / 6 mice were inoculated with 5 × 10 6 Diluted in PBS to a concentration of 5 x 10 cells / mL, 100 μl (5 x 10 5 B16F10 cells (cells) were implanted subcutaneously into both flanks of each subject, except for the right hind flank where cells were implanted, and the left front flank where cells were implanted.

[0104] Preparation Example 1.5. Grouping of mouse tumor models Approximately 7 days before and after implantation of the B16F10 tumor cell line of Preparation Example 1.4, the tumor size of the C57BL / 6 mice was measured using an electronic caliper, as described above, and the mice were divided into a total of 5 groups, with 10 mice per group. Specifically, the tumor volume of the majority of subjects implanted with the tumor cell line was approximately 50-120 mm. 3At the time of reaching the target size, the tumors implanted on both sides of one subject were measured, and the subjects were divided into groups according to the Z-array method based on the average tumor size.

[0105] Preparation Example 1.6. Raising Mice as Experimental Subjects The test subjects, C57BL / 6 mice, were housed in polycarbonate cages measuring 200 (width, mm) x 260 (depth, mm) x 130 (height, mm), with five mice per box. The temperature conditions were 20°C to 25°C and humidity 50±20%. The cage was ventilated 10 to 15 times per hour, maintaining a 12-hour day-night cycle. The illuminance was 150 to 300 lux.

[0106] During rearing, the animals were allowed free access to food and water. Tap water was filtered through a flow-through sterilizer, irradiated with ultraviolet light, and provided in polycarbonate drinking water bottles (250 mL). The rearing boxes and feeders were replaced once a week, and the water bottles were replaced twice a week. Rearing materials were washed with disinfectant and then sterilized in a UV sterilizer before being reused.

[0107] Experimental Example 1. Therapeutic effect of combined irradiation and mGI-101 in a mouse tumor model Experimental Example 1.1. Intraperitoneal administration of mGI-101 and irradiation The test substance, mGI-101 prepared in Preparation Example 1, was administered intraperitoneally to mouse tumor models at 8 weeks of age at the time of administration. Specifically, mGI-101 was first administered to experimental groups G2 and G4 on the day of grouping (day 1). For experimental group G5, mGI-101 was first administered on day 4, followed by radiation the following day. Subsequently, experimental groups G2, G4, and G5 received additional intraperitoneal administration of mGI-101 once a week, for a total of two doses. For control group G1, PBS was administered on the day of grouping (day 1), followed by additional intraperitoneal administration of PBS once a week, for a total of two doses.

[0108] For radiation, the mice were placed on their right side under injection anesthesia so that the right side of the tumor implantation site could be irradiated. Radiation was administered once on day 3 at an intensity of 6 Gy. The results are summarized in Table 1 below. [Table 1]

[0109] Experimental Example 1.2. Measurement of tumor volume and tumor growth inhibition in mouse tumor models After intraperitoneal injection of mGI-101 and irradiation, the long axis (maximum length, L) and short axis (perpendicular width, W) of tumors implanted in both flanks of mice were measured twice weekly using electronic calipers. These measurements were then used to calculate tumor volume. [Formula 1] Tumor volume (mm 3 ) = [L (mm)×W (mm)×W (mm)]×0.5

[0110] On the other hand, tumor growth inhibition (TGI) was calculated by substituting the values ​​into the following [Equation 2]. [Formula 2] TGI = (1-(T i -T0) / (V i -V0))×100 T i = Tumor volume before administration of experimental group T0 = ​​tumor volume after administration of the experimental group V i = Tumor volume before administration of the control group V0 = tumor volume after treatment with the control group The tumor volume of each subject before administration was measured at the time of group division.

[0111] Experimental Example 1.3. Tumor growth inhibition effect of radiation exposure and mGI-101 administration For irradiated right-sided tumors, G3 significantly inhibited tumor growth on day 15 compared with G1 and G2, and both G4 and G5 significantly inhibited tumor growth on days 11 and 15 compared with G1 and G2 (Figures 1a and 2a to 2e).

[0112] For left-sided tumors that were not irradiated, G2 significantly inhibited tumor growth compared to G1 on day 15. G4 significantly inhibited tumor growth compared to G1 and G3 on day 15. G5 significantly inhibited tumor growth compared to G1 and G3 on day 11, and compared to G1, G2, and G3 on day 15 (Figures 1b and 3a to 3e).

[0113] On average for both tumors, G2 significantly inhibited tumor growth compared to G1 on day 15. Both G4 and G5 significantly inhibited tumor growth compared to G1 on day 11. Also, both G4 and G5 significantly inhibited tumor growth compared to G1, G2, and G3 on day 15 (Figure 1c).

[0114] Experimental Example 1.4. Analysis of tumor growth inhibition by irradiation and mGI-101 administration The tumor growth inhibition (TGI) of the irradiated right-sided tumors was as follows: G1: 1 mouse showed ≥30% inhibition, 1 mouse showed ≥50% inhibition, and 0 mice showed ≥80% inhibition. G2: 4 mice showed ≥30% inhibition, 3 mice showed ≥50% inhibition, and 0 mice showed ≥80% inhibition. G3: 5 mice showed ≥30% inhibition, 4 mice showed ≥50% inhibition, and 2 mice showed ≥80% inhibition. G4: 7 mice showed ≥30% inhibition, 6 mice showed ≥50% inhibition, and 2 mice showed ≥80% inhibition. G5: 10 mice showed ≥30% inhibition, 10 mice showed ≥50% inhibition, and 4 mice showed ≥80% inhibition (Figure 4a and Table 2).

[0115] [Table 2]

[0116] For tumor growth inhibition in the unirradiated left side, G1 showed 0 mice with ≥30% inhibition, 0 mice with ≥50% inhibition, and 0 mice with ≥80% inhibition. G2 showed ≥30% inhibition in 5 mice, ≥50% inhibition in 2 mice, and ≥80% inhibition in 2 mice. G3 showed ≥30% inhibition in 0 mice, ≥50% inhibition in 0 mice, and ≥80% inhibition in 0 mice. G4 showed ≥30% inhibition in 6 mice, ≥50% inhibition in 3 mice, and ≥80% inhibition in 0 mice. G5 showed ≥30% inhibition in 8 mice, ≥50% inhibition in 6 mice, and ≥80% inhibition in 4 mice (Figure 4b and Table 3).

[0117] [Table 3]

[0118] For mean tumor growth inhibition of both tumors, G1 showed ≥30% inhibition in 1 mouse, ≥50% inhibition in 0 mice, and ≥80% inhibition in 0 mice. G2 showed ≥30% inhibition in 4 mice, ≥50% inhibition in 2 mice, and ≥80% inhibition in 2 mice. G3 showed ≥30% inhibition in 1 mouse, ≥50% inhibition in 0 mice, and ≥80% inhibition in 0 mice. G4 showed ≥30% inhibition in 6 mice, ≥50% inhibition in 4 mice, and ≥80% inhibition in 0 mice. G5 showed ≥30% inhibition in 10 mice, ≥50% inhibition in 7 mice, and ≥80% inhibition in 4 mice (Figure 4c and Table 4).

[0119] [Table 4]

[0120] These results demonstrate that the combined use of GI-101 and radiation therapy exerts a synergistic anticancer effect not only at the irradiated tumor site, but also at distant tumor sites that have not been irradiated. Further aspects of the present invention are described below: [Section 1] A pharmaceutical composition for enhancing cancer radiotherapy, comprising a fusion protein dimer comprising an IL-2 protein and a CD80 protein. [Section 2] The pharmaceutical composition of claim 1, wherein the IL-2 protein and the CD80 protein are linked via a linker. [Section 3] Item 1. The pharmaceutical composition of Item 1, wherein the IL-2 protein has the amino acid sequence of SEQ ID NO:10. [Section 4] Item 1. The pharmaceutical composition according to Item 1, wherein the IL-2 protein is an IL-2 mutant. [Section 5] Item 5. The pharmaceutical composition according to Item 4, wherein the IL-2 mutant is a mutant in which at least one amino acid selected from the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 is substituted. [Section 6] Item 5. The pharmaceutical composition according to Item 4, wherein the IL-2 mutant is obtained by at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R and L72G in the amino acid sequence of SEQ ID NO: 10. [Section 7] Item 5. The pharmaceutical composition according to Item 4, wherein the IL-2 mutant is obtained by combining any one of the following substitutions (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R (d) R38A / F42A / L72G. [Section 8] Item 5. The pharmaceutical composition of Item 4, wherein the IL-2 mutant has the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24. [Section 9] The pharmaceutical composition of item 1, wherein CD80 has the amino acid sequence of SEQ ID NO:11. [Section 10] Item 1. The pharmaceutical composition according to Item 1, wherein the CD80 protein is a fragment of CD80. [Section 11] Item 11. The pharmaceutical composition of Item 10, wherein the CD80 fragment consists of amino acids 35 to 242 of the amino acid sequence of SEQ ID NO:11. [Section 12] Item 3. The pharmaceutical composition according to Item 2, wherein the linker is albumin or an Fc domain of an immunoglobulin. [Section 13] Item 13. The pharmaceutical composition according to Item 12, wherein the Fc domain is a wild-type or mutant Fc domain. [Section 14] Item 13. The pharmaceutical composition of Item 12, wherein the Fc domain has the amino acid sequence of SEQ ID NO:4. [Section 15] Item 14. The pharmaceutical composition of Item 13, wherein the Fc domain mutant has the amino acid sequence of SEQ ID NO: 12. [Section 16] The pharmaceutical composition according to item 1, wherein the fusion protein is represented by the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain-[linker (2)]mY-C'(I) N'-Y-[linker (1)]n-Fc domain-[linker (2)]mX-C'(II) wherein in structural formulas (I) and (II): N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is the CD80 protein, Y is the IL-2 protein, Linkers (1) and (2) are peptide linkers; n and m each independently represent 0 or 1. [Section 17] Item 17. The pharmaceutical composition according to Item 16, wherein the linker (1) is a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3. [Section 18] Item 17. The pharmaceutical composition according to Item 16, wherein the linker (2) is a peptide linker consisting of the amino acid sequence of SEQ ID NO:5. [Section 19] Item 17. The pharmaceutical composition according to Item 16, wherein the fusion protein consists of structural formula (I). [Section 20] Item 1. The pharmaceutical composition according to Item 1, wherein the fusion protein has 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. [Section 21] Item 1. The pharmaceutical composition according to Item 1, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. [Section 22] irradiating a cancer site in a non-human mammal suffering from cancer with radiation; Administering the pharmaceutical composition according to any one of items 1 to 21 to the mammal. Radiation therapy for cancer, including: [Section 23] Item 23. The method according to Item 22, wherein the radiation is administered at a dose of 0.1 Gy to 100 Gy. [Section 24] Item 23. The method according to Item 22, wherein the pharmaceutical composition is administered before or after radiation exposure. [Section 25] Item 25. The method according to Item 24, wherein the pharmaceutical composition is administered 6 to 48 hours before or after radiation exposure. [Section 26] 23. The method of claim 22, wherein the pharmaceutical composition is administered 1 to 20 times per week.

Claims

1. A pharmaceutical composition for enhancing cancer radiotherapy, comprising a fusion protein dimer comprising an IL-2 protein and a CD80 protein.

2. The pharmaceutical composition of claim 1, wherein the IL-2 protein and the CD80 protein are linked via a linker.

3. 2. The pharmaceutical composition of claim 1, wherein the IL-2 protein has the amino acid sequence of SEQ ID NO:

10.

4. The pharmaceutical composition according to claim 1, wherein the IL-2 protein is an IL-2 mutant, and the IL-2 mutant is a mutant in which at least one amino acid selected from the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 is substituted.

5. 5. The pharmaceutical composition according to claim 4, wherein the IL-2 mutant is obtained by at least one substitution in the amino acid sequence of SEQ ID NO: 10 selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

6. The pharmaceutical composition according to claim 4, wherein the IL-2 mutant is obtained by combining any one of the following substitutions (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R (d) R38A / F42A / L72G.

7. The pharmaceutical composition of claim 4, wherein the IL-2 mutant has the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.

8. 2. The pharmaceutical composition of claim 1, wherein CD80 has the amino acid sequence of SEQ ID NO:

11.

9. The pharmaceutical composition of claim 1 , wherein the CD80 protein is a fragment of CD80.

10. The pharmaceutical composition according to claim 9, wherein the fragment of CD80 consists of amino acids 35 to 242 of the amino acid sequence of SEQ ID NO:

11.

11. The pharmaceutical composition of claim 2 , wherein the linker is albumin or an Fc domain of an immunoglobulin.

12. The pharmaceutical composition of claim 11, wherein the Fc domain is a wild-type or mutant Fc domain, and the mutant Fc domain has the amino acid sequence of SEQ ID NO:

12.

13. The pharmaceutical composition of claim 11, wherein the Fc domain has the amino acid sequence of SEQ ID NO:

4.

14. 2. The pharmaceutical composition of claim 1, wherein the fusion protein has the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain-[linker (2)]m-Y-C'(I) N'-Y-[linker (1)]n-Fc domain-[linker (2)]m-X-C'(II) wherein in structural formulas (I) and (II): N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is the CD80 protein, Y is the IL-2 protein, Linkers (1) and (2) are peptide linkers; n and m each independently represent 0 or 1.

15. The pharmaceutical composition according to claim 14, wherein the linker (1) is a peptide linker consisting of the amino acid sequence of SEQ ID NO:

3.

16. The pharmaceutical composition according to claim 14, wherein the linker (2) is a peptide linker consisting of the amino acid sequence of SEQ ID NO:

5.

17. 15. The pharmaceutical composition of claim 14, wherein the fusion protein comprises structural formula (I):

18. 2. The pharmaceutical composition of claim 1, wherein the fusion protein has 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30.

19. 2. The pharmaceutical composition of claim 1, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, malignant melanoma, and lymphoma.

20. irradiating a cancer site in a non-human mammal suffering from cancer with radiation; 20. Administering to said mammal a pharmaceutical composition according to any one of claims 1 to 19. Radiation therapy for cancer, including:

21. 21. The method of claim 20, wherein the radiation is administered at a dose of 0.1 Gy to 100 Gy.

22. 21. The method of claim 20, wherein the pharmaceutical composition is administered before or after radiation.

23. 23. The method of claim 22, wherein the pharmaceutical composition is administered 6 to 48 hours before or after the time of radiation administration.

24. 21. The method of claim 20, wherein the pharmaceutical composition is administered from 1 to 20 times per week.

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