Antibody-based conjugates for enhancing the therapeutic effect of targeted therapeutics

Abstract

The present invention relates to a conjugate comprising an antibody for treating cancer, a linker, and a block copolymer comprising PEO and PPO, and further comprising a low molecular weight compound. The conjugate has excellent cancer cell targeting ability, and can effectively increase cancer cells by increasing the half-life of the antibody, and thus can be effectively used for treating cancer.

Description

Technical Field

[0001] This invention relates to conjugates capable of enhancing the therapeutic effect of targeted therapeutic agents, the conjugates comprising antibodies, linkers, and block copolymers, the block copolymers comprising poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), and conjugates further incorporating low molecular weight compounds into the above conjugates. Background Technology

[0002] Since traditional chemotherapy is one of the fundamental methods for treating cancer, and the benefit of most chemotherapeutic anticancer agents used in anticancer clinical trials lies in their ability to target the cell cycle, toxicity depends on the degree of cancer cell proliferation. Furthermore, chemotherapeutic anticancer agents are typically used at near-maximum tolerated doses to achieve clinical therapeutic effects, and treatment using various drugs has become the standard therapy for cancer. However, anticancer agents only kill rapidly proliferating cells and cannot distinguish between normal cells and tumor cells or tumor tissue. Due to these drawbacks, systemic toxicity and cytotoxicity occur, and long-term treatment can lead to resistance to anticancer agents. Therefore, there is an urgent need for improved therapies using cytotoxic drugs that target and kill cancer cells specifically.

[0003] Unlike cytotoxic drugs, monoclonal antibodies that bind to specific antigens on the surface of tumor cells offer an alternative therapeutic approach that reduces systemic toxicity because they bind specifically to the tumor. In fact, antigens preferentially or specifically expressed on the surface of cancer cells have been identified through expression profiling studies, and monoclonal antibodies that specifically bind to tumor-associated antigens can be designed and manufactured. Some form of targeted therapy, delivered at the molecular level, blocks cancer cell proliferation by blocking signals associated with oncogenicity and tumor growth. Cancer cell-targeted therapies promise to be more effective than existing methods without harming normal cells. These monoclonal antibodies are under ongoing development, and some have already been approved by the U.S. Food and Drug Administration (FDA). Examples of approved monoclonal antibodies include rituximab, trastuzumab, alemtuzumab, cetuximab, bevacizumab, and ipilimumab.

[0004] However, only a few antibodies are useful for cancer treatment because most are not very effective at killing cancer cells. For antibody-based targeted anticancer agents to be used effectively, they need to induce effective cancer cell killing through a single injection and maintain antigen-antibody binding with strong affinity over a long period. Summary of the Invention

[0005] Technical issues

[0006] In these cases, the inventors have investigated a method that can effectively kill cancer cells with a single injection by inducing antibody-based targeted anticancer drugs and maintain antigen-antibody binding with strong binding force for a long time, thereby making it effective.

[0007] As a result, the inventors prepared a conjugate in which the linker and the block copolymer containing PEO and PPO are linked to a monoclonal antibody that selectively binds to a target factor overexpressed in cancer cells, and demonstrated that this conjugate can be retained in vivo for a longer period of time and induce better anticancer effects than existing therapeutic agents through interaction with the cell membrane.

[0008] Furthermore, the inventors prepared an additional conjugate in which a low molecular weight compound was conjugated to one end of the conjugate, and demonstrated that the additionally prepared conjugate overcame the limitations of monoclonal antibodies and low molecular weight compounds, and that due to the synergistic effect of the block copolymer containing PEO and PPO, it could induce better anticancer effects, thus completing the present invention.

[0009] Therefore, one object of the present invention is to provide an antibody-based conjugate for treating cancer, having an enhanced in vivo half-life and therapeutic effect, and a pharmaceutical composition for treating cancer comprising the conjugate.

[0010] Technical solutions

[0011] To achieve this objective, one aspect of the present invention provides a conjugate comprising: (a) an antibody for treating cancer; (b) a linker covalently linked to the antibody; and (c) a block copolymer comprising PEO and PPO covalently linked to the linker.

[0012] As used in this article, the term "antibody for cancer therapy" refers to an antibody used to treat cancer, and can be classified as an animal-derived antibody, chimeric antibody, humanized antibody, or human antibody based on its origin.

[0013] Animal-derived antibodies are antibodies produced by injecting antigens into non-human animals. Chimeric antibodies are those in which the constant region of the animal-derived antibody that induces the greatest immunogenicity is replaced by the constant region of the human antibody.

[0014] Humanized antibodies are antibodies in which the rest of the sequence of an animal-derived antibody, except for the complementarity-determining region (CDR) sequence (which is the antigen-binding site), is replaced by a human antibody sequence, thereby eliminating the immunogenicity of the animal-derived antibody.

[0015] In addition, human antibodies are produced by selecting antibodies against specific antigens using phage display technology on a human antibody library, and then introducing the corresponding antibody genes into mice.

[0016] In addition, antibodies used to treat cancer can be classified into receptor-targeting antibodies and immune checkpoint inhibitors or immune checkpoint blockers based on their mechanism of action.

[0017] Receptor-targeting antibodies are antibodies that specifically bind to specific receptors on the surface of cancer cells. According to an exemplary embodiment of the present invention, the specific receptor may be selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and vascular endothelial growth factor receptor 2 (VEGFR2).

[0018] Immune checkpoint inhibitors maintain the immune function of T cells by preventing programmed death ligand 1 (PD-L1) in cancer cells from binding to PD-1 on T cells.

[0019] Epidermal growth factor receptor (EGFR) is a group of cell membrane receptors that regulate cell growth, division, and death. EGFR is a 170 kDa type 1 membrane protein known to be overexpressed in various types of tumors (solid tumors such as lung cancer, head and neck tumors, colorectal cancer, pancreatic cancer, and breast cancer) due to its amplification and expression. Tumors overexpressing EGFR tend to be more aggressive, more metastatic, and more resistant to anticancer therapies, thus having a worse prognosis. To overcome this, targeted therapies have been developed using antibodies that target EGFR. Antibodies bind to EGFR to inhibit the binding of EGFR, thereby suppressing cancer cell signaling and growth to treat cancer.

[0020] Cetuximab is a chimeric antibody that binds to the epidermal growth factor receptor (EGFR) on the cell surface, interfering with ligand binding, thereby inhibiting receptor activation, increasing receptor internalization within the cell, and reducing receptor expression. As a result, cetuximab arrests the cell cycle at G0-G1, induces Rb gene dephosphorylation, inhibits cell proliferation, induces apoptosis, and inhibits the production of angiogenic factors (such as VEGF).

[0021] Human epidermal growth factor receptor 2 (HER2) is a tyrosine-phosphorylated growth factor receptor with a molecular weight of 185 kDa, located on the cell surface. Although HER2 lacks a ligand-binding site in its molecule, it forms dimers with other receptors such as EGFR, HER3, and HER4. Receptor-ligand conjugation manifests effects such as cell proliferation, cell survival, metastasis, and angiogenesis through various cell signaling pathways. HER2 is overexpressed in various cancer types, including 20-30% of breast, gastric, ovarian, lung, and prostate cancers. Its overexpression further enhances its function in promoting cell survival, proliferation, angiogenesis, and metastasis.

[0022] Trastuzumab is a humanized antibody, a recombinant human monoclonal antibody targeting the extracellular domain of the HER2 protein, and was the first such antibody to receive FDA approval. It induces apoptosis by inhibiting signaling pathways in HER2-overexpressing tumor cells and suppressing the intracellular G1 / S cell cycle, and increases susceptibility to anticancer agents such as platinum-based drugs, taxanes, doxorubicin, and cyclophosphamide. Furthermore, HER2 receptor activity decreases when the antibody binds to the extracellular domain of HER2.

[0023] Programmed death-ligand 1 (PD-L1) is a 40 kDa type I transmembrane protein that is highly expressed in cancer cells. PD-L1 plays a role in evading immune cell attack by interacting with the PD-1 receptor on the surface of T cells. Their interaction simultaneously reduces apoptosis in regulatory cells and decreases the proliferation of antigen-specific T cells in lymph nodes, allowing cancer cells to evade anti-cancer immune responses.

[0024] Avelumab is a human antibody, an immune checkpoint inhibitor, and a human monoclonal antibody that targets PD-L1 overexpressed in cancer cells. This antibody activates the immunosuppressive environment formed in cancer cells by blocking the interaction between PD-1 on T cells and PD-L1 on cancer cells. The advantage of avelumab is that it exhibits anti-cancer effects by blocking the activity of the PD-L1 protein (which acts as an immune checkpoint), regardless of the type of cancer that expresses PD-L1. Avelumab is known to be effective against non-small cell lung cancer, melanoma, colorectal cancer, kidney cancer, and hepatocellular carcinoma.

[0025] Vascular endothelial growth factor receptor 2 (VEGFR2) is expressed in vascular and lymphatic endothelial cells and binds to VEGF-A, VEGF-E, etc., to promote angiogenesis and vascular endothelial cell migration.

[0026] Ramucirumab is a human antibody, a VEGFR2 antagonist that blocks ligand binding and thereby inhibits receptor activation. It is used to treat colorectal cancer, non-small cell lung cancer, and gastric cancer.

[0027] As used herein, the term "connector" refers to a substance that links an antibody for targeting cancer to a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer. According to an exemplary embodiment of the invention, the connector may be selected from the group consisting of maleimide, succinic anhydride, and N-hydroxysuccinimide ester, and may preferably be maleimide or succinic anhydride.

[0028] As used herein, the term "block copolymer comprising PEO and PPO (hereinafter referred to as PEO-PPO block copolymer)" means a copolymer that alternately comprises polypropylene oxide blocks and polyethylene oxide blocks.

[0029] According to an exemplary embodiment of the present invention, the PEO-PPO block copolymer may be a PEO-PPO-PEO block copolymer, which is a terpolymer that alternately contains polypropylene oxide blocks and polyethylene oxide blocks.

[0030] According to an exemplary embodiment of the present invention, the PEO-PPO block copolymer may be selected from the group consisting of poloxamer 68, poloxamer 124, poloxamer 127, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. Preferably, the PEO-PPO-PEO block copolymer may be poloxamer 188. Or Polosham 407

[0031] In this invention, the antibody and the linker for treating cancer, as well as the linker and the PEO-PPO-PEO block copolymer, can each be covalently linked. The covalent bond can be selected from the group consisting of amide bonds, carbonyl bonds, ester bonds, thioester bonds, sulfonamide bonds, and carbamate bonds.

[0032]

[0033] In an exemplary embodiment of the present invention, the conjugate can be prepared by first combining a linker and a PEO-PPO block copolymer, and then further combining an antibody for targeting cancer, or by first combining an antibody for targeting cancer and a linker, and then combining a PEO-PPO block copolymer.

[0034] Furthermore, according to an exemplary embodiment of the invention, the conjugate may also contain a low molecular weight compound at one end, and preferably, the low molecular weight compound may be bound to the end of the PEO-PPO block copolymer.

[0035] In this invention, the low molecular weight compound can be an anticancer agent or a photosensitizer. The anticancer agent can be a cytotoxic anticancer agent, and the photosensitizer can be selected from the group consisting of dihydroporphyrins, bacteriochlorins, porphyrins, porphyrines, and phthalocyanines. For example, mesotetraaminophenylporphyrin, zinc protoporphyrin, protoporphyrin, and hematoporphyrin can be used as porphyrin photosensitizers, and aluminum phthalocyanine can be used as phthalocyanine photosensitizers.

[0036] According to an exemplary embodiment of the present invention, the dihydroporphyrin photosensitizer may be dihydroporphyrin e6. As described above, dihydroporphyrin e6 can be bound to the ends of the PEO-PPO block copolymer.

[0037] The inventors have demonstrated that conjugates linking cancer-targeting antibodies, linkers, and PEO-PPO block copolymers enhance the antibody's ability to target cancer cells to induce effective apoptosis and increase the antibody's in vivo half-life. Furthermore, they have demonstrated that these conjugates can effectively kill cancer cells, as, in addition to the aforementioned effects, the further binding of low molecular weight compounds to the conjugates exhibits a cancer-killing effect due to photosensitizer properties.

[0038] Therefore, another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising the conjugate as an active ingredient. Since either an antibody-connector-PPO block copolymer conjugate or an antibody-connector-PEO-PPO block copolymer-low molecular weight compound conjugate for treating cancer is used as an active ingredient in the pharmaceutical composition, descriptions of any overlap between the two will be omitted to avoid overcomplicating the specification.

[0039] In addition to the active ingredient, the pharmaceutical composition of the present invention may also include a pharmaceutically acceptable carrier. In this case, a pharmaceutically acceptable carrier is a carrier commonly used in the formulation process, examples of which include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. Furthermore, in addition to the components described above, the pharmaceutically acceptable carrier may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0040] Depending on the desired method, the pharmaceutical compositions of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, or intraperitoneally, or topically). When the active ingredients of the present invention are formulated into formulations for oral administration such as tablets, capsules, chewable tablets, powders, liquids, and suspensions, they may include binders (such as rubber arabic, corn starch, microcrystalline cellulose, or gelatin), excipients (such as calcium diphosphate or lactose), disintegrants (such as alginate, corn starch, or potato starch), lubricants (such as magnesium stearate), sweeteners (such as sucrose or saccharin), and flavoring agents (such as peppermint, methyl salicylate, or fruit flavorings).

[0041] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts. In this invention, a "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level can be determined based on factors including the patient's disease type, disease severity, drug activity, drug sensitivity, timing of administration, route of administration, excretion rate, treatment duration, and concurrently used drugs, as well as other factors known in the medical field. The pharmaceutical compositions according to the invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with therapeutic agents in the related art, and can be administered in single or multiple doses. Considering all the foregoing factors, it is important to administer the composition in the minimum amount that yields the maximum effect without any side effects, and this amount can be readily determined by those skilled in the art.

[0042] Beneficial effects

[0043] Containing antibodies, linkers, and block copolymers containing PEO and PPO for cancer treatment, as well as conjugates further containing low molecular weight compounds, these conjugates exhibit excellent cancer cell targeting capabilities and can effectively kill cancer cells by increasing the half-life of the antibodies, thus making them effective for cancer treatment. Attached Figure Description

[0044] Figure 1 This demonstrates maleimide-Planick F68 (Mal-PF68) as confirmed by an exemplary embodiment of the present invention. 1 Results of H-NMR spectroscopy.

[0045] Figure 2 This demonstrates maleimide-Planick F127 (Mal-PF127) as confirmed by an exemplary embodiment of the present invention. 1 Results of H-NMR spectroscopy.

[0046] Figure 3 This demonstrates succinyl-Plancic F68 (Suc-PF68) according to an exemplary embodiment of the present invention. 1Results of H-NMR spectroscopy.

[0047] Figure 4 This demonstrates succinyl-Plancic F127 (Suc-PF127) according to an exemplary embodiment of the present invention. 1 Results of H-NMR spectroscopy.

[0048] Figure 5 This demonstrates the confirmation of maleimide-polyethylene glycol 2k according to an exemplary embodiment of the present invention. 1 Results of H-NMR spectroscopy.

[0049] Figure 6 This demonstrates the confirmation of maleimide-polyethylene glycol 6k according to an exemplary embodiment of the present invention. 1 Results of H-NMR spectroscopy.

[0050] Figure 7 The results show the molecular weight of cetuximab-maleimide-Pranic F68 (CTX-Mal-PF68) as confirmed by MALDI-TOF / MS spectroscopy.

[0051] Figure 8 The results show the molecular weights of trastuzumab-maleimide-Planick F68 (TRA-Mal-PF68) and trastuzumab-succinyl-Planick F68 (TRA-Suc-PF68) confirmed by MALDI-TOF / MS spectroscopy.

[0052] Figure 9 The results show the molecular weights of avermab-maleimide-pranic F68 (AVE-Mal-PF68) and avermab-succinyl-pranic F68 (AVE-Suc-PF68) confirmed by MALDI-TOF / MS spectroscopy.

[0053] Figure 10 The results show the molecular weight of ramucirumab-maleimide-Pranic F68 (RAM-Mal-PF68) as confirmed by MALDI-TOF / MS spectroscopy.

[0054] Figure 11The circular dichroism measurements of cetuximab-maleimide-Plancic F68 / F127 conjugates (CTX-Mal-PF68 / PF127) (A), trastuzumab-maleimide-Plancic F68 / F127 conjugates (TRA-Mal-PF68 / PF127) (B), avermectin-maleimide-Plancic F68 / F127 conjugates (AVE-Mal-PF68 / PF127) (C), and ramucirumab-maleimide-Plancic F68 / F127 conjugates (RAM-Mal-PF68 / PF127) (D) are shown.

[0055] Figures 12A to 12D show the results of confirmed cytotoxicity after treatment of normal cell lines that do not express epidermal growth factor receptor (EGFR) with maleimide-Plannic conjugate, maleimide-polyethylene glycol conjugate, cetuximab-maleimide-Plannic conjugate, or cetuximab-maleimide-polyethylene glycol conjugate.

[0056] Figures 13A and 13B show the results confirming cytotoxicity after treatment of EGFR-expressing ovarian cancer cell lines with maleimide-Prännick conjugate, cetuximab, or cetuximab-maleimide-Prännick conjugate.

[0057] Figures 14A to 14D show the results of confirmed cytotoxicity after treatment of normal cell lines that do not express human epidermal growth factor receptor 2 (HER2) with maleimide-Plannic conjugate, maleimide-polyethylene glycol conjugate, trastuzumab-maleimide-Plannic conjugate, or trastuzumab-maleimide-polyethylene glycol conjugate.

[0058] Figures 15A to 15F show the results of confirming cytotoxicity after treatment of HER2-expressing breast cancer cell lines with maleimide-Prännick conjugate and trastuzumab-maleimide-Prännick conjugate.

[0059] Figures 16A to 16D show the results of confirmed cytotoxicity after treatment of normal cell lines that do not express programmed death ligand 1 (PD-L1) with maleimide-Plannic conjugate, maleimide-polyethylene glycol conjugate, avermab-maleimide-Plannic conjugate, or avermab-maleimide-polyethylene glycol conjugate.

[0060] Figures 17A to 17F show the results of confirming cytotoxicity after treatment of PD-L1-expressing cancer cell lines with maleimide-Prännick conjugate, avermab, or avermab-maleimide-Prännick conjugate.

[0061] Figures 18A to 18D show the results of confirmed cytotoxicity after treatment of normal cell lines that do not express vascular endothelial growth factor receptor 2 (VEGFR2) with maleimide-Plannic conjugate, maleimide-polyethylene glycol conjugate, ramucirumab-maleimide-Plannic conjugate, or ramucirumab-maleimide-polyethylene glycol conjugate.

[0062] Figures 19A to 19D show the results of confirming cytotoxicity after treatment of PD-L1-expressing cancer cell lines with maleimide-Plannic conjugate, ramucirumab, or ramucirumab-maleimide-Plannic conjugate.

[0063] Figures 20A and 20B show the results confirming cytotoxicity after treatment of HER2-expressing breast cancer cell lines with succinyl-Plancic conjugate, succinyl-polyethylene glycol conjugate, trastuzumab-succinyl-Plancic conjugate, or trastuzumab-succinyl-polyethylene glycol conjugate.

[0064] Figures 21A to 21D show the results of confirming cytotoxicity after treatment of PD-L1-expressing cancer cell lines with succinyl-Plancic conjugate, succinyl-polyethylene glycol conjugate, avermab-succinyl-Plancic conjugate, or avermab-succinyl-polyethylene glycol conjugate.

[0065] Figure 22 The results show the presence or absence of EGFR expression in conjugated target cells by flow cytometry after treatment with maleimide-Prannick F68-dihydroporphyrin e6, maleimide-polyethylene glycol 2K-dihydroporphyrin e6, cetuximab-maleimide-Prannick F68-dihydroporphyrin e6, or cetuximab-maleimide-polyethylene glycol 2K-dihydroporphyrin e6 conjugates.

[0066] Figure 23 The results show the presence or absence of EGFR expression in conjugated target cells after treatment with maleimide-Prannicke F68-dihydroporphyrin e6, maleimide-polyethylene glycol 2K-dihydroporphyrin e6, cetuximab-maleimide-Prannicke F68-dihydroporphyrin e6, or cetuximab-maleimide-polyethylene glycol 2K-dihydroporphyrin e6 conjugates, confirmed by confocal laser scanning microscopy.

[0067] Figures 24A and 24B show the results confirming the in vivo behavior of the conjugates after injection into mice with avermab-dihydroporphyrin e6, avermab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6, or avermab-maleimide-Prannick F68-dihydroporphyrin e6 conjugates.

[0068] Figure 25The behavior of the conjugates in cancer tissues was shown when cetuximab-maleimide-polyethylene glycol 2k-dihydroporphyrin 36 or cetuximab-maleimide-Prannick F68-dihydroporphyrin e6 conjugates were injected after inducing cancer formation in mice.

[0069] Figures 26A and 26B show the results confirming changes in cancer tissue size when cetuximab, cetuximab-maleimide-PEG 2k, cetuximab-maleimide-PEG 6k, cetuximab-maleimide-Planick F68, or cetuximab-maleimide-Planick 127 conjugates were injected into mice after inducing cancer formation.

[0070] Figure 27 The study demonstrated the results of inducing cancer formation in mice, followed by injection of cetuximab, cetuximab-maleimide-PEG 2k, cetuximab-maleimide-PEG 6k, cetuximab-maleimide-Planick F68, or cetuximab-maleimide-Planick 127 conjugates, and confirmed the changes in cancer tissue size in each mouse.

[0071] Figure 28 Results showed that the conjugates of cetuximab-maleimide-polyethylene glycol / Planick-dihydroporphyrin e6 (A) or trastuzumab-maleimide-polyethylene glycol / Planick-dihydroporphyrin e6 (B) can generate singlet oxygen in aqueous solution.

[0072] Figures 29A to 29C show the results confirming the cytotoxicity of the cetuximab-maleimide-polyethylene glycol / Prannicke-dihydroporphyrin e6 conjugate itself in NIH-3T3(A), SKOV-3(B), and A-2780(C) cells.

[0073] Figure 30 The results demonstrate the photodynamic cytotoxicity of the cetuximab-maleimide-polyethylene glycol / Pranick-dihydroporphyrin e6 conjugate in NIH-3T3(A), A-2780(B), and SKOV-3(C) cells. Detailed Implementation

[0074] In the following, one or more specific exemplary embodiments will be described in more detail by way of examples. However, these embodiments are provided only to illustrate one or more specific exemplary embodiments, and the scope of the invention is not limited to these embodiments.

[0075] Example 1: Preparation of the connector-Prönnicke conjugate

[0076] 1-1. Maleimide-Prunic F68 conjugate

[0077] 253 mg of 6-maleimide hexanoic acid (Mal), 297 mg of dicyclohexylcarbodiimide (DCC), and 317 mg of butylated hydroxytoluene (BHT) were dissolved in 3 mL of dimethylformamide (DMF). 1 g of Pranic 68 polymer was dissolved in 15 mL of DMF. After stirring for 6 hours, the solution containing maleimide hexanoic acid was added to the aqueous solution containing Pranic 68 polymer, and the resulting mixture was stirred at room temperature for 48 hours. After the reaction was complete, the product was purified by crystallization in 40 mL of diethyl ether. The supernatant, except for the precipitate, was discarded, and diethyl ether was added again during recrystallization. The recrystallization process was repeated three times in total to remove unreacted byproducts. Subsequently, the precipitate was dried under reduced pressure to obtain maleimide-Pranic 68 powder (Mal-PF68). The synthesis results were analyzed by nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 H-NMR confirmed ( Figure 1 ).

[0078] 1-2. Maleimide-Prunic F127 conjugate

[0079] 169 mg of 6-maleimide hexanoic acid, 198 mg of dicyclohexylcarbodiimide, and 212 mg of butylated hydroxytoluene were dissolved in 3 ml of dimethylformamide. 1 g of Pranic 127 polymer was dissolved in 15 ml of dimethylformamide. All solutions were stirred for 6 hours and the same process as in Examples 1-1 was performed to obtain maleimide-Pranic 127 powder (Mal-PF127). The synthesis results were analyzed by nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 H-NMR confirmed ( Figure 2 ).

[0080] 1-3. Preparation of succinyl-Planick F68 conjugates

[0081] 240 mg of succinic anhydride (Suc) and 260 mg of 4-dimethylaminopyridine (DMAP) were dissolved in 10 mL of dimethyl sulfoxide (DMSO). 1 g of Pluronic F68 was dissolved in 50 mL of DMSO. The solutions were stirred for 6 hours. The solution containing the succinic anhydride was added to the aqueous solution containing the Pluronic 68 polymer, and the resulting mixture was stirred at room temperature for 24 hours. After the reaction was complete, the solution was purified by dialysis using a dialysis membrane (MWCO 3,500) for 2 days. Subsequently, the succinyl-Pluronic F68 dissolved in water was lyophilized to obtain the final powder (Suc-PF68). The synthesis results were analyzed by nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 H-NMR confirmed ( Figure 3 ).

[0082] 1-4. Preparation of succinyl-Planick F127 conjugates

[0083] 163 mg of succinic anhydride (Suc) and 179 mg of 4-dimethylaminopyridine (DMAP) were dissolved in 10 mL of dimethyl sulfoxide (DMSO). 1 g of Pranic F127 was dissolved in 50 mL of DMSO. The final powder (Suc-PF127) was then obtained using the same method as in Examples 1-3. The synthesis results were analyzed by nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 H-NMR confirmed ( Figure 4 ).

[0084] Example 2: Preparation of antibody-linker-Prönnicke conjugate

[0085] 2-1. Cetuximab-maleimide-Planick F68, Planick F127 conjugate

[0086] To remove salt, the cetuximab (CTX) injection formulation was passed through a PD10 column with 0.1 M PBS buffer (pH 7.4) as the mobile phase solvent to remove excipients or additives. 1 ml of the obtained substance was collected, and the antibody was quantified using the dicaprylic acid (BCA) method. Only 18 μl of the 2 mg Traut reagent was taken and dispersed in an aqueous solution of 4 mg cetuximab, and the antibody was aminothiolized for 1 hour. After 1 hour, the antibody was purified using a PD10 column to remove the Traut reagent. Subsequently, 1.13 mg maleimide-Prönnicke F68 (Mal-PF68) or 1.63 mg maleimide-Prönnicke F127 (Mal-PF127) was added to an aqueous solution containing 1 mg cetuximab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction products were centrifuged (14,000 g, 10 min) in Amicon Ultra (15 ml, molecular weight cutoff: 100,000 Da) tubes to remove unreacted substances. The final products (CTX-Mal-PF68 and CTX-Mal-PF127) were stored under refrigeration.

[0087] 2-2. Trastuzumab-maleimide-Planick F68, Planick F127 conjugate

[0088] The salt was removed from the trastuzumab (Tra) injection formulation in the same manner as in Example 2-1, the aminothiolized trastuzumab was then removed, and the Traut reagent was removed. Subsequently, 1.21 mg of maleimide-Prönnick F68 or 1.75 mg of maleimide-Prönnick F127 was added to an aqueous solution containing 1 mg of trastuzumab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (Tra-Mal-PF68 and Tra-Mal-PF127) were stored refrigerated.

[0089] 2-3. Acimetidine-maleimide-Planick F68, Planick F127 conjugates

[0090] Salts were removed from the avermab (AVE) injection formulation in the same manner as in Example 2-1, the aminothiolized avermab was then removed, and the Traut reagent was removed. Subsequently, 1.2 mg of maleimide-Plancic F68 or 1.72 mg of maleimide-Plancic F127 was added to an aqueous solution containing 1 mg of avermab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (AVE-Mal-PF68 and AVE-Mal-PF127) were stored refrigerated.

[0091] 2-4. Ramucirumab-maleimide-Planick F68, Planick F127 conjugates

[0092] The salt was removed from the ramucirumab injection formulation in the same manner as in Example 2-1, the aminothiolized ramucirumab was then removed, and the Traut reagent was removed. Subsequently, 1.2 mg of maleimide-Pranic F68 or 1.72 mg of maleimide-Pranic F127 was added to an aqueous solution containing 1 mg of ramucirumab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (RAM-Mal-PF68 and RAM-Mal-PF127) were stored refrigerated.

[0093] 2-5. Trastuzumab-succinyl-Planick F68, Planick F127 conjugates

[0094] Salts were removed from the trastuzumab injection formulation in the same manner as in Example 2-1, the antibody was quantified, and 1 mg of antibody was dissolved in 0.5 M MES buffer. 0.031 mg of 1,3-dimethylaminopropyl-3-ethylcarbodiimide (EDC), 0.0188 mg of N-hydroxysuccinimide (NHS), and 1.2 mg of succinyl-Prönnicke F68 or 1.9 mg of succinyl-Prönnicke F127 were stirred in 0.5 M MES buffer for 1 hour. This solution was added to the antibody-dissolved solution, and the resulting mixture was stirred at 4°C for 12 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (Tra-Suc-PF68 and Tra-Suc-PF127) were stored refrigerated.

[0095] 2-6. Preparation of acimetidine-succinyl-Planick F68 and Planick F127 conjugates

[0096] Salts were removed from the avermab injection formulation in the same manner as in Example 2-1, the antibody was quantified, and 1 mg of antibody was dissolved in 0.5 M MES buffer. Subsequently, the final products (AVE-Suc-PF68 and AVE-Suc-PF127) were prepared in the same manner as in Examples 2-5 and stored under refrigeration.

[0097] Example 3: Preparation of antibody-linker Prönnicke-photosensitizer conjugate

[0098] 3-1. Prönkel F68-dihydroporphyrin e6 conjugate

[0099] In a 20 mL flask, 110 mg of dihydroporphyrin e6 (Ce6), 45 mg of dicyclohexylcarbodiimide (DCC), and 26 mg of 4-dimethylaminopyridine (DMAP) were dissolved in 5 mL of dichloromethane (DCM). 1 g of Pranic F68 (PF68, 8400 g / mol) was dissolved in 10 mL of dichloromethane. After stirring each solution for 6 hours, the two solutions were mixed and stirred at room temperature for 48 hours, followed by crystallization in 45 mL of diethyl ether. The supernatant, except for the precipitate, was discarded. Diethyl ether was added again during recrystallization, and the recrystallization process was repeated three times in total to remove unreacted byproducts. The product was then dried under reduced pressure to obtain a powder. This powder was redissolved in methanol at a concentration of 20 mg / mL and purified by open column chromatography to obtain the synthesized Pranic F68-dihydroporphyrin e6 (PF68-Ce6).

[0100] 3-2. Maleimide-Pranic F68-dihydroporphyrin e6 conjugate

[0101] 23.2 mg of 6-maleimide hexanoic acid (Mal), 27.2 mg of dicyclohexylcarbodiimide (DCC), and 29.1 mg of butylated hydroxytoluene (BHT) were dissolved in 2 ml of dimethylformamide. 200 mg of the Pranic F68-dihydroporphyrin e6 (PF68-Ce6) conjugate obtained in Example 3-1 was dissolved in 3 ml of dimethylformamide. After stirring each solution for 6 hours, the solution containing maleimide hexanoic acid was added to the aqueous solution containing the Pranic F68-dihydroporphyrin e6 conjugate, and the resulting mixture was stirred at room temperature for 48 hours. After the reaction was complete, the product was purified by crystallization in 45 ml of diethyl ether. The supernatant, except for the precipitate, was discarded. Diethyl ether was added again during recrystallization, and the recrystallization process was repeated three times in total to remove unreacted byproducts. The product was then dried under reduced pressure to obtain the final powder (Mal-PF68-Ce6).

[0102] 3-3. Cetuximab-maleimide-Pranic F68-dihydroporphyrin e6 conjugate

[0103] The salt was removed from the cetuximab injection formulation in the same manner as in Example 2-1, the aminothiolized cetuximab was then removed, and the Traut reagent was removed. Subsequently, 3.63 mg of maleimide-Pranic F68-dihydroporphyrin e6 (Mal-PF68-Ce6) prepared in Example 3-2 was added to an aqueous solution containing 1 mg of cetuximab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final product (CTX-Mal-PF68-Ce6) was stored refrigerated.

[0104] 3-4. Trastuzumab-maleimide-Prunic F68-dihydroporphyrin e6 conjugate

[0105] The salt was removed from the trastuzumab injection formulation in the same manner as in Example 2-1, the aminothiolized trastuzumab was then removed, and the Traut reagent was removed. Subsequently, 2.72 mg of maleimide-Pranic F68-dihydroporphyrin e6 prepared in Example 3-2 was added to an aqueous solution containing 5 mg of trastuzumab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final product (Tra-Mal-PF68-Ce6) was stored refrigerated.

[0106] 3-5. Acimetidine-maleimide-Pranic F68-dihydroporphyrin e6 conjugate

[0107] The salt was removed from the avermab injection formulation in the same manner as in Example 2-1, the aminothiolized avermab was then removed, and the Traut reagent was removed. Subsequently, 1.28 mg of maleimide-Pranic F68-dihydroporphyrin e6 prepared in Example 3-2 was added to an aqueous solution containing 2 mg of avermab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final product (AVE-Mal-PF68-Ce6) was stored refrigerated.

[0108] Comparative Example 1: Preparation of Connector-Polyethylene Glycol Conjugate

[0109] 1-1. Maleimide-polyethylene glycol 2K conjugate

[0110] 211 mg of 6-maleimide hexanoic acid, 247 mg of dicyclohexylcarbodiimide, and 138 mg of NHS were dissolved in 5 ml of dimethylformamide. 0.2 g of polyethylene glycol 2K polymer was dissolved in 10 ml of dimethylformamide. After stirring each solution for 4 hours, the solution containing maleimide hexanoic acid was added to the aqueous solution containing polyethylene glycol 2K polymer, and the resulting mixture was stirred at room temperature for 24 hours. After the reaction was complete, the product was purified by crystallization with 40 ml of diethyl ether. The supernatant, excluding the precipitate, was discarded, and diethyl ether was added again during recrystallization. The recrystallization process was repeated three times in total to remove unreacted byproducts. Subsequently, the precipitate was dried under reduced pressure to obtain maleimide-polyethylene glycol 2K powder (Mal-PEG2K). The synthesis results were analyzed by nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 H-NMR confirmed ( Figure 5 ).

[0111] 1-2. Maleimide-polyethylene glycol 6K conjugate

[0112] 70.3 mg of 6-maleimide hexanoic acid, 82.5 mg of dicyclohexylcarbodiimide, and 40.6 mg of NHS were dissolved in 5 ml of dimethylformamide. 0.2 g of polyethylene glycol 6K polymer was dissolved in 10 ml of dimethylformamide. After stirring each solution for 4 hours, the solution containing maleimide hexanoic acid was added to the aqueous solution containing polyethylene glycol 6K polymer, and the resulting mixture was stirred at room temperature for 24 hours. After the reaction was complete, the product was purified by crystallization in 40 ml of diethyl ether. The supernatant, except for the precipitate, was discarded, and diethyl ether was added again during recrystallization. The recrystallization process was repeated three times in total to remove unreacted byproducts. Subsequently, the precipitate was dried under reduced pressure to obtain maleimide-polyethylene glycol 6K powder (Mal-PEG6K). The synthesis results were analyzed by nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1H-NMR confirmed ( Figure 6 ).

[0113] Comparative Example 2: Preparation of Antibody-Linker-Polyethylene Glycol Conjugate

[0114] 2-1. Cetuximab-maleimide-polyethylene glycol 2K, polyethylene glycol 6K conjugate

[0115] The salt was removed from the cetuximab injection formulation in the same manner as in Example 2-1, the aminothiolized cetuximab was then removed, and the Traut reagent was removed. Subsequently, 0.44 mg of maleimide-PEG2K or 1.24 mg of maleimide-PEG6K was added to an aqueous solution containing 3 mg of cetuximab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (CTX-Mal-PEG2K and CTX-Mal-PEG6K) were stored refrigerated.

[0116] 2-2. Trastuzumab-maleimide-polyethylene glycol 2K, polyethylene glycol 6K conjugate

[0117] The salt was removed from the trastuzumab injection formulation in the same manner as in Example 2-1, the aminothiolized trastuzumab was then removed, and the Traut reagent was removed. Subsequently, 0.75 mg of maleimide-PEG2K or 2.1 mg of maleimide-PEG6K was added to an aqueous solution containing 5 mg of trastuzumab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (Tra-Mal-PEG2K and Tra-Mal-PEG6K) were stored refrigerated.

[0118] 2-3. Acimetidine-maleimide-polyethylene glycol 2K, polyethylene glycol 6K conjugate

[0119] The salt was removed from the avermab injection formulation in the same manner as in Example 2-1, the aminothiolized avermab was then removed, and the Traut reagent was removed. Subsequently, 0.301 mg of maleimide-PEG2K or 0.845 mg of maleimide-PEG6K was added to an aqueous solution containing 2 mg of avermab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (AVE-Mal-PEG2K and AVE-Mal-PEG6K) were stored refrigerated.

[0120] 2-4. Ramucirumab-maleimide-polyethylene glycol 2K, polyethylene glycol 6K conjugate

[0121] The salt was removed from the ramucirumab injection formulation in the same manner as in Example 2-1, the aminothiolized ramucirumab was then removed, and the Traut reagent was removed. Subsequently, 0.301 mg of maleimide-PEG2K or 0.845 mg of maleimide-PEG6K was added to an aqueous solution containing 2 mg of ramucirumab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (RAM-Mal-PEG2K and RAM-Mal-PEG6K) were stored refrigerated.

[0122] 2-5. Trastuzumab-succinyl-polyethylene glycol 2K, polyethylene glycol 5K conjugate

[0123] Salts were removed from the trastuzumab injection formulation in the same manner as in Example 2-1, the antibody was quantified, and 1 mg of antibody was dissolved in 0.5 M MES buffer. 0.031 mg EDC, 0.0188 mg NHS, and 0.27 mg succinyl-PEG 2K or 0.82 mg succinyl-PEG 5K were stirred in 0.5 M MES buffer for 1 hour. This solution was added to the antibody-dissolved solution, and the resulting mixture was stirred at 4°C for 12 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final products (Tra-Suc-PEG 2K and Tra-Suc-PEG 6K) were stored refrigerated.

[0124] 2-6. Acimetidine-succinyl-polyethylene glycol 2K, polyethylene glycol 5K conjugate

[0125] Salts were removed from the avermab injection formulation in the same manner as in Example 2-1, the antibody was quantified, and 1 mg of antibody was dissolved in 0.5 M MES buffer. 0.031 mg EDC, 0.0188 mg NHS, and 0.27 mg succinyl-PEG 2K or 0.82 mg succinyl-PEG 5K were stirred in 0.5 M MES buffer for 1 hour. This solution was added to the antibody-dissolved solution, and the resulting mixture was stirred at 4°C for 12 hours. After the reaction was complete, the reaction product was centrifuged (14,000 g, 10 min) in an Amicon Ultra tube to remove unreacted material. The final products (AVE-Suc-PEG 2K and AVE-Suc-PEG 6K) were stored refrigerated.

[0126] Comparative Example 3: Preparation of Antibody-Polyethylene Glycol-Photosensitizer Conjugate

[0127] 3-1. Cetuximab-maleimide-polyethylene glycol 2K-dihydroporphyrin E6 conjugate

[0128] The salt was removed from the cetuximab injection formulation in the same manner as in Example 2-1, the aminothiolized cetuximab was then removed, and the Traut reagent was removed. Subsequently, 1.85 mg of maleimide-PEG2K-dihydroporphyrin E6 was added to an aqueous solution containing 1 mg of cetuximab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged (14,000 g, 10 min) in an Amicon Ultra tube to remove unreacted substances. The final product (CTX-Mal-PEG2K-Ce6) was stored refrigerated.

[0129] 3-2. Trastuzumab-maleimide-polyethylene glycol 2K-dihydroporphyrin e6 conjugate

[0130] The salt was removed from the trastuzumab injection formulation in the same manner as in Example 2-1, the aminothiolized trastuzumab was then removed, and the Traut reagent was removed. Subsequently, 0.949 mg of maleimide-PEG2K-Ce6 was added to an aqueous solution containing 5 mg of trastuzumab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged (14,000 g, 10 min) in an Amicon Ultra tube to remove unreacted substances. The final product (Tra-Mal-PEG2K-Ce6) was stored refrigerated.

[0131] 3-3. Acimetidine-maleimide-polyethylene glycol 2K-dihydroporphyrin E6 conjugate

[0132] The salt was removed from the avermab injection formulation in the same manner as in Example 2-1, the aminothiolized avermab was then removed, and the Traut reagent was removed. Subsequently, 0.764 mg of maleimide-polyethylene glycol 2K-dihydroporphyrin E6 was added to an aqueous solution containing 2 mg of avermab, and the resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction product was centrifuged in an Amicon Ultra tube (14,000 g, 10 min) to remove unreacted substances. The final product (AVE-Mal-PEG2K-Ce6) was stored refrigerated.

[0133] Experimental Example 1: MALDI-TOF Analysis of Antibody-Linker-Prönnicke Conjugates

[0134] To confirm whether the antibodies prepared in Examples 2-1 to 2-6 were conjugated with Prönnik F68, the molecular weight of each conjugate was measured using a MALDI-TOF analyzer.

[0135] As a result, the molecular weights of cetuximab-maleimide-Plannic F68, trastuzumab-maleimide-Plannic F68, acitumab-maleimide-Plannic F68, and ramucirumab-maleimide-Plannic F68 were determined to be approximately 161, 157, 155, and 156 kDa, respectively. This was obtained by adding the molecular weights of cetuximab (152 kDa), trastuzumab (148 kDa), acitumab (147 kDa), and ramucirumab (147 kDa) to those of maleimide-Plannic F68 (8611 g / mol), respectively. Figure 7 , Figure 8 A, Figure 9 A and Figure 10 ).

[0136] Furthermore, the molecular weights of trastuzumab-succinyl-Plancic F68 and avermab-succinyl-Plancic F68 were determined to be approximately 157 kDa and 156 kDa, respectively. This was obtained by adding the molecular weights of trastuzumab (148 kDa) and avermab (147 kDa) to the succinyl-Plancic F68 conjugate (9033 g / mol), respectively. Figure 8 B and Figure 9 (B).

[0137] from Figures 7 to 10 The results confirmed the successful synthesis of antibody-based Prönnik conjugates.

[0138] Experimental Example 2: Evaluation of the structural stability of antibody-linker-Prönnicke conjugate

[0139] To verify the structural stability of the antibody-maleimide-Prännicke F68 and antibody-maleimide-Prännicke F127 conjugates prepared in Examples 2-1 to 2-4 by examining their secondary structures, circular dichroism was measured. Immunoglobulin G, which constitutes the monoclonal antibody, has an inherent secondary structure, and due to this structure, it exhibits positive circular heterochromia at 202 nm and a negative value at 218 nm. Therefore, the retention of the antibody structure can be indirectly confirmed by measuring circular dichroism.

[0140] The measurement results confirm that the secondary structure of the monoclonal antibody, as the original material, is well preserved in the antibody-maleimide-Pranic conjugate. Figure 11 ).

[0141] Experimental Example 3: Confirmation of the Cancer Cell Killing Efficacy of Antibody-Connector-Prönnick Conjugate

[0142] To compare the efficacy of the antibody-linker-Prönnicke F68 and F127 conjugates prepared in Examples 2-1 to 2-6 with the efficacy of the antibodies alone, autonomous killing ability was demonstrated in cancer cells with different epidermal growth factor receptor expression levels. As a control group (Comparative Examples 2-1 to 2-6), antibody-polyethylene glycol 2K, 5K, or 6K conjugates containing only the PEO block were used.

[0143] 3-1. Cetuximab-maleimide-Planick F68, Planick F127 conjugate

[0144] L929 and NIH3T3 normal cells that do not express epidermal growth factor receptor (EGFR) and the EGFR-expressing ovarian cell line SKOV3 were cultured and treated for 4 hours with cetuximab (CTX), cetuximab-maleimide-pranic F68 (CTX-Mal-PF68), or cetuximab-maleimide-pranic F127 (CTX-Mal-PF127). Afterward, the cells were washed with buffer and cultured for another day by adding clean culture medium.

[0145] Cultured cells were treated with MTT reagent and cultured for another 3 hours. The culture medium and MTT reagent were then removed, and dimethyl sulfoxide (DMSO) was added to dissolve the formazan formed in the cells. Absorbance was then measured at 570 nm to compare the amount of formazan formed and to analyze the viability and cytotoxicity of the cancer cells.

[0146] The analysis confirmed that maleimide-Plancic conjugates (Mal-PF68 and Mal-PF127), maleimide-polyethylene glycol conjugates (Mal-PEG2K and Mal-PEG6K), cetuximab-maleimide-Plancic conjugates (CTX-Mal-PF68 / PF127), and cetuximab-maleimide-polyethylene glycol conjugates (CTX-Mal-PEG2K / PEG6K) are non-cytotoxic in L929 and NIH3T3 cells (normal cells that do not express EGFR) (Figure 12, A to D).

[0147] Maleimide-Pluronic conjugates (Mal-PF68 / PF127) are non-cytotoxic even in EGFR-expressing ovarian cancer cell lines. However, the cetuximab-maleimide-Pluronic F68 (CTX-Mal-PF68) or cetuximab-maleimide-Pluronic F127 (CTX-Mal-PF127) treatment groups induced more apoptosis than the cetuximab antibody (CTX) treatment group (Figure 13A and B).

[0148] It can be seen from the above results that the use of cetuximab-maleimide-Pluronic F68 or cetuximab-maleimide-Pluronic F127 may be more effective in cancer treatment than the use of cetuximab antibody alone.

[0149] 3-2. Trastuzumab-maleimide-Pluronic F68 and Pluronic F127 conjugates

[0150] L929 and NIH3T3 normal cells that do not express human epidermal growth factor receptor 2 (HER2) and breast cancer cell lines expressing HER2 (HER2 expression level: MDA-MB-231 < MCF-7 < SK-BR3) were cultured and then treated with different concentrations of trastuzumab-maleimide-Pluronic conjugates. Thereafter, cell viability and cytotoxicity were analyzed in the same manner as in Experimental Example 2-1.

[0151] As a result, it was confirmed that all maleimide-Pluronic conjugates (Mal-PF68 and Mal-PF127), maleimide-polyethylene glycol conjugates (Mal-PEG2K and Mal-PEG6K), trastuzumab-maleimide-polyethylene glycol (Tra-Mal-PEG2K and Tra-Mal-PEG6K) and trastuzumab-maleimide-polyethylene glycol conjugates (Tra-Mal-PF68 and Tra-Mal-PF127) are non-cytotoxic in L929 and NIH3T3 cells that do not express EGFR (Figure 14A to D).

[0152] Furthermore, the maleimide-pranyl chloride (Mal-PF68 and Mal-PF127) and maleimide-polyethylene glycol (Mal-PEG2K and Mal-PEG6K) conjugates themselves were not toxic even in HER2-expressing breast cancer cell lines (Figure 15, A, C, and E). However, the increased cytotoxicity of the trastuzumab-maleimide-pranyl chloride conjugate treatment group compared to either trastuzumab alone or the trastuzumab-maleimide-polyethylene glycol conjugate treatment group confirmed the possibility of effective cancer therapy (Figure 15, B, D, and F). Moreover, the antigen-specific therapeutic effect was confirmed by the proportional increase in toxicity of the trastuzumab-maleimide-pranyl chloride conjugate with the HER2 expression rate of the cell lines.

[0153] 3-3. Acimetidine-maleimide-Planick F68, Planick F127 conjugate

[0154] L929 and NIH3T3 cell lines, which are normal cells that do not express programmed death-ligand 1 (PD-L1), and melanoma, colorectal cancer, and lung cancer cell lines that express PD-L1 (B16F10, HCTE116, and A549, respectively) were cultured and then treated with ancimumab-maleimide-prolactin conjugate. Cell viability and cytotoxicity were then analyzed in the same manner as in Experimental Example 2-1.

[0155] Maleimide-Planc (Mal-PF68 and Mal-PF127) or maleimide-polyethylene glycol conjugates (Mal-PEG2K and Mal-PEG6K) are themselves non-toxic in L929 and NIH3T3 cells that do not express PD-L1 (Fig. 16, A and C). All avermab-maleimide-Planc (AVE-Mal-PF68 and AVE-Mal-PF127) or avermab-maleimide-polyethylene glycol (AVE-Mal-PEG2K and AVE-Mal-PEG6K) conjugates were also confirmed to be non-toxic at concentrations below 10 μl / ml (Fig. 17, B and D).

[0156] Furthermore, the maleimide-Planc (Mal-PF68 and Mal-PF127) and maleimide-polyethylene glycol (Mal-PEG2K and Mal-PEG6K) conjugates themselves were non-toxic even in PD-L1-expressing breast cancer cell lines (Figure 17, A, C, and E). However, the avermab-maleimide-Planc conjugate treatment group showed toxicity at concentrations of 5 μg / ml or lower, exhibiting a greater increase in cytotoxicity compared to the avermab antibody alone (AVE) or the avermab-maleimide-polyethylene glycol treatment group (Figure 17, B, D, and F), thus confirming that effective cancer treatment is possible.

[0157] 3-4. Ramucirumab-maleimide-Planick F68, Planick F127 conjugates

[0158] L929 and NIH3T3 normal cells, gastric cancer cell line (AGS), and non-small cell lung cancer (HCC15) cells that do not express vascular endothelial growth factor receptor 2 (VEGFR2) were cultured and then treated with different concentrations of ramucirumab-maleimide-pranic conjugate. Cell viability and cytotoxicity were then analyzed in the same manner as in Experimental Example 2-1.

[0159] As a confirmatory result, it was confirmed that maleimide-Prannick (Mal-PF68 and Mal-PF127) and maleimide-polyethylene glycol (Mal-PEG2K and Mal-PEG6K) conjugates were non-cytotoxic in normal cells that do not express VEGFR2 (Figure 18, A and C), as were ramucirumab-maleimide-Prannick (RAM-Mal-PF68 and RAM-Mal-PF127) and ramucirumab-maleimide-polyethylene (RAM-Mal-PEG2K and RAM-Mal-PEG6K) conjugates (Figure 18, B and D).

[0160] Furthermore, maleimide-Pranic (Mal-PF68 and Mal-PF127) and maleimide-polyethylene glycol (Mal-PEG2K and Mal-PEG6K) conjugates were non-cytotoxic even in VEGFR2-expressing cancer cell lines (Figure 19, A and C). However, the cytotoxicity of the ramucirumab-maleimide-Pranic conjugate treatment group was further increased compared to the ramucirumab antibody alone (RAM) or the ramucirumab-maleimide-polyethylene glycol treatment group (Figure 19, B and D), thus confirming that effective cancer treatment is possible.

[0161] 3-5. Trastuzumab-succinyl-Pranic conjugate

[0162] A breast cancer cell line expressing HER2 (SK-BR3) was cultured and then treated with trastuzumab-succinyl-Pranic conjugate. Cell viability and cytotoxicity were analyzed in the same manner as in Experimental Example 2-1.

[0163] The results confirmed that in the HER2-expressing breast cancer cell line SK-BR3, the succinyl-Planc conjugates (Suc-PF68 and Suc-PF127) and the succinyl-polyethylene glycol (Suc-PEG2K and Suc-PEG5K) conjugates were non-toxic at concentrations below 50 μg / ml (Figure 20, A). Conversely, the trastuzumab-succinyl-Planc treatment group showed further increased cytotoxicity compared to either the trastuzumab-only treatment group or the trastuzumab-succinyl-polyethylene glycol conjugate treatment group, suggesting that effective cancer treatment is possible (Figure 20, B).

[0164] 3-6. Acimetidine-succinyl-Pranic conjugate

[0165] Lung cancer and colorectal cancer cell lines expressing PD-L1 (A549 and HCT116, respectively) were cultured and then treated with ancimumab-succinyl-Pranic conjugate. Cell viability and cytotoxicity were then analyzed in the same manner as in Experimental Example 2-1.

[0166] The results confirmed that succinyl-Planc (Suc-PF68 and Suc-PF127) and succinyl-polyethylene glycol (Suc-PEG2K and Suc-PEG5K) conjugates were non-cytotoxic in PD-L1-expressing cancer cells (Figure 21, A and C). However, the avermab-succinyl-Planc conjugate (AVE-Suc-PF68 / PF127) treatment group exhibited toxicity at concentrations of 5 μg / ml or lower, showing a greater increase in cytotoxicity compared to the avermab antibody alone (AVE) or the avermab-succinyl-polyethylene glycol (AVE-Suc-PEG2K / PEG5K) treatment group (Figure 21, B and D), thus confirming the possibility of effective cancer treatment.

[0167] Experimental Example 4: Quantitative assessment of the targeting ability of antibody-linker-Prnik-photosensitizer conjugates based on the expression level of cancer cell receptors.

[0168] The targeting ability of the cetuximab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6 conjugate was quantitatively demonstrated in cells with different levels of epidermal growth factor receptor (EGFR) expression, compared with the control group cetuximab-maleimide-Prunic F68-dihydroporphyrin e6 conjugate.

[0169] After culturing NIH-3T3 cells (normal cells that do not express EGFR), A2780 cells (cancer cells with low levels of EGFR expression), and SKOV3 cells (cancer cells with high levels of EGFR expression), each cell type was treated with an antibody-adaptor-Prönkel-fluorescent substance for 2 hours. Subsequently, the cells were washed with buffer solution, and their targeting ability for each cell type was analyzed by flow cytometry.

[0170] As a result, since NIH-3T3 cells do not express EGFR and the conjugates (maleimide-Plancidine F68-dihydroporphyrin e6 and maleimide-polyethylene glycol 2K-dihydroporphyrin e6) that are not specifically linked to antibodies flow into the cells, the fluorescence signal is increased in the maleimide-Plancidine F68-dihydroporphyrin e6 (CTX-Mal-PF68-Ce6) or maleimide-polyethylene glycol 2K-dihydroporphyrin e6 (CTX-Mal-PEG2K-Ce6) conjugate treatment groups compared to the cetuximab-maleimide-Plancidine F68-dihydroporphyrin e6 (CTX-Mal-PEG2K-Ce6) conjugate treatment groups. Figure 22 ).

[0171] In EGFR-expressing A2780 and SKOV3 cells, the cetuximab-maleimide-Pranix F68-dihydroporphyrin e6 treatment group showed enhanced fluorescence compared to the cetuximab-maleimide-PEG 2kJ-dihydroporphyrin e6 treatment group, thus confirming enhanced targeting ability. Figure 22 This confirms that, compared to polyethylene polymers, cetuximab-maleimide-Prunic F68 further enhances the antibody's targeting ability.

[0172] Based on the above results, it is confirmed that the antibody-maleimide-Pranic conjugate can more effectively target cancer cells with epidermal growth factor receptors.

[0173] Experimental Example 5: Confirmation of the cell-targeting properties of the antibody-linker-Prönnicke-photosensitizer conjugate based on the expression level (fluorescence intensity) of cancer cell receptors.

[0174] The cell targeting of cetuximab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6, compared with cetuximab-maleimide-Pranick F68-dihydroporphyrin e6, was visually confirmed by confocal laser scanning microscopy based on cells with different levels of EGFR expression.

[0175] After culturing NIH-3T3 cells (normal cells that do not express EGFR), A2780 cells (cancer cells with low levels of EGFR expression), and SKOV3 cells (cancer cells with high levels of EGFR expression), each cell type was treated with an antibody-adaptor-Prönkel-fluorescent substance for 2 hours. Subsequently, the treated cells were washed with a buffer solution and fixed with 4% paraformaldehyde.

[0176] The results confirmed that almost no fluorescence signal was detected in NIH-3T3 cells (which are normal cells) before and after the introduction of cetuximab. Figure 23 C), and in cancer cells, the fluorescence signal was significantly different before and after antibody introduction, proportional to the EGFR expression level. Specifically, it was clearly visually confirmed that SKOV3 cells with the highest EGFR expression levels had increased fluorescence intensity after cetuximab introduction compared to A2780 cells. Furthermore, stronger fluorescence intensity was confirmed in the cetuximab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6 (CTX-Mal-PEG2K-Ce6) treatment group compared to the cetuximab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6 (CTX-Mal-PF68-Ce6) treatment group. Figure 23 (A and B).

[0177] Based on the above results, it is confirmed that antibody-based Pranic polymer compositions can more effectively target cancer cells with epidermal growth factor receptors.

[0178] Experimental Example 6: In vivo distribution of antibody-linker-Prönnicke-photosensitizer conjugate

[0179] Following intravenous injection of avermab-maleimide-Pregnik F68-dihydroporphyrin e6 (AVE-Mal-PF68-Ce6) at a concentration of 1.78 mg / kg into male athymic nude mice (BALB / c nude mice, 5 weeks old), fluorescence images were acquired for 144 hours using a fluorescently labeled bioimaging system (FOBI, ​​NeoScience, Suwon, South Korea) to confirm the behavior of the conjugate. As a control group, avermab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6 (AVE-Mal-Peg2K-Ce6) and avermab-dihydroporphyrin e6 (AVE-Ce6) conjugates were used.

[0180] As a confirmatory result, compared to the avermab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6 and avermab-dihydroporphyrin e6 injection groups, stronger fluorescence was observed in the avermab-maleimide-Prannick F68-dihydroporphyrin e6 injection group for a significantly longer duration (Figure 24A). After representing the fluorescence signal numerically as 1 at time 0, as a result of quantifying the fluorescence signal according to each time period, it was confirmed that at 144 hours, the fluorescence observed in the avermab-maleimide-Prannick F68-dihydroporphyrin e6 injection group was approximately 2.7 times that observed in either the avermab-dihydroporphyrin e6 or avermab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6 injection groups (Figure 24B).

[0181] Based on the above results, it is confirmed that the antibody-maleimide-Pranic conjugate is retained in vivo for a longer period of time due to its increased half-life.

[0182] Experimental Example 7: Evaluation of the in vivo cancer targeting ability of antibody-linker-Prnik-photosensitizer conjugates

[0183] 1×10⁻⁶ molluscs was subcutaneously injected into male athymic nude mice (BALB / c nude mice, 5 weeks old). 7 One ASPC-1 cancer cell was detected, and after 15 days the cancer size reached 80 mm. 3 At that time, cetuximab-maleimide-Pranic F68-dihydroporphyrin e6 conjugate (CTX-Mal-PF68-Ce6) was administered intravenously at a concentration of 0.5 mg / kg. Subsequently, fluorescent images of cancerous tissue were acquired for 120 hours using a fluorescently labeled bioimaging system (FOBI, ​​NeoScience, Suwon, South Korea) to confirm the behavior of the conjugate.

[0184] As a confirmatory result, stronger photosensitizer fluorescence was observed in cancer tissues of mice injected with cetuximab-maleimide-polyethylene glycol 2k-dihydroporphyrin e6 (CTX-Mal-PEG2K-Ce6) compared to the control group. Even in the quantification results, the fluorescence signal was confirmed to be approximately twice that of the control group. Figure 25 (A and B).

[0185] The results confirm that the conjugates accumulated in cancer tissue for a longer period of time due to the enhanced targeting ability of the antibody-based Prönnick polymer composition.

[0186] Experimental Example 8: Inhibition of Cancer Cell Growth in Vivo by Antibody-Connector-Prnik-Photosensitizer Conjugate

[0187] 1x10 molts were subcutaneously injected into BALB / c nude mice (5 weeks old). 7 A431 cancer cells were detected, and the tumor size reached 200 mm after 15 days. 3 At the time of administration, cetuximab-maleimide-Prunic F68 or Prunic F127 conjugates were administered intravenously five times at a concentration of 0.5 mg / kg based on dihydroporphyrin e6. Subsequently, the effect of inhibiting cancer cell growth was confirmed by measuring the size and weight of cancer tissue every 2 to 3 days. PBS, cetuximab alone, and cetuximab-maleimide-PEG 2K and 6K were used as control groups.

[0188] As a confirmatory result, rapid tumor growth was observed in the control PBS injection group after the first intravenous injection. Tumor growth was confirmed to be slower in the cetuximab (CTX) and cetuximab-maleimide-PEG 2K and 6K injection groups (CTX-Mal-PEG2K and CTX-Mal-PEG6K) compared to the control, while cancer growth was inhibited in the cetuximab-maleimide-Pranick F-68 and F127 injection groups (CTX-Mal-PF68 and CTX-Mal-PF127) (Figure 26A and 26B). Figure 27 This confirms that the cetuximab-maleimide-Pranic F68 and F127 groups further enhanced the antibody's targeting ability compared to the polyethylene polymer. Furthermore, the absence of significant changes in mouse body weight indirectly confirms the lack of toxicity in any injection group (Figure 26, B).

[0189] Experimental Example 9: Analysis of the ability of antibody-linker-Prönnicke-photosensitizer conjugates to generate singlet oxygen.

[0190] Using the antibody-polyethylene glycol-photosensitizer conjugate prepared in Comparative Example 3, the ability of the cetuximab / trastuzumab-maleimide-Prnik-photosensitizer conjugate prepared in Example 3 to generate singlet oxygen was analyzed by fluorescence spectrophotometry (RF).

[0191] A 2 μM solution of a singlet oxygen green fluorescent probe (SOSG) was prepared to react with singlet oxygen to enhance fluorescence, and 1 ml of this solution was mixed with 1 ml of the conjugate sample. The intensity of a 671 nm laser was set to 50 mW / cm². 2 Furthermore, SOSG fluorescence was measured at Ex 504 nm and Em 525 nm while the mixed sample was irradiated with laser at 10-second intervals.

[0192] As a result of the measurements, it was confirmed that the conjugate containing Prönkel exhibited more efficient photoactivity than the control group, demonstrating a superior ability to generate singlet oxygen. Figure 28 ).

[0193] Experimental Example 10: Analysis of the ability of antibody-linker-Prönnicke-photosensitizer conjugates to generate singlet oxygen.

[0194] The ability of the cetuximab-maleimide-pronil-dihydroporphyrin e6 conjugate to kill cells was demonstrated in cancer cells with different levels of epidermal growth factor receptor (HER2) expression.

[0195] After culturing NIH-3T3 cells (normal cells without epidermal growth factor receptor), A2780 cells (cancer cells with low levels of epidermal growth factor receptor expression), and SKOV3 cells (cancer cells with high levels of EGFR expression), each cell type was treated with a cetuximab-maleimide-pronil-dihydroporphyrin e6 conjugate for 4 hours. Subsequently, the cells were washed with buffer and cultured for an additional 24 hours with fresh medium.

[0196] Cultured cells were treated with MTT reagent and cultured for 3 hours. The culture medium, MTT reagent, etc., were then removed, and dimethyl sulfoxide was added to dissolve the formazan formed in the cells. Subsequently, absorbance was measured at 570 nm, and the amount of formazan formed was compared to analyze the viability of each cell type and the cytotoxicity of the cetuximab-based photosensitizer composition.

[0197] The results confirmed that there was no significant difference in cytotoxicity before and after the introduction of cetuximab in NIH-3T3 cells (which are normal cells). Figure 30 (A), but cancer showed a large cytotoxic difference proportional to HER2 expression levels before and after antibody introduction. Figure 30 (B and C). In particular, compared with A2780 cells, SKOV3 cells expressing the highest level of epidermal growth factor receptor expression exhibited self-cytotoxicity after the introduction of cetuximab, even though they were not exposed to light.

[0198] Experimental Example 11: Confirmation of the phototoxicity of antibody-linker-Prönnicke-photosensitizer conjugates

[0199] Based on previous experimental results, the epidermal growth factor receptor (HER2)-specific distribution pattern of the cetuximab-maleimide-pranick-dihydroporphyrin e6 conjugate prepared in Example 3 was confirmed. Furthermore, cells were treated with a concentration of the conjugate that did not exhibit cytotoxic effects and irradiated with a laser, and changes in cell viability were then analyzed in normal or cancer cells.

[0200] After culturing NIH-3T3 cells (normal cells) and A2780 and SKOV3 cancer cells expressing epidermal growth factor receptor, cells were treated for 4 hours with an antibody-based photosensitizer composition and maleimide-polyethylene glycol 2k-dihydroporphyrin e6 (Mal-PEG 2k-Ce6) as a control, with an intensity of 2 J / cm². 2 The cells were irradiated with laser light and then incubated in an incubator for another day.

[0201] Cultured cells were treated with MTT reagent and cultured for 3 hours. The culture medium, MTT reagent, etc., were then removed, and dimethyl sulfoxide was added to dissolve the formazan formed in the cells. Subsequently, absorbance was measured at 570 nm, and the amount of formazan formed was compared to analyze the viability of each cell type and the cytotoxicity of the antibody-based photosensitizer composition.

[0202] The results confirmed that no phototoxicity occurred in NIH-3T3 cells (which are normal cells) before or after the introduction of cetuximab. Figure 30 In cancer cells, phototoxicity increased based on HER2 expression levels, cetuximab treatment, and laser irradiation. Figure 30 (B and C).

Claims

1. A conjugate comprising: (a) An antibody for the treatment of cancer, said antibody being selected from the group consisting of anti-EGFR antibody, anti-HER2 antibody, anti-VEGFR2 antibody and anti-PD-L1 antibody; (b) a connector selected from the group consisting of maleimide, succinic anhydride, and N-hydroxysuccinimide ester, which is covalently linked to the antibody; and (c) A block copolymer selected from the group consisting of poloxamer 188 and poloxamer 407 and covalently connected to the connector.

2. The conjugate according to claim 1, wherein the antibody for treating cancer in (a) is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies and human antibodies.

3. The conjugate according to claim 1, wherein the covalent bond is selected from the group consisting of amide bonds and thioether bonds.

4. The conjugate according to claim 1, wherein one end of the conjugate is further bound with an anticancer agent or a photosensitizer.

5. The conjugate according to claim 4, wherein the photosensitizer is selected from the group consisting of dihydroporphyrin, chlorophyll, porphyrin, porphyrinene and phthalocyanine.

6. The conjugate according to claim 5, wherein the dihydroporphyrin photosensitizer is dihydroporphyrin E6.

7. A pharmaceutical composition for treating cancer, comprising the conjugate of claim 1 as an active ingredient.

8. The pharmaceutical composition of claim 7, wherein the cancer expresses a gene selected from the group consisting of epidermal growth factor receptor, human epidermal growth factor receptor 2, programmed death ligand 1, and vascular endothelial growth factor receptor 2 on the surface of the cancer cells.

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