Ph-sensitive nanoparticle-based drug delivery system

Abstract

The present invention relates to a pH-sensitive nanoparticle-based drug delivery system which can be used for cancer treatment. The drug delivery system comprises: nanoparticles comprising a drug and having a linker on the surface thereof; and a fusion protein comprising a polypeptide which binds to the linker and an antibody which specifically binds to a tumor, wherein the drug is characterized by being eluted from the nanoparticles in a tumor microenvironment, thereby making it possible to minimize side effects such as toxicity to normal tissues and maximize therapeutic effects on the tumor.

Description

pH-sensitive nanoparticle-based drug delivery system

[0001] The present invention relates to a pH-sensitive nanoparticle-based drug delivery system that can be used for cancer treatment.

[0002] Recently, various methods for selectively targeting cancer cells have been developed for cancer treatment. Among these, antibody-drug conjugates (ADCs) are a key method for delivering cytotoxic drugs by specifically binding to cancer cells. However, existing ADCs fail to fully address issues such as nonspecific toxicity, immunogenicity, and drug resistance. To overcome these issues, the development of novel immunotherapeutic agents that are activated only in the tumor microenvironment is essential.

[0003] Meanwhile, numerous research papers using cancer cells and tissues have demonstrated that interferon-gamma (IFN-γ) induces cancer cell death (Ebbinghaus et al., 2005). However, due to the nature of proteins, IFN-γ has low stability and can be rapidly degraded in the body. Therefore, despite its potent anticancer effects, it has played a supporting role in immunotherapy rather than being developed as an ADC. Furthermore, since IFN-γ is a potent immune modulator, and many IFN-γ receptors are expressed on the surface of non-target normal cells, direct delivery of IFN-γ without tumor selectivity can cause systemic side effects such as inflammatory responses due to excessive immune stimulation (Lamaze and Blouin, 2013).

[0004] Tumors possess a mildly acidic microenvironment (pH 6.8 or lower) compared to normal tissues due to rapid cellular metabolism and incomplete vascularization (Ferreira et al., 2013). Therefore, the present invention utilizes this tumor microenvironment to provide a pH-sensitive nanoparticle-based drug delivery system capable of selectively releasing drugs within the tumor microenvironment.

[0005] In addition, the present invention is intended to provide a novel drug delivery system that can be applied to various types of cancer and can exhibit a synergistic effect, particularly in combination therapy with immune checkpoint inhibitors.

[0006] To solve the above-described problem, the present invention provides a drug delivery system comprising a nanoparticle comprising a drug and a linker on the surface; and a fusion protein comprising a polypeptide binding to the linker and an antibody specifically binding to a tumor; wherein the drug is released from the nanoparticle in the tumor microenvironment.

[0007] In the above drug delivery system, the nanoparticles may include a pH-sensitive resin.

[0008] In the above drug delivery system, the pH-sensitive resin may include at least one selected from the group consisting of polylactic-co-glycolic acid (PLGA) and polylactic-co-glycolic acid (PLGA)-oligohistidine copolymer.

[0009] In the above drug delivery system, the drug may include at least one selected from the group consisting of cytokines, anticancer agents, antibiotics, anti-inflammatory agents, and immune checkpoint inhibitors.

[0010] In the above drug delivery system, the antibody may be an antibody fragment.

[0011] In the above drug delivery system, the antibody fragment may be at least one selected from the group consisting of a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a scFv-scFv fragment, a minibody, a diabody, and a single-domain antibody (sdAb) fragment.

[0012] In the above drug delivery system, the polypeptide binding to the linker may be irreversibly bound to the linker.

[0013] The drug delivery system of the present invention comprises a nanoparticle designed to release a drug only in the tumor microenvironment and a fusion protein comprising an antibody that specifically binds to a tumor, which is connected by a linker, thereby minimizing side effects such as toxicity to normal tissues and maximizing the therapeutic effect on the tumor, thereby providing an important strategy for overcoming the limitations of existing drugs and increasing the stability of treatment.

[0014] In addition, the drug delivery system of the present invention can provide an advantage of omitting the tumor cell influx and lysosomal degradation process required by existing antibody-drug conjugates by selectively accumulating within the tumor microenvironment without internalization into tumor cells and acting directly or indirectly from the outside of the tumor cells.

[0015] In addition, the drug delivery system of the present invention can be applied to the treatment of various cancers through combinations of various anticancer substances and antibodies, and can exhibit a synergistic effect, particularly in combination therapy with immune checkpoint inhibitors.

[0016] FIG. 1 is a diagram schematically illustrating the structure of a drug delivery system according to one embodiment of the present invention.

[0017] FIG. 2 is a diagram showing the results of SDS-PAGE analysis of a scFv-streptavidin fusion protein included in a drug delivery system according to one embodiment of the present invention.

[0018] FIG. 3 is a diagram showing the results of SEC-HPLC analysis of a scFv-streptavidin fusion protein included in a drug delivery system according to one embodiment of the present invention.

[0019] FIG. 4 is a diagram showing the results of SDS-PAGE analysis of recombinant interferon-gamma included in a drug delivery system according to one embodiment of the present invention.

[0020] FIG. 5 is a diagram showing the SEC-HPLC analysis results of recombinant interferon-gamma included in a drug delivery system according to one embodiment of the present invention.

[0021] Figure 6 is a PLGA-CGGH15βA copolymer included in a drug delivery system according to one embodiment of the present invention. 1 This diagram shows the results of H-NMR analysis.

[0022] Figure 7a is a diagram showing the results of evaluating the liver cancer cell targeting ability of a liver cancer targeting antibody included in a drug delivery system according to one embodiment of the present invention using a fluorescence microscope and an optical microscope.

[0023] Figure 7b is a diagram showing the average fluorescence intensity of each experimental group and control group in specific numerical values ​​based on the fluorescence microscopy evaluation results of Figure 7a.

[0024] Figure 8 is a diagram showing the results of evaluating whether a drug delivery system according to one embodiment of the present invention induces immune cell stimulation.

[0025] Figure 9 is a diagram showing the results of evaluating the anticancer efficacy of a drug delivery system according to one embodiment of the present invention against the liver cancer cell line SNU886.

[0026] Fig. 10 is an enlarged view showing each part of Fig. 9 of the present invention.

[0027] Figure 11 is a diagram showing the results of evaluating whether a drug delivery system according to one embodiment of the present invention exhibits toxicity to normal hepatocytes.

[0028] Figure 12 is a diagram showing the results of evaluating the survival rate of cancer cells according to the treatment concentration of a drug delivery system according to one embodiment of the present invention.

[0029] Figure 13 is a diagram showing the results of evaluating the survival rate of normal hepatocytes according to the treatment concentration of a drug delivery system according to one embodiment of the present invention.

[0030] Figure 14 is a diagram showing the results of evaluating the degree of induction of expression of PD-1 and PD-L1 in liver cancer cells (HepG2) by a drug delivery system according to one embodiment of the present invention.

[0031] Figure 15 is a diagram showing the results of evaluating the degree of induction of expression of PD-1 and PD-L1 in T cells (HSB2) by a drug delivery system according to one embodiment of the present invention.

[0032] The present invention will be described in more detail. Details not described herein are readily apparent to those skilled in the technical field of the present invention or similar fields, and therefore, their descriptions will be omitted. Each description and embodiment disclosed in this application may also be applied to other descriptions and embodiments. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions set forth below.

[0033] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0034]

[0035] Drug delivery system

[0036] FIG. 1 is a schematic diagram illustrating the structure of a drug delivery system according to one embodiment of the present invention. Referring to FIG. 1, the drug delivery system of the present invention comprises a nanoparticle comprising a drug and a linker on its surface; and a fusion protein comprising a polypeptide binding to the linker and an antibody specifically binding to a tumor.

[0037] Hereinafter, each component of the present invention will be described in detail.

[0038]

[0039] nanoparticles

[0040] Normal tissues generally have a microenvironment of approximately pH 7.4, whereas tumors have a characteristic of a slightly acidic microenvironment of pH 7 or lower, and this slightly acidic tumor microenvironment can weaken the immune effect.

[0041] Nanoparticles included in the drug delivery system of the present invention contain a drug within them, and the drug is physically encapsulated within the nanoparticles and contained within the nanoparticles. The drug is released from the nanoparticles in the tumor microenvironment, thereby exerting a therapeutic effect on the tumor. However, the drug is not released in the normal tissue microenvironment, thereby minimizing adverse effects such as toxicity to normal tissues.

[0042] In one embodiment of the present invention, the tumor microenvironment may have a pH of 7 or lower, preferably a pH of 6.8 or lower, and more preferably a pH of 5.6 to 6.8. That is, the drug contained within the nanoparticle may be eluted from the nanoparticle in a tumor microenvironment having a pH of 7 or lower, preferably may be eluted from the nanoparticle in a tumor microenvironment having a pH of 6.8 or lower, and more preferably may be eluted from the nanoparticle in a tumor microenvironment having a pH of 5.6 to 6.8.

[0043]

[0044] In one embodiment of the present invention, the nanoparticle may include a pH-sensitive resin. The drug delivery system of the present invention, in which nanoparticles including a pH-sensitive resin are applied, is administered into the body of a subject and reaches the tumor microenvironment by the targeting function of the tumor-specific antibody on the fusion protein described below. The pH-sensitive resin is sensitive to the tumor microenvironment of pH 7 or lower, and as the structure collapses, the drug loaded inside the nanoparticle is eluted from the nanoparticle, and the eluted drug can thus induce an immune response against the tumor or play a role in directly attacking the tumor.

[0045] The pH-sensitive resin is not particularly limited as long as its structure collapses in response to a tumor microenvironment of pH 7 or lower, but may include at least one selected from the group consisting of polylactic-co-glycolic acid (PLGA) and a polylactic-co-glycolic acid (PLGA)-oligohistidine copolymer, and preferably includes polylactic-co-glycolic acid (PLGA) and a polylactic-co-glycolic acid (PLGA)-oligohistidine copolymer. The pH-sensitive resin including polylactic-co-glycolic acid (PLGA) and / or polylactic-co-glycolic acid (PLGA)-oligohistidine copolymer has the advantage of being able to more sensitively react to a tumor microenvironment of pH 7 or lower, thereby preventing side effects on normal tissues and further enhancing the therapeutic effect on tumors.

[0046]

[0047] In one embodiment of the present invention, the polylactic-co-glycolic acid (PLGA) may be a compound represented by the following chemical formula 1.

[0048] [Chemical Formula 1]

[0049]

[0050] In the above chemical formula 1, x and y represent the molar ratio of each repeating unit, x+y=1, and 0.1≤x≤0.9.

[0051]

[0052] In one embodiment of the present invention, the polylactic acid-co-glycolic acid (PLGA)-oligohistidine copolymer may be a compound represented by the following chemical formula 2.

[0053] [Chemical Formula 2]

[0054]

[0055] In the above chemical formula 2, x and y represent the molar ratio of each repeating unit, x+y=1, and 0.1≤x≤0.9.

[0056]

[0057] The diameter of the above nanoparticles may be 50 to 500 nm, preferably 75 to 250 nm, and more preferably 100 to 200 nm. When the nanoparticles satisfy the above diameter range, the nanoparticles that reach the tumor microenvironment can pass through the abnormally wide endothelial cell gap of about 100 to 600 nm of the tumor blood vessels, selectively accumulate in the tumor tissue, and are rapidly eliminated from normal tissues, thereby minimizing non-target toxicity to normal tissues, etc. In addition, when the nanoparticles satisfy the above diameter range, they can avoid rapid elimination through the liver and kidneys while remaining in the tumor microenvironment for a long time, thereby improving drug persistence and action intensity at the target site, which is preferable.

[0058]

[0059] The drug included in the above nanoparticles is not particularly limited as long as it exhibits a therapeutic effect on a tumor, but may include at least one selected from the group consisting of cytokines, anticancer agents, antibiotics, anti-inflammatory agents, and immune checkpoint inhibitors, and preferably may include a cytokine and further include at least one selected from the group consisting of anticancer agents, antibiotics, anti-inflammatory agents, and immune checkpoint inhibitors.

[0060] The cytokine may include, but is not limited to, one or more selected from the group consisting of interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), tumor necrosis factor-beta (TNF-β), interleukin-2 (IL-2), interleukin-15 (IL-15), GM-CSF, interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-18 (IL-18), and interleukin-21 (IL-21), and preferably may include interferon-gamma (IFN-γ).

[0061] The above interferon-gamma (IFN-γ) may be composed of an amino acid sequence of SEQ ID NO: 10, or may be composed of a base sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 10.

[0062] Interferon-gamma enhances the anticancer effect by activating immune cells, enhancing the activation of T cells and natural killer (NK) cells, and the function of antigen-presenting cells. Specifically, when cancer patients' cytotoxic T cells are immunodepleted, interferon-gamma can reactivate them, promoting cancer cell attack and increasing the expression of immune checkpoint factors (Rotman et al., 2020). This enhances the immune system's anticancer response, enabling effective tumor cell elimination, in addition to its direct anticancer effects of inhibiting tumor cell growth and inducing apoptosis.

[0063] Additionally, combined therapy with interferon-gamma and immune checkpoint inhibitors may offer new therapeutic possibilities for cancer patients who are refractory to immune checkpoint inhibitors due to minimal expression of immune checkpoint proteins.

[0064] The above anticancer agents include, but are not limited to, cabozantinib, bevacizumab, epirubicin, oxaliplatin, capecitabine, 5-fluorouracil (5-FU), leucovorin, paclitaxel, and albumin-bound paclitaxel.

[0065] The above antibiotics include, but are not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.

[0066] The above anti-inflammatory agents include, but are not limited to, dexamethasone, indomethacin, ibuprofen, clobetasol propionate, diflorasone diacetate, halobetasol propionate, amcinonide, fluocinonide, mometasone furoate, deoxymethasone, diclofenac, and piroxicam, for example.

[0067] The above immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors (pembrolizumab, nivolumab, cemiplimab, tislelizumab, dostarlimab, retipanlimab, toripalimab, etc.), PD-L1 inhibitors (atezolizumab, avelumab, durvalumab, cosibelimab, etc.), CTLA-4 inhibitors (ipilimumab, etc.), and LAG-3 inhibitors.

[0068]

[0069] The above nanoparticle has a linker on its surface, and the linker binds to a complementary polypeptide on the fusion protein described below. The linker may be any linker known in the art without limitation, and for example, O 6 Benzylguanine (O 6-benzylguanine), SpyCatcher, GGG sequence and biotin, preferably biotin. When the linker includes biotin, there is an advantage of further enhancing biocompatibility and stability.

[0070] In one embodiment of the present invention, the linker may further include polyethylene glycol (PEG) for easy binding to nanoparticles.

[0071]

[0072] fusion protein

[0073] The fusion protein included in the drug delivery system of the present invention includes a polypeptide that binds to the linker and an antibody that specifically binds to a tumor.

[0074]

[0075] The polypeptide that binds to the linker may include a peptide sequence complementary to the linker, thereby binding to the linker.

[0076] In one embodiment of the present invention, the polypeptide that binds to the linker may be one that is irreversibly bound to the linker. The polypeptide that is irreversibly bound to the linker may include, but is not limited to, one or more selected from the group consisting of SNAP-tag, SpyTag, Sortag (LPETG), avidin, streptavidin, neutravidin, and captavidin, and preferably, streptavidin.

[0077] In one embodiment of the present invention, the bond between the linker and the polypeptide irreversibly bound to the linker is O 6It may be any one selected from the group consisting of benzylguanine-SnapTag bond, Spycatcher-SpyTag bond, GGG sequence-SorTag bond and biotin-streptavidin bond.

[0078] In one embodiment of the present invention, the streptavidin may be composed of an amino acid sequence of SEQ ID NO: 3, or may be composed of a base sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 3.

[0079]

[0080] The above antibody may include a binding domain (or binding moiety) having a specific binding affinity for an antigen or marker specifically expressed in a tumor, and is meant to include both an intact antibody and an antibody fragment.

[0081] The antigen or marker specifically expressed in the above tumor may be applied without any particular limitation as long as it is an antigen or marker known in the art, and for example, it may be at least one selected from the group consisting of integrin alpha 4, Ang2, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CD19, HER2, HER2 neu, HER3, HER4, HER1(EGFR), PD-L1, PSMA, CEA, TROP-2, MUC1, Lewis-Y, CD20, CD33, CD38, mesothelin, VEGFR1, VEGFR2, ROR1, EphA2, ENPP3, GPC3, FGFR4 and TPBG / 5T4, preferably at least one selected from the group consisting of HER2, PD-L1, CD20, FGFR4 and GPC3, and more preferably GPC3.

[0082] In one embodiment of the present invention, the antibody may be an antibody fragment. The antibody fragment may be at least one selected from the group consisting of a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a scFv-scFv fragment, a minibody, a diabody, and a single-domain antibody (sdAb) fragment, and preferably a single-chain Fv (scFv) fragment.

[0083] The single-chain variable fragment (scFv) may include, but is not limited to, a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 1; and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0084]

[0085] The fusion protein may comprise an scFv antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 1 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 2; and streptavidin consisting of the amino acid sequence of SEQ ID NO: 3, or consisting of a base sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 3.

[0086] Additionally, the fusion protein may be composed of an amino acid sequence of SEQ ID NO: 7, or may be composed of a base sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 7.

[0087]

[0088] <Mechanism of action of drug delivery system>

[0089] When the drug delivery system of the present invention is administered into a subject's body and reaches the target tumor or cancer cells through the bloodstream, tumor-specific antibodies recognize and bind to antigens or markers specifically expressed on the surface of the tumor or cancer cells. At this time, the nanoparticles respond to the tumor microenvironment, which exhibits a lower pH than the microenvironment of normal tissue, and their structure collapses, releasing the drug contained within the nanoparticles. The drug released into the tumor microenvironment directly attacks the tumor or cancer cells or activates immune cells to eliminate the tumor or cancer cells.

[0090] In particular, when interferon-gamma (IFN-γ) is used as the above drug, IFN-gamma released from nanoparticles restores the anti-cancer immunity of impaired immune cells, thereby inducing the immune system to effectively carry out anti-cancer actions. Furthermore, IFN-gamma increases PD-L1 expression on tumors or cancer cells, potentially enabling treatment with immune checkpoint inhibitors like Keytruda and Opdivo for patients who have been refractory to immune checkpoint inhibitor therapy due to low PD-L1 expression.

[0091] Meanwhile, when the drug delivery system of the present invention utilizes biotin-streptavidin as a linker and a polypeptide bound thereto, the streptavidin-biotin bond can offer several outstanding advantages in connecting antibodies and nanoparticles. First, the bond between streptavidin and biotin is well known to be highly biocompatible and stable (Dundas et al., 2013). In particular, the streptavidin-biotin bond is highly potent and irreversible, which maximizes the stability of the drug delivery system. This provides a significant advantage in maintaining long-term stability compared to other existing linker systems, and ensures stable binding of the antibody to the targeted tumor or cancer cells.

[0092] Meanwhile, in the design of antibody-drug conjugates (ADCs), the linker that connects the antibody and the drug must be controllable so that the drug is separated and released under specific circumstances. Therefore, irreversible binding between the linker and the drug is considered unsuitable in antibody-drug conjugates. However, in the drug delivery system according to the present invention, the linker is intended to connect a nanoparticle containing the drug and a fusion protein containing the antibody, which is different from the linker in conventional antibody-drug conjugates that connect the drug and the antibody.

[0093] Conventionally known ADC linkers generally control drug release through reversible binding, which poses a risk of side effects as it may result in unexpected drug release under certain circumstances.

[0094] In contrast, the drug delivery system of the present invention is controlled so that the nanoparticles disintegrate and release the drug only when they reach the targeted tumor or cancer cells and are in a tumor microenvironment with a pH of 7 or lower. This control method focuses on drug release through physical disintegration of the nanoparticles, rather than controlling the bond between the drug and the linker. Therefore, the irreversible bond between the linker and the polypeptide bound to the linker (e.g., streptavidin-biotin bond, etc.) can significantly contribute to improving the stability, safety, and effectiveness of the drug delivery system.

[0095] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended only to illustrate the present invention, and the scope of the present invention is not limited by the examples.

[0096]

[0097] <Example>

[0098] Example 1. Preparation of a recombinant single-chain antibody variable region (scFv)-streptavidin fusion protein

[0099] This example describes a method for producing a recombinant single-chain antibody variable region (scFv) using CHO cells. However, the present invention is not limited thereto, and other processes known in the art, such as using 293 cells and Escherichia coli, can be used instead of CHO cells (Jδger et al., 2013; Sarker et al., 2019; Ying et al., 2023).

[0100]

[0101] 1.1 Synthesis of a gene linking the light chain variable region (VL) and heavy chain variable region (VH) of a GPC3-specific antibody with streptavidin.

[0102] 1) The amino acid sequences of the heavy chain variable region (VH_anti-GPC3, SEQ ID NO: 1) and light chain variable region (VL_anti-GPC3, SEQ ID NO: 2) of the anti-GPC3 antibody were obtained from Patent Publication Nos. 10-2010-0056467 and 10-2008-0068023.

[0103] 2) The amino acid sequence of streptavidin (SEQ ID NO: 3) was obtained from the protein DB (GenBank: AAM49066.1) listed in NCBI.

[0104] 3) A flexible linker (SEQ ID NO: 4) was inserted to allow flexible connection between independent domains, and a leader sequence (leader sequence_1, SEQ ID NO: 5) was added to the beginning of the sequence to increase the efficiency of protein synthesis. In addition, six histidine residues (His-tag, SEQ ID NO: 6) were added to the C-terminus to facilitate purification of the recombinant protein.

[0105] 4) The amino acid sequences for designing a recombinant single-chain antibody variable region (scFv)-streptavidin fusion protein (scFv-streptavidin, SEQ ID NO: 7) are shown in Table 1 below.

[0106]

[0107] 서열번호명칭아미노산 서열1VH_anti-GPC3EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEWMGIIFPGDSDTRYSPSFQGQVTISADRSIRTAYLQWSSLKASDTASYYCARTREGYFDYWGQGTLVTVSS2VL_anti-GPC3EIVLTQSPGTLSLSPGERATLSCRAVQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPTFGGGTKVEIK3streptavidinAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAA4flexible LinkerGGGGSGGGGSGGGGS5leader sequence_1MGSTAILALLLAILRGVCA6His-tagHHHHHH7scFv-streptavidinMGSTAILALLLAILRGVCAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEWMGIIFPGDSDTRYSPSFQGQVTISADRSIRTAYLQWSSLKASDTASYYCARTREGYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRAVQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPTFGGGTKVEIKGGGGSGGGGSGGGGSAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAHHHHHH

[0108]

[0109] 5) cDNA encoding the amino acid sequence of the recombinant scFv-streptavidin fusion protein consisting of the amino acid sequence of the above sequence number 7 was produced using the PCR technique (Marsic et al., 2008).

[0110] To ensure efficient translation of the cDNA produced above, a kozak sequence (CGGACC) was added to the 5' end, and a restriction enzyme recognition sequence (EcoRI recognition sequence: GAATTC; XhoI recognition sequence: CTCGAG) was included for cloning into a vector, thereby producing a gene encoding a recombinant scFv-streptavidin fusion protein (hereinafter also referred to as 'scFv-streptavidin gene').

[0111]

[0112] 1.3 Gene cloning using pcDNA3.1 vector

[0113] After linearizing the pcDNA3.1 vector with restriction enzymes EcoRI and XhoI, the scFv-streptavidin gene sequence prepared above and the pcDNA3.1 vector were mixed at a molar ratio of 3:1, and a ligation reaction was performed using T4 DNA ligase at 4°C for 16 hours. The ligation reaction product was injected into competent E. coli DH5α, and transformation was performed using a heat shock method at 42°C for 1.5 minutes, followed by the addition of LB medium and incubation at 37°C for 1 hour. The cultured bacteria were plated on LB medium, and the transformed clones were selected using an antibiotic (Ampicillin 100 μg / mL), and 100 mL of LB medium was added to the selected clones and incubated at 37°C for 12 to 16 hours. Plasmid DNA (hereinafter referred to as 'pcDNA3.1-scFv-streptavidin plasmid') was extracted from the culture medium, and Sanger sequencing was performed to confirm whether the insertion sequence was introduced correctly.

[0114]

[0115] 1.4 Transformation into CHO cell lines and analysis of fusion protein expression

[0116] CHO (Chinese Hamster Ovary) cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS at 37°C and 5% CO2. When CHO cells reached 70-80% confluency, pcDNA3.1-scFv-streptavidin plasmid was introduced into CHO (Chinese Hamster Ovary) cells using lipofection to transform them.

[0117] Specifically, 3 μg of pcDNA3.1-scFv-streptavidin plasmid was mixed with 250 μg of Opti-MEM medium, and 7.5 μg of lipofectamine 3000 mixed with 250 μg of Opti-MEM medium was further mixed, followed by incubation for 20 minutes to prepare a transduction mixture. The prepared transduction mixture was added to CHO cells that reached 70 to 80% confluent, and cultured for 6 hours at 37°C and 5% CO2, and then replaced with fresh medium. Successfully transduced cells were selected by culturing in a medium containing G418 antibiotic for 48 hours after transduction. Transformed cell clones classified as antibiotic resistant were established, mass-cultivated to express the recombinant protein, and expression of the recombinant protein was confirmed using an ELISA technique, and the cell culture supernatant containing the recombinant scFv-streptavidin fusion protein was collected.

[0118]

[0119] 1.5 Harvesting and purification of recombinant fusion proteins

[0120] The collected cell culture supernatant was filtered through a 0.45 μm nitrocellulose filter to remove cell debris, and the recombinant scFv-streptavidin fusion protein was purified through nickel affinity chromatography using a His-tag.

[0121] The specific process for purifying the recombinant scFv-streptavidin fusion protein is as follows.

[0122] ① Column preparation: A column using Ni-NTA (nickel-nitrilotriacetic acid) resin was equilibrated with binding buffer (NaH2PO450 mM, NaCl 300 mM; pH 8.0).

[0123] ② Protein binding: The collected culture supernatant was slowly passed through a Ni-NTA column, flowing at low speed to allow the recombinant scFv-streptavidin fusion protein containing the His-tag to bind to the resin.

[0124] ③ To remove nonspecifically bound proteins and impurities, the column was washed by flowing 5-10 times the volume of washing buffer (NaH2PO450mM, NaCl 300mM, imidazole 20-50 mM; pH 8.0) containing 20-50 mM imidazole.

[0125] ④ Washing monitoring was performed by measuring the absorbance (280 nm) of the washing solution that was flowed during the washing process to compare and confirm whether non-specific proteins were removed.

[0126] ⑤ The recombinant scFv-streptavidin fusion protein containing a His-tag was eluted from the column using an elution buffer (NaH2PO450 mM, NaCl 300 mM, imidazole 20-50 mM; pH 8.0).

[0127] ⑥ The eluted protein was automatically collected in 1 mL units through a fraction collector in the FPLC (Fast Protein Liquid Chromatography) system and purified.

[0128] ⑦ The collected fractions were analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and SEC-HPLC (size exclusion-high-performance liquid chromatography) to evaluate the purity and concentration of the protein (Figs. 2 and 3), and the fraction containing the highest concentration of recombinant scFv-streptavidin fusion protein was selected and stored at -80°C.

[0129]

[0130] Example 2. Preparation of biotin-tagged interferon-gamma loaded PLGA nanoparticles

[0131] 2.1 Preparation of recombinant interferon-gamma

[0132] The amino acid sequence of human interferon-gamma (SEQ ID NO: 8) for the production of recombinant interferon-gamma was selected with reference to the publicly available protein sequence listed in NCBI. To increase the efficiency of protein synthesis, a leader sequence (leader sequence_2, SEQ ID NO: 9) was added to the very beginning of the sequence. In addition, six histidine residues (His-tag, SEQ ID NO: 6) were added to the C-terminus to facilitate the purification of interferon-gamma.

[0133] The amino acid sequences for designing recombinant interferon-gamma (SEQ ID NO: 10) are shown in Table 2 below.

[0134]

[0135] Sequence number Name Amino acid sequence 6 His-tag HHHHHH 8 IFNg_wt QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKE DMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ 9 leader sequence_2 MGWSCIILFLVATATGVHS 10 IFNg_mo MGWSCIILFLVATATGVHSQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKE DMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQHHHHHH

[0136]

[0137] A cDNA encoding the amino acid sequence of recombinant interferon-gamma consisting of the amino acid sequence of the above sequence number 10 was produced by PCR technique, and the production of recombinant interferon-gamma using the same was performed in the same manner as the production method of the recombinant scFv-streptavidin fusion protein described in Example 1.

[0138] For the recombinant interferon-gamma manufactured above, purity and concentration were evaluated by SDS-PAGE and SEC-HPLC analysis, and the results are shown in Figures 4 and 5, respectively.

[0139]

[0140] 2.2 Synthesis of PLGA-CGGH15βA copolymer

[0141] 0.20 g of PLGA-Mal-PEG (PLGA 10k, PEG 2k, NSP) was dissolved in 2 mL of anhydrous DMF (dimethylformamide), and oligohistidine (CGGH15βA, GenScript) was dissolved in phosphate-buffered saline (PBS). After adding the CGGH15βA solution to the PLGA-Mal-PEG solution, the reaction vessel was placed under a nitrogen atmosphere, and the polymerization reaction was performed at room temperature using a magnetic stirrer for about 3 hours. After the reaction was completed, the reaction mixture was poured into 10 mL of a cold ethyl ether / methanol mixture (1:1) to precipitate. The precipitated PLGA-CGGH15βA was washed several times with a ethyl ether / methanol mixture (1:1) to remove impurities, and dried in a vacuum to obtain a PLGA-CGGH15βA copolymer.

[0142] Regarding the PLGA-CGGH15βA copolymer obtained above 1 H-NMR analysis was performed to confirm the composition of the copolymer and the presence of the CGGH15βA sequence (Fig. 6).

[0143]

[0144] 2.3 Preparation of biotin-tagged interferon-gamma loaded PLGA nanoparticles

[0145] The synthesis of PLGA nanoparticles was performed by surface modification using the W1 / O / W2 (Water / Oil / Water) technique.

[0146] PLGA solution was prepared by dissolving 12.5 mg of PLGA (Resomer® RG 502 H, Sigma-aldrich) and 12.5 mg of PLGA-CGGH15βA copolymer in 0.5 ml of ethyl acetate (EA; volume ratio EA:compound = 3:1). Biotin solution was prepared by dissolving 2.2 mg of DSPE-PEG(2000)-Biotin (NSP) in EA. IFN-γ solution was prepared by dissolving 3 mg of the recombinant interferon-gamma (IFN-γ) prepared above in PBS containing 5% trehalose.

[0147] The above-prepared IFN-γ solution, PLGA solution, and biotin solution were mixed and sonicated (4°C, 1 min, 4 times, 100%) to form a W1 / O emulsion. This was dissolved in 0.5 ml of EA (volume ratio EA:compound = 3:1) solution, and then added dropwise to 3% PVA, mixed with a magnetic stirrer, and centrifuged at 8000 rpm for 10 minutes to obtain a precipitate. The obtained precipitate was suspended in triple-distilled water and sonicated (4°C, 1 min, 4 times, 100%) to form a W1 / O / W2 emulsion, after which the nanoparticles in the emulsion were freeze-dried. The nanoparticles were dialyzed against 2 L isotonic NaCl / Hepes buffer (HBS) at 4°C using a dialysis membrane with a molecular weight cutoff of 14,000 Da, and washed using a Vivaspin 20 filter (molecular weight cutoff of 1,000 kDa). The biotin-tagged interferon-gamma-loaded PLGA nanoparticles obtained after washing were stored frozen in a solution that was adjusted to a final volume of 3 mL and protected from light.

[0148]

[0149] Example 3. Preparation of drug delivery system comprising biotin-tagged interferon-gamma loaded PLGA nanoparticles and scFv-streptavidin fusion protein

[0150] Biotin-tagged interferon-gamma loaded PLGA nanoparticle solution and scFv-streptavidin fusion protein solution were prepared respectively, and the fusion protein solution was mixed with the PLGA nanoparticle solution at a molar ratio of 1:1. The mixture was stirred at 4°C for 2 hours to perform a conjugation reaction, and then centrifuged at 10,000 g for 10 minutes to recover the conjugate of biotin-tagged interferon-gamma loaded PLGA nanoparticle and scFv-streptavidin fusion protein (hereinafter referred to as “the drug delivery system of the present invention”). The recovered conjugate was washed by suspending it in PBS and repeating the centrifugation process three times, and the washed conjugate was redispersed in PBS.

[0151]

[0152] <Experimental Example>

[0153] The types of T cell lines and GPC3-expressing liver cancer cell lines used in this experimental example are shown in Table 3 below.

[0154]

[0155] Cell line derived from human HSB2T lymphoblastSNU886HCCHepG2HCC

[0156] (HCC: Hepatocellular carcinoma)

[0157]

[0158] 1. Evaluation of liver cancer cell targeting ability

[0159] The drug delivery system of the present invention manufactured above was treated and cultured in the GPC3-expressing liver cancer cell line SNU886 (experimental group). In addition, as a control, a conjugate of biotin-tagged interferon-gamma loaded PLGA nanoparticles and streptavidin was treated and cultured in the liver cancer cell line SNU886. After 24 hours of incubation, a detection antibody targeting streptavidin, Biotin-4-Fluorescein (B10570, ThermoFisher), was attached, and the result was compared with the control using the fluorescence function of a fluorescence microscope (GFP 488 nm, a) and the optical function of an optical microscope (bright field, b) (Figs. 7a and 7b). As a result, the drug delivery system of the present invention including the scFv-streptavidin fusion protein exhibited very high fluorescence intensity on the surface of the treated liver cancer cells, so that streptavidin fused with the antibody was detected, whereas no fluorescence intensity was detected at all in the liver cancer cells treated as the control group. Through this, it can be confirmed that the drug delivery system of the present invention specifically binds to the surface of liver cancer cells and exhibits targeting ability.

[0160] In this way, the drug delivery system of the present invention selectively accumulates within the tumor microenvironment without being internalized into the tumor or cancer cells, thereby directly or indirectly exerting a therapeutic effect outside the tumor or cancer cells, thereby providing a technical advantage of omitting the tumor cell influx and lysosomal degradation process required in conventional antibody-drug conjugates.

[0161]

[0162] 2. Evaluation of immune cell stimulation

[0163] The drug delivery system of the present invention was treated with a concentration of 500 ng / ㎖ based on interferon-gamma in a co-culture of a GPC3-expressing hepatoma cell line (SNU886) and a T cell line (HSB2), and the expansion of T cells was observed after 5 days of culture (experimental group). In addition, as a control group, the drug delivery system of the present invention was not treated in a co-culture of SNU886 and HSB2 (control group 1), treated with IFN-γ instead of the drug delivery system of the present invention (control group 2), or treated with a conjugate of PLGA nanoparticles not loaded with IFN-γ and scFv-streptavidin fusion protein (control group 3), and cultured for 5 days.

[0164] As a result, T cell expansion was observed only in the control group 2 treated with IFN-γ and the experimental group treated with the drug delivery system of the present invention (Fig. 8). This indicates that the nanoparticles included in the drug delivery system of the present invention were disintegrated in the microenvironment of the liver cancer cell line, releasing the IFN-γ within the nanoparticles, thereby activating T cells.

[0165]

[0166] 3. Anticancer efficacy evaluation

[0167] 3.1 Evaluation of anticancer efficacy against SNU886 cell line

[0168] The drug delivery system of the present invention was treated with a concentration of 500 ng / ㎖ based on interferon-gamma in a co-culture of a GPC3-expressing hepatoma cell line (SNU886) and a T cell line (HSB2), and the survival of the hepatoma cells was confirmed after 9 days of culture (experimental group). In addition, as a control group, the drug delivery system of the present invention was not treated in a co-culture of SNU886 and HSB2 (control group 1), or treated with IFN-γ instead of the drug delivery system of the present invention (control group 2), or treated with a conjugate of PLGA nanoparticles not loaded with IFN-γ and scFv-streptavidin fusion protein (control group 3), and cultured for 9 days.

[0169] As a result, on the 4th day after culture, no death of liver cancer cells was observed in either the experimental group or the control group 1 to 3, but on the 9th day after culture, death of liver cancer cells was observed in the control group 2 treated with IFN-γ and the experimental group treated with the drug delivery system of the present invention (Figs. 9 and 10).

[0170]

[0171] 3.2 Toxicity assessment for normal hepatocytes

[0172] Meanwhile, the experiment was performed in the same manner as in 3.1 above, except that the GPC3-expressing liver cancer cell line was replaced with normal hepatocytes (HL-7702), to determine whether there was toxicity to normal hepatocytes.

[0173] As a result, no death of normal hepatocytes was observed in either experimental or control groups 1 to 3 even after 9 days of culture (Fig. 11). This suggests that the drug delivery system of the present invention can exhibit anticancer efficacy specifically against target cancer cells without exhibiting toxicity toward normal hepatocytes.

[0174]

[0175] 3.3 Evaluation of cancer cell survival rate according to treatment concentration

[0176] The drug delivery system of the present invention was treated with interferon-gamma-based concentrations of 0, 0.25, 0.5, 1, and 5 μg / ml in co-culture of GPC3-expressing hepatoma cell line (SNU886) and T cell line (HSB2), and the survival rate of cancer cells was confirmed after 9 days of culture (experimental group). In addition, as a control group, co-culture of SNU886 and HSB2 was treated with IFN-γ instead of the drug delivery system of the present invention (control group 1) or treated with a conjugate of PLGA nanoparticles without IFN-γ and scFv-streptavidin fusion protein (control group 2), and cultured for 9 days.

[0177] As a result, in the case of the control group 1 treated with IFN-γ and the experimental group treated with the drug delivery system of the present invention, the survival rate of cancer cells was 30% or less at all treatment concentrations, confirming that excellent anticancer effects could be exhibited at very low treatment concentrations (Fig. 12).

[0178]

[0179] Meanwhile, the GPC3-expressing liver cancer cell line was replaced with normal hepatocytes (HL-7702) and the same experiment was performed as above to confirm the survival rate of normal hepatocytes.

[0180] As a result, the control group 1 treated with IFN-γ showed a tendency for the cell viability of normal hepatocytes to decrease as the treatment concentration increased, but it was confirmed that the experimental group treated with the drug delivery system of the present invention did not affect the cell viability of normal hepatocytes at all treatment concentrations (Fig. 13).

[0181]

[0182] 4. Evaluation of combination therapy with immune checkpoint inhibitors

[0183] Human hepatoma cell line HepG2 (1Х10 5 Cells) were seeded into 60 mm cell culture dishes and cultured under the following conditions.

[0184] (i) Control group 1: HepG2 liver cancer cell line monoculture group;

[0185] (ii) Control group 2: Co-culture group of hepatoma cell line HepG2 and T cell line HSB2; and

[0186] (iii) Experimental group: A co-culture group of the liver cancer cell line HepG2 and the T cell line HSB2, treated with the drug delivery system of the present invention after 24 hours of culture.

[0187] The above co-culture group was cultured by inoculating HepG2 cells and HSB2 cells together in the same cell culture dish.

[0188] For the above experimental group, the drug delivery system of the present invention was treated at a concentration of 200 ng / ㎖ based on interferon-gamma after 24 hours of culture, and the drug delivery system of the present invention was not treated for control groups 1 and 2. After 48 hours of culture, floating HSB2 cells were removed, and only adherent HepG2 cells were collected, and total RNA was extracted using Trizol reagent. cDNA was synthesized through reverse transcription using the extracted total RNA as a template, and this was applied to real-time quantitative PCR (qPCR) to quantitatively analyze the mRNA expression levels of PD-1 and PD-L1, respectively.

[0189] As a result, in the experimental group treated with the drug delivery system of the present invention, it was confirmed that the mRNA expression level of PD-L1 in the liver cancer cell line HepG2 was significantly increased by interferon-gamma eluted from the drug delivery system (Fig. 14), and the mRNA expression levels of PD-1 and PD-L1 in the T cell line HSB2 were significantly increased (Fig. 15).

[0190] This suggests that interferon-gamma can eliminate tumors or cancer cells within the tumor microenvironment through its inherent anticancer activity, while simultaneously inducing immune evasion mechanisms in tumors or cancer cells. In particular, its ability to induce increased expression of PD-1 and / or PD-L1 provides the basis for anticipated synergistic effects in combination therapy with immune checkpoint inhibitors, such as PD-1 or PD-L1 inhibitors.

[0191] Therefore, the drug delivery system of the present invention is expected to have high value not only as a single anticancer agent but also as an anticancer agent for combination therapy with an immune checkpoint inhibitor.

[0192]

[0193] <References>

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[0195] 2. Diagnostics | Free Full-Text | Recombinant Protein Expression and Purification of N, S1, and RBD of SARS-CoV-2 from Mammalian Cells and Their Potential Applications [WWW Document], n.d. URL https: / www.mdpi.com / 2075-4418 / 11 / 10 / 1808 (accessed 8.23.24).

[0196] 3. Dundas, C.M., Demonte, D., Park, S., 2013. Streptavidin-biotin technology: improvements and innovations in chemical and biological applications. Appl Microbiol Biotechnol 97, 9343-9353. https: / doi.org / 10.1007 / s00253-013-5232-z

[0197] 4. Ebbinghaus, C., Ronca, R., Kaspar, M., Grabulovski, D., Berndt, A., Kosmehl, H., Zardi, L., Neri, D., 2005. Engineered vascular-targeting antibody-interferon-γ fusion protein for cancer therapy. International Journal of Cancer 116, 304-313. https: / doi.org / 10.1002 / ijc.20952

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[0200] 7. J gger, V., B ssow, K., Wagner, A., Weber, S., Hust, M., Frenzel, A., Schirrmann, T., 2013. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnology 13, 52. https: / doi.org / 10.1186 / 1472-6750-13-52

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[0202] 9. Lamaze, C., Blouin, C.M., 2013. Interferon Gamma Receptor: The Beginning of the Journey. Front. Immunol. 4. https: / doi.org / 10.3389 / fimmu.2013.00267

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[0204] 11. Marsic, D., Hughes, R.C., Byrne-Steele, M.L., Ng, J.D., 2008. PCR-based gene synthesis to produce recombinant proteins for crystallization. BMC Biotechnology 8, 44. https: / doi.org / 10.1186 / 1472-6750-8-44

[0205] 12. Rotman, J., den Otter, L.A.S., Bleeker, M.C.G., Samuels, S.S., Heeren, A.M., Roemer, M.G.M., Kenter, G.G., Zijlmans, H.J.M.A.A., van Trommel, N.E., de Gruijl, T.D., Jordanova, E.S., 2020. PD-L1 and PD-L2 Expression in Cervical Cancer: Regulation and Biomarker Potential. Front Immunol 11, 596825. https: / doi.org / 10.3389 / fimmu.2020.596825

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Claims

1. Nanoparticles containing a drug and having a linker on the surface; and A fusion protein comprising a polypeptide binding to the linker and an antibody specifically binding to a tumor; The above drug is a drug delivery vehicle that is released from nanoparticles in the tumor microenvironment.

2. In claim 1, The above nanoparticles are drug delivery vehicles comprising a pH-sensitive resin.

3. In claim 2, A drug delivery system wherein the pH-sensitive resin comprises at least one selected from the group consisting of polylactic-co-glycolic acid (PLGA) and polylactic-co-glycolic acid (PLGA)-oligohistidine copolymers.

4. In claim 1, The drug delivery system comprises at least one drug selected from the group consisting of cytokines, anticancer agents, antibiotics, anti-inflammatory agents, and immune checkpoint inhibitors.

5. In claim 1, The above antibody is a drug delivery vehicle that is an antibody fragment.

6. In claim 5, A drug delivery system wherein the antibody fragment is at least one selected from the group consisting of a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a scFv-scFv fragment, a minibody, a diabody, and a single-domain antibody (sdAb) fragment.

7. In claim 1, A drug delivery system wherein the polypeptide binding to the above linker is irreversibly bound to the linker.

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