Single domain antibody specifically binding to sox2, fusion protein including same, and use thereof

Abstract

The present invention relates to a single domain antibody specifically binding to STAT3 or an antigen-binding fragment thereof, a fusion protein comprising the single domain antibody or the antigen-binding fragment thereof, and a use of the fusion protein.

Description

Single-domain antibody specifically binding to STAT3, fusion protein containing the same, and uses thereof

[0001] The present invention relates to a single-domain antibody or an antigen-binding fragment thereof that specifically binds to STAT3, a fusion protein comprising said single-domain antibody or said antigen-binding fragment thereof, and a use of said fusion protein.

[0002] Proteins perform various physiological and pathological functions within cells, and regulating their expression and inhibiting their function are key strategies in new drug development. In particular, the abnormal activation or overexpression of specific proteins is known to play a core role in the pathological mechanisms of various diseases, such as cancer, autoimmune diseases, and inflammatory diseases. Existing inhibitor-based therapies work by suppressing the activity of target proteins, but they have the limitation of not being able to eliminate the proteins themselves.

[0003] Targeted Protein Degradation (TPD) technology has been developed as a new approach to overcome these problems. A representative technology, PROTAC (Proteolysis Targeting Chimera), is a dual-functional small molecule compound formed by linking a small molecule ligand that binds to a target protein (POI; Protein of Interest) with a small molecule ligand that binds to E3 ubiquitin ligase (E3 ligase) via a linker. PROTAC induces ubiquitination by bringing the target protein into close proximity with the E3 ligase, ultimately causing it to be degraded by the proteasome. This technology offers higher efficacy and specificity than existing inhibitor-based approaches and has the advantage of being applicable to so-called 'undruggable targets,' for which drug development has been difficult.

[0004] However, small molecule PROTAC technology is extremely limited in the available E3 ubiquitin ligases, such as Cereblon (CRBN), VHL, MDM2, and cIAP, and fails to fully utilize the hundreds of types of E3 ligases present in the human body. Furthermore, due to their large molecular weight and complex structures, PROTACs often exhibit unfavorable physicochemical properties as drugs, such as cell permeability and pharmacokinetic stability.

[0005] bioPROTAC technology has been proposed as an alternative to overcome these limitations. BioPROTAC is a protein-based degradation induction technology that creates a single fusion protein by fusing a target protein binding domain (e.g., nanobodies, DARPin, protein scaffolds, peptides, etc.) with the substrate recognition domain of an E3 ubiquitin ligase. BioPROTAC expressed within cells artificially brings the target protein and the E3 ligase close together to induce ubiquitination, thereby selectively degrading the target protein. This technology has also been reported under various names such as 'biodegrader', 'ubiquibody', and 'AdPROM', and unlike small molecule PROTACs, it has the advantage of being able to flexibly combine the binding domain and the E3 ligase. In fact, vhhGFP4 nanobody, DARPin, αRep, monobody, peptide (Con1), etc. were used as target binding domains, and it was proven that various types of E3 ligases such as CUL1 (βTrCP, FBW7, SKP2), CUL2 (VHL), CUL3 (SPOP), CUL5 (SOCS2), and CHIP can be utilized.

[0006] This bioPROTAC technology has been utilized for the degradation of intractable target proteins that were previously difficult to drugize. For example, various cases have been reported, including cancer research through the degradation of KRAS protein, cell cycle regulation through PCNA degradation, and tumor suppression through the degradation of β-catenin and c-Myc. BioPROTACs are also attracting attention as a new paradigm for therapeutic development due to their ability to achieve sustained degradation even in small quantities through catalytic action. However, the technical hurdle of intracellular delivery still exists, and mRNA-based delivery technologies to overcome this are recently gaining attention.

[0007] Meanwhile, the Signal Transducer and Activator of Transcription 3 (STAT3) protein is well known to be overactivated and play a pathological role in various cancers and inflammatory diseases. As a transcription factor, STAT3 has long been studied as an attractive therapeutic target because it is critically involved in cell growth, survival, and immune evasion. However, due to the structural characteristics of STAT3, it is difficult to find binding sites for small molecules, and consequently, the development of existing inhibitors has yielded limited results.

[0008] Against this technical background, the inventors discovered a single-domain antibody that specifically binds to STAT3 and confirmed that fusing it with an E3 ubiquitin ligase induces the selective degradation of STAT3. Furthermore, the present invention was completed by demonstrating the therapeutic effect of STAT3 degradation on the respective diseases in atopic and tumor models using STAT3 bioPROTAC.

[0009] One objective of the present invention is to provide an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof. Another objective is to provide a polynucleotide encoding said antibody or antigen-binding fragment, an expression vector containing said antibody or antigen-binding fragment, and a transformed cell. Additionally, the invention is to provide a method for manufacturing said antibody or antigen-binding fragment.

[0010] Furthermore, the present invention aims to provide a fusion protein comprising an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof, and to provide a polynucleotide encoding the same, an expression vector, and a transformed cell. Additionally, the invention aims to provide a method for manufacturing the fusion protein.

[0011] In addition, another objective of the present invention is to provide the use of the fusion protein for the treatment of atopic or tumor diseases.

[0012] To achieve the above objective, the present invention provides an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof comprising: HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO 50, SEQ ID NO 12, SEQ ID NO 19, SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 40, and SEQ ID NO 46; HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO 14, SEQ ID NO 21, SEQ ID NO 28, SEQ ID NO 36, SEQ ID NO 42, and SEQ ID NO 47; and HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 44, SEQ ID NO 48, and SEQ ID NO 51.

[0013] In the present invention, the anti-STAT3 single-domain antibody or its antigen-binding fragment is

[0014] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO. 50, HCDR2 comprising the amino acid sequence of SEQ ID NO. 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 16;

[0015] (ii) HCDR1 having the amino acid sequence of SEQ ID NO. 12, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 16;

[0016] (iii) HCDR1 having the amino acid sequence of SEQ ID NO. 19, HCDR2 having the amino acid sequence of SEQ ID NO. 21, and HCDR3 having the amino acid sequence of SEQ ID NO. 23;

[0017] (iv) HCDR1 having the amino acid sequence of SEQ ID NO. 26, HCDR2 having the amino acid sequence of SEQ ID NO. 28, and HCDR3 having the amino acid sequence of SEQ ID NO. 30;

[0018] (v) HCDR1 having the amino acid sequence of SEQ ID NO. 34, HCDR2 having the amino acid sequence of SEQ ID NO. 36; and HCDR3 having the amino acid sequence of SEQ ID NO. 38;

[0019] (vi) HCDR1 comprising the amino acid sequence of SEQ ID NO. 40, HCDR2 comprising the amino acid sequence of SEQ ID NO. 42, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 44;

[0020] (vii) HCDR1 comprising the amino acid sequence of SEQ ID NO. 46, HCDR2 comprising the amino acid sequence of SEQ ID NO. 47, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 48;

[0021] (viii) HCDR1 comprising the amino acid sequence of SEQ ID NO. 12, HCDR2 comprising the amino acid sequence of SEQ ID NO. 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 51; or

[0022] (ix) may include HCDR1 having the amino acid sequence of SEQ ID NO. 50, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 51.

[0023] In the present invention, the anti-STAT3 single-domain antibody or the antigen-binding fragment thereof may comprise any one amino acid sequence selected from the group consisting of SEQ ID NOs 90 to 92 and SEQ ID NOs 4 to 10.

[0024] The present invention also provides an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof, comprising: HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO. 88, SEQ ID NO. 53, SEQ ID NO. 59, SEQ ID NO. 65, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 79 and SEQ ID NO. 84; HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO. 55, SEQ ID NO. 61, SEQ ID NO. 67, SEQ ID NO. 75, SEQ ID NO. 81 and SEQ ID NO. 86; and HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 63, SEQ ID NO. 69, SEQ ID NO. 77, SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 89.

[0025] In the present invention, the anti-STAT3 single-domain antibody or its antigen-binding fragment is

[0026] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO. 88, HCDR2 comprising the amino acid sequence of SEQ ID NO. 55, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 57;

[0027] (ii) HCDR1 comprising the amino acid sequence of SEQ ID NO. 53, HCDR2 comprising the amino acid sequence of SEQ ID NO. 55, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 57;

[0028] (iii) HCDR1 having the amino acid sequence of SEQ ID NO. 59, HCDR2 having the amino acid sequence of SEQ ID NO. 61, and HCDR3 having the amino acid sequence of SEQ ID NO. 63;

[0029] (iv) HCDR1 having the amino acid sequence of SEQ ID NO. 65, HCDR2 having the amino acid sequence of SEQ ID NO. 67, and HCDR3 having the amino acid sequence of SEQ ID NO. 69;

[0030] (v) HCDR1 having the amino acid sequence of SEQ ID NO. 71, HCDR2 having the amino acid sequence of SEQ ID NO. 55, and HCDR3 having the amino acid sequence of SEQ ID NO. 57;

[0031] (vi) HCDR1 comprising the amino acid sequence of SEQ ID NO. 73, HCDR2 comprising the amino acid sequence of SEQ ID NO. 75, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 77;

[0032] (vii) HCDR1 comprising the amino acid sequence of SEQ ID NO. 79, HCDR2 comprising the amino acid sequence of SEQ ID NO. 81, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 83;

[0033] (viii) HCDR1 having the amino acid sequence of SEQ ID NO. 84, HCDR2 having the amino acid sequence of SEQ ID NO. 86, and HCDR3 having the amino acid sequence of SEQ ID NO. 87;

[0034] (ix) HCDR1 comprising the amino acid sequence of SEQ ID NO. 71, HCDR2 comprising the amino acid sequence of SEQ ID NO. 55, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 89; or

[0035] (x) It may include HCDR1 containing the amino acid sequence of SEQ ID NO. 88, HCDR2 containing the amino acid sequence of SEQ ID NO. 55, and HCDR3 containing the amino acid sequence of SEQ ID NO. 89.

[0036] The present invention also provides a polynucleotide encoding the anti-STAT3 single-domain antibody or its antigen-binding fragment.

[0037] The present invention also provides an expression vector loaded with a polynucleotide.

[0038] The present invention also provides a transformed cell into which the expression vector is introduced.

[0039] The present invention also provides a method for producing an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof, comprising: i) a step of culturing the transformed cells; and ii) a step of obtaining an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof from a culture medium in which the transformed cells were cultured.

[0040] The present invention also provides a fusion protein comprising the anti-STAT3 single-domain antibody or its antigen-binding fragment; and a fragment of E3 ubiquitin ligase or a variant thereof.

[0041] In the present invention, the fragment of the E3 ubiquitin ligase or a variant thereof may be a fragment of SPOP (speckle-type BTB-POZ) or a variant thereof.

[0042] In the present invention, the fragment of SPOP (speckle-type BTB-POZ) or a variant thereof may comprise the amino acid sequence of SEQ ID NO. 100.

[0043] In the present invention, the fusion protein may include a linker.

[0044] In the present invention, the anti-STAT3 single-domain antibody or its antigen-binding fragment may be located at the N-terminus or C-terminus of a fragment of E3 ubiquitin ligase or a variant thereof.

[0045] In the present invention, the fusion protein may comprise any one amino acid sequence selected from the group consisting of SEQ ID NOs 90 to 92 and SEQ ID NOs 4 to 10 and the amino acid sequence of SEQ ID NO. 100.

[0046] The present invention also provides a polynucleotide encoding the fusion protein.

[0047] The present invention also provides an expression vector loaded with the polynucleotide.

[0048] The present invention also provides a transformed cell into which the expression vector is introduced.

[0049] The present invention also provides a method for producing a fusion protein comprising: i) a step of culturing the transformed cells; and ii) a step of obtaining a fusion protein from a culture medium in which the transformed cells were cultured.

[0050] The present invention also provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising the fusion protein; the polynucleotide; or the expression vector.

[0051] The present invention also provides a quasi-drug composition for preventing or improving atopic dermatitis comprising the fusion protein; the polynucleotide; or the expression vector.

[0052] The present invention also provides a food composition for preventing or improving atopic dermatitis comprising the fusion protein; the polynucleotide; or the expression vector.

[0053] The present invention also provides a cosmetic composition for preventing or improving atopic dermatitis comprising the fusion protein; the polynucleotide; or the expression vector.

[0054] The present invention also provides a method for preventing or treating atopic dermatitis, comprising the step of administering the fusion protein; the polynucleotide; or the expression vector to a subject in need thereof.

[0055] The present invention also provides a use for the manufacture of a drug for the prevention or treatment of atopic dermatitis of the fusion protein; the polynucleotide; or the expression vector.

[0056] The present invention also provides the fusion protein; the polynucleotide; or the expression vector for use in preventing or treating atopic dermatitis.

[0057] The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein; the polynucleotide; or the expression vector as an active ingredient.

[0058] In the present invention, the cancer may be one or more selected from the group consisting of breast cancer, colorectal cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, glioma, ovarian cancer, cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0059] In the present invention, the pharmaceutical composition may be administered in combination with one or more other preparations effective as anticancer agents.

[0060] In the present invention, the above formulation is

[0061] (i) sorafenib, regorafenib, lenvatinib, cabozantinib, sunitinib, pazopanib, vandetanib, axitinib, nintedanib, tivozanib, apatinib, ponatinib, ripretinib, foretinib, anlotinib, or donafenib;

[0062] (ii) cisplatin, carboplatin, oxaliplatin, doxorubicin, epirubicin, paclitaxel, docetaxel, irinotecan, topotecan, gemcitabine, 5-fluorouracil (5-FU), capecitabine, cyclophosphamide, ifosfamide, methotrexate, bleomycin, vincristine, vinblastine or vinorelbine; or

[0063] (iii) It may be an anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG-3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-4-1BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-VISTA, anti-NKG2A, anti-PVRIG, anti-CD73, anti-B7-H3, anti-IDO-1, anti-CD200, or anti-TIGIT antibody.

[0064] The present invention also provides a method for preventing or treating cancer, comprising the step of administering the fusion protein; the polynucleotide; or the expression vector to a subject in need thereof.

[0065] The present invention also provides a use for the fusion protein; the polynucleotide; or the expression vector for the manufacture of a drug for the prevention or treatment of cancer.

[0066] The present invention also provides the fusion protein; the polynucleotide; or the expression vector for use in cancer prevention or treatment.

[0067] The anti-STAT3 single-domain antibody of the present invention possesses a small molecular size and high stability, as well as excellent intracellular accessibility and tissue permeability. This enables high selectivity and binding affinity for target proteins. The STAT3 bioPROTAC based on the antibody selectively degrades the STAT3 protein, restores skin barrier protein expression under atopic-induced conditions, and exhibits a significant therapeutic effect on atopic dermatitis in animal models. Furthermore, by inhibiting cancer cell growth through the selective degradation of STAT3 in various cancer cell lines and animal models, it offers the advantage of being applicable to anticancer treatment.

[0068] Figure 1 shows the results of discovering a total of six single-domain antibodies (A4, 1D6, 2D1, 3E5, 4E8, and 5A5) that bind to STAT3 using phage display and confirming their binding affinity to STAT3 by flow cytometry.

[0069] Figure 2 shows the results of identifying three variants (4E8_A8, 4E8_A9, 4E8_A10) of the anti-STAT3 single-domain antibody 4E8 and confirming their binding affinity to STAT3 through flow cytometry analysis.

[0070] Figure 3 shows the results of NanoBRET analysis of STAT3 binding after fusing Fc to the anti-STAT3 single-domain antibody and its variant. 4E8-Fc and 4E8_A8-Fc were labeled as 4E8 and 4E8_A8, respectively, and MDM2-p53 was used as a positive control.

[0071] Figure 4 shows the results of FACS analysis of STAT3 binding after fusing Fc to an anti-STAT3 single-domain antibody and its variant and expressing them on the surface of yeast. 4E8-Fc and 4E8_A8-Fc were labeled as 4E8 and 4E8_A8, respectively.

[0072] Figure 5 shows the results of ELISA analysis of STAT3-specific binding of anti-STAT3 single-domain antibodies and their variants after fusing Fc (4E8-Fc, 4E8_A8-Fc).

[0073] Figure 6 shows the results of BLI analysis of STAT3 binding after fusing Fc to the anti-STAT3 single-domain antibody and its variant (4E8-Fc, 4E8_A8-Fc). a shows the BLI analysis results of 4E8-Fc, b shows the BLI analysis results of 4E8_A8-Fc, and c shows the binding strength values ​​of the two types of STAT3 nanobody-Fc.

[0074] Figure 7 shows the results of confirming the STAT3 resolution of six STAT3 bioPROTACs (A4.005, 1D6.005, 2D1.005, 3E5.005, 4E8.005, and 5A5.005) prepared using six anti-STAT3 single-domain antibodies by Western blotting (a, plasmid DNA; b, mRNA form of bioPROTAC).

[0075] Figure 8 shows the results of confirming the STAT3 resolution of four STAT3 bioPROTACs (4E8.005, 4E8_A8.005, 4E8_A9.005, 4E8_A10.005) produced using the anti-STAT3 single-domain antibody 4E8 and three variants thereof by Western blotting (a, plasmid DNA; b, mRNA form bioPROTAC).

[0076] Figure 9 shows the results of confirming the ubiquitin-proteasome-dependent STAT3 degradation ability of STAT3 bioPROTAC (A4.005, 4E8.005, 4E8_A8.005) by Western blotting.

[0077] Figure 10 shows the results of confirming the mechanism of action of STAT3 bioPROTAC by Western blotting.

[0078] Figure 11 shows the results of confirming the STAT3 selective resolution of two STAT3 bioPROTACs (4E8.005, 4E8_A8.005) by Western blotting.

[0079] Figure 12 shows the results of quantitative proteomic analysis confirming the STAT3 selective degradation ability of two STAT3 bioPROTACs (4E8.005, 4E8_A8.005).

[0080] Figure 13 shows the results of comparing the STAT3 degradation ability between two types of STAT3 bioPROTACs (4E8.005, 4E8_A8.005) and a STAT3 PROTAC compound (SD-36) in two types of skin keratinocyte cell lines (HEKn, HaCaT).

[0081] Figure 14 shows the results of comparing the recovery efficacy of skin barrier proteins (Filaggrin, Involucrin) by STAT3 bioPROTAC (4E8_A8.005) and competing drugs (KT-333, Upadacitinib, Dupilumab) under atopic dermatitis induction conditions.

[0082] Figure 15 shows the results of comparing the recovery efficacy of skin barrier proteins (Filaggrin, Loricrin) by STAT3 bioPROTAC (4E8_A8.005) and a competitor drug (Dupilumab) under different atopic induction conditions.

[0083] Figure 16 shows the results of confirming the STAT3 resolution of STAT3 bioPROTAC (4E8.A8.005, 4E8.A8.005(circ)) in the form of linear or circular mRNA in skin keratinocyte cell lines (HEKn).

[0084] Figure 17 shows the results of comparing the efficacy of restoring skin barrier proteins (Filaggrin, Loricrin) by STAT3 bioPROTAC (4E8_A8.005, 4E8_A8.005(circ)) in linear or circular mRNA form and a competitor drug (Dupilumab) under atopic induction conditions.

[0085] Figure 18 shows the results of comparing the efficacy of restoring skin barrier proteins (Filaggrin, Loricrin) by STAT3 bioPROTAC (4E8_A8.005, 4E8_A8.005(circ)) in linear or circular mRNA form and a competitor drug (Upadacitinib) under atopic induction conditions.

[0086] Figure 19 shows the results of confirming the STAT3 degradation ability of two STAT3 bioPROTACs (4E8.005, 4E8_A8.005) in mouse cell lines (Neuro-2a).

[0087] Figure 20 shows the results of confirming the therapeutic efficacy of STAT3 bioPROTAC (4E8_A8.005) and a competitor drug (Dupilumab) in an atopic dermatitis-induced animal model using MC903, expressed as a, sensory evaluation index; b, mouse total IgE; c, blood CCL17 (TARC) concentration; d, blood IL-22 concentration; e, epidermal hyperplasia evaluation index; and f, epidermal thickness. Data are presented as mean ± standard error (SEM) or median (for mouse total IgE) and were analyzed using Tukey's test of multiple comparisons following one-way ANOVA (*p < 0.05, **p < 0.01).

[0088] Figure 21 shows the results of analyzing the therapeutic efficacy of STAT3 bioPROTAC (4E8_A8.005) and a competitor drug (Dupilumab) in an atopic dermatitis-induced animal model using MC903 by H&E staining. Double arrows indicate epidermal hyperplasia, and single arrows indicate hyperkeratosis and parakeratosis.

[0089] Figure 22 shows the results of confirming the STAT3 degradation ability of two STAT3 bioPROTAC compounds (4E8.005, 4E8_A8.005) and a STAT3 PROTAC compound (SD-36) in human cancer cell lines (SiHa cell lines).

[0090] Figure 23 shows the results of confirming the cancer cell growth inhibitory activity of STAT3 bioPROTAC (4E8_A8.005) in various cancer cell lines (SiHa, NCI-H1703, FaDu, SW900). Data are presented as mean ± standard error (SEM). Statistical analysis was performed using an unpaired t-test with Welch correction (*p < 0.05, ** p < 0.01).

[0091] Figure 24 shows the results of comparing the concentration-dependent in vitro anticancer efficacy of STAT3 bioPROTAC (4E8_A8.005) with various STAT3 inhibitors (KT-333, siSTAT3, Oxaliplatin).

[0092] Figure 25 shows the results of confirming the STAT3 degradation ability of STAT3 bioPROTAC in linear or circular mRNA form and STAT3 PROTAC compound (SD-36) in various cancer cell lines (Huh-7, SiHa).

[0093] Figure 26 shows the results of confirming the combined effect of STAT3 bioPROTAC (4E8_A8.005) and sorafenib in an animal model of liver cancer.

[0094] Figure 27 shows the FR and CDR sequences of each anti-STAT3 single-domain antibody.

[0095] FIG. 28 shows an E3 ubiquitin ligase fragment sequence (005) constituting a STAT3 bioPROTAC as one embodiment of the present invention and two types of STAT3 bioPROTAC sequences (4E8.005, 4E8_A8.005) containing the same.

[0096] The following detailed description of the present invention will be described with reference to specific drawings (where drawings are available) regarding specific embodiments in which the present invention may be practiced, but the present invention is not limited thereto and is limited only by the appended claims to all scopes identical or equivalent to those described in the claims. It should be understood that various embodiments of the present invention are different but need not be mutually exclusive. For example, specific shapes, structures, and characteristics described in this specification may be modified from one embodiment to another or realized by combining multiple embodiments without departing from the technical spirit and scope of the present invention. Technical and academic terms used in this specification have the same meaning as commonly used in the field to which the present invention belongs, unless otherwise defined. For the purpose of interpreting this specification, the following definitions shall apply, and terms expressed in the singular form shall be interpreted as also referring to the plural form (i.e., at least one) unless inappropriate in the context, and vice versa.

[0097] In the numerical ranges described in this specification, “to” is used to mean including both threshold ranges (greater than or equal to and less than), and when not including both threshold ranges, the numerical range is described as “greater than” and “less than.” In this specification, the term “about” used for numerical values ​​is used to mean a range that is expected to produce an effect substantially equivalent to the stated numerical value by a person skilled in the art; for example, it may be ±20%, ±10%, ±5%, etc. of the stated numerical value, but is not limited thereto.

[0098] The present invention relates to a new therapeutic technology that selectively degrades the STAT3 protein, which is known to be a major cause of cancer and inflammatory diseases. In the present invention, targeting STAT3, which was difficult to target with existing small molecule compounds, a single-domain antibody that specifically binds to STAT3 was discovered, and a novel STAT3 bioPROTAC was produced by fusing an E3 ubiquitin ligase fragment to it. Furthermore, it was confirmed that the STAT3 bioPROTAC is effective in treating atopic dermatitis and tumors.

[0099]

[0100] Anti-STAT3 monodomain antibody or its antigen-binding fragment

[0101] In the present invention, six anti-STAT3 single-domain antibodies (A4, 1D6, 2D1, 3E5, 4E8, 5A5) that specifically bind only to STAT3 and do not bind to other STAT family proteins (STAT1, STAT6) were discovered through phage display technology. In addition, three anti-STAT3 single-domain antibodies (4E8_A8, 4E8_A9, 4E8_A10) with improved binding affinity to STAT3 were additionally discovered through random mutation of the discovered anti-STAT3 single-domain antibodies.

[0102] Accordingly, in one aspect, the present invention relates to an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof.

[0103] As one embodiment, the present invention provides an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof.

[0104] In one embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise an HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 50, SEQ ID NO 12, SEQ ID NO 19, SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 40, and SEQ ID NO 46 based on Kabat numbering; an HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 14, SEQ ID NO 21, SEQ ID NO 28, SEQ ID NO 36, SEQ ID NO 42, and SEQ ID NO 47; and an HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 44, SEQ ID NO 48, and SEQ ID NO 51.

[0105] In one embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 50, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 16.

[0106] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 12, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 16.

[0107] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 19, HCDR2 having the amino acid sequence of SEQ ID NO. 21, and HCDR3 having the amino acid sequence of SEQ ID NO. 23.

[0108] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 26, HCDR2 having the amino acid sequence of SEQ ID NO. 28, and HCDR3 having the amino acid sequence of SEQ ID NO. 30.

[0109] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 34, HCDR2 having the amino acid sequence of SEQ ID NO. 36, and HCDR3 having the amino acid sequence of SEQ ID NO. 38.

[0110] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 40, HCDR2 having the amino acid sequence of SEQ ID NO. 42, and HCDR3 having the amino acid sequence of SEQ ID NO. 44.

[0111] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 46, HCDR2 having the amino acid sequence of SEQ ID NO. 47, and HCDR3 having the amino acid sequence of SEQ ID NO. 48.

[0112] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 12, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 51.

[0113] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 50, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 51.

[0114] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise, based on IMGT numbering, HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO. 88, SEQ ID NO. 53, SEQ ID NO. 59, SEQ ID NO. 65, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 79 and SEQ ID NO. 84; HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO. 55, SEQ ID NO. 61, SEQ ID NO. 67, SEQ ID NO. 75, SEQ ID NO. 81 and SEQ ID NO. 86; and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 63, SEQ ID NO. 69, SEQ ID NO. 77, SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 89.

[0115] In one embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 88, HCDR2 having the amino acid sequence of SEQ ID NO. 55, and HCDR3 having the amino acid sequence of SEQ ID NO. 57.

[0116] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 53, HCDR2 having the amino acid sequence of SEQ ID NO. 55, and HCDR3 having the amino acid sequence of SEQ ID NO. 57.

[0117] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 59, HCDR2 having the amino acid sequence of SEQ ID NO. 61, and HCDR3 having the amino acid sequence of SEQ ID NO. 63.

[0118] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 65, HCDR2 having the amino acid sequence of SEQ ID NO. 67, and HCDR3 having the amino acid sequence of SEQ ID NO. 69.

[0119] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 71, HCDR2 having the amino acid sequence of SEQ ID NO. 55, and HCDR3 having the amino acid sequence of SEQ ID NO. 57.

[0120] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 73, HCDR2 having the amino acid sequence of SEQ ID NO. 75, and HCDR3 having the amino acid sequence of SEQ ID NO. 77.

[0121] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 79, HCDR2 having the amino acid sequence of SEQ ID NO. 81, and HCDR3 having the amino acid sequence of SEQ ID NO. 83.

[0122] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 84, HCDR2 having the amino acid sequence of SEQ ID NO. 86, and HCDR3 having the amino acid sequence of SEQ ID NO. 87.

[0123] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 71, HCDR2 having the amino acid sequence of SEQ ID NO. 55, and HCDR3 having the amino acid sequence of SEQ ID NO. 89.

[0124] As another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 88, HCDR2 having the amino acid sequence of SEQ ID NO. 55, and HCDR3 having the amino acid sequence of SEQ ID NO. 89.

[0125] In one embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 4.

[0126] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 5.

[0127] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 6.

[0128] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 7.

[0129] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 8.

[0130] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 9.

[0131] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 10.

[0132] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 90.

[0133] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 91.

[0134] In another embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may comprise or be composed of the amino acid sequence of SEQ ID NO. 92.

[0135] The specific sequences of FR1 to FR4 and CDR1 to CDR3 constituting the anti-STAT3 single-domain antibody or its antigen-binding fragment of the present invention are described in Table 5, Table 6 or FIG. 27, and the anti-STAT3 single-domain antibody or its antigen-binding fragment of the present invention may comprise a combination of the CDR1 to CDR3 sequences of each anti-STAT3 single-domain antibody described in Table 5, Table 6 or FIG. 27, or may be composed of each anti-STAT3 single-domain antibody sequence described in Table 5, Table 6 or FIG. 27.

[0136] The term "Signal Transducer and Activator of Transcription 3 (STAT3)" refers to a transcription factor encoded by the STAT3 gene in humans, belonging to the STAT protein family. STAT3 is activated by various extracellular signals, particularly cytokines and growth factors, and translocates to the nucleus via JAK-mediated phosphorylation to act as a transcription regulator. Through these processes, it is involved in various physiological functions, including cell growth, differentiation, survival, and the regulation of immune responses.

[0137] In the present invention, STAT3 may be included without limitation as long as it is mammalian STAT3, but preferably refers to human STAT3 or primate STAT3. Furthermore, in the present invention, the STAT3 protein includes all of the wild type, fragments thereof, or variants thereof, but is not limited thereto. The "wild type" includes all proteins found in nature or nucleic acids encoding them, and may be described interchangeably with the wild type. The wild type STAT3 protein generally refers to a polypeptide containing the amino acid sequence of the wild type STAT3 protein, and the amino acid sequence of the wild type protein generally refers to the amino acid sequence found in naturally occurring STAT3. The STAT3 gene may be identified as NCBI Entrez Gene 6774 or HGNC:11364, and the STAT3 protein may be identified as UniProtKB P40763. In the present invention, the STAT3 protein may include the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0138] The term "anti-STAT3 single-domain antibody" refers to an antibody molecule that specifically binds to STAT3 having a single variable region.

[0139] The above "single domain antibody (sdAb)" generally refers to an antibody having binding activity against an antigen that contains only a single heavy chain variable (VH). While sdAbs derived from heavy chains are primarily used, single variable region fragments derived from light chains are also reported to bind specifically to antigens. In the present invention, the single domain antibody may be an antibody that does not contain a light chain and comprises a single chain FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4 in the order from the N-terminus to the C-terminus. The single domain antibody is used interchangeably with heavy chain antibodies, VHH, nanobody VHH, nanobody, or HCAb (heavy chain only antibody); although it lacks a light chain, HCAb possesses a distinct antigen-binding mechanism. The variable region (VHH region) of a heavy chain antibody represents the smallest known antigen-binding unit generated by an adaptive immune response. The above antibody may include not only the complete antibody form but also antigen-binding fragments of the antibody molecule.

[0140] The term "antigen-binding fragment" refers to one or more fragments of an intact antibody that possess the ability to specifically bind to a given antigen, namely STAT3. The antibody fragments in the present invention include, but are not limited to, single-chain antibodies, bispecific antibodies, trispecific antibodies, multispecific antibodies such as diabodies, triabodidies, tetrabodies, Fab fragments, F(ab')2 fragments, Fd, scFv, domain antibodies, minibodies, scaps (sterol regulatory binding protein cleavage activating protein), chelating recombinant antibodies, bibodies, intrabodies, nanobodies, SMIPs (small modular immunopharmaceuticals), binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, antibody constant domain derivatives, and synthetic antibodies based on protein scaffolds having the ability to bind to STAT3.

[0141] It is obvious to a person skilled in the art that any fragment of the antibody according to the present invention will exhibit the same characteristics as the antibody according to the present invention as long as the binding function to STAT3 is maintained.

[0142] The term “heavy chain (HC)” refers to the entire length heavy chain and fragments thereof, including a variable region domain VH containing an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3.

[0143] The term "variable" (V) indicates that specific parts of the variable region differ significantly in sequence among antibodies. The variable region mediates antigen binding and defines the specificity of a particular antibody for a specific antigen. Variability in all heavy chain variable regions is concentrated in three segments called the hypervariable region (HVR), or CDR. A more highly conserved part of the variable region is called the framework (FR) region. The heavy chain variable region has structures FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus.

[0144] The term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region (HVR) of the immunoglobulin heavy chain. The heavy chain variable region contains three CDRs (HCDR1, HCDR2, and HCDR3). CDRs provide key contact residues for the binding of antibodies to antigens or epitopes.

[0145] In some embodiments, the anti-STAT3 single-domain antibody of the present invention or its antigen-binding fragment may comprise or be composed of an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity with any one of the amino acid sequences of SEQ ID NOs 4 to 10 and SEQ ID NOs 90 to 92. In other embodiments, the anti-STAT3 single-domain antibody of the present invention or its antigen-binding fragment may comprise an amino acid sequence in which some amino acid residues in any one of the amino acid sequences of SEQ ID NOs 4 to 10 and SEQ ID NOs 90 to 92 are conserved amino acid substituted.

[0146] In some embodiments, each CDR sequence according to the present invention may comprise or be composed of an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity with the proposed amino acid sequence. In other embodiments, each CDR sequence may comprise a sequence in which some amino acid residues in the amino acid sequence are substituted with conservative amino acids.

[0147] The term “conservative amino acid substitution” means that one class of amino acids is replaced by another amino acid of the same class. Conservative amino acid substitution does not alter the structure, function, or both of the polypeptide. Conservative amino acid substitutions are provided in Table 1 under the item “preferred substitutions” and are further described below in relation to amino acid side chain classes (1) through (6). Amino acid substitutions can be introduced into products screened for molecules of interest and desired activities, e.g., maintained / improved antigen binding, reduced immunogenicity, or improved physical properties.

[0148] [Table 1]

[0149]

[0150] Amino acids may be grouped as follows according to their common side chain properties, except where otherwise stated in this specification:

[0151] (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

[0152] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0153] (3) Acids: Asp, Glu;

[0154] (4) Basics: His, Lys, Arg;

[0155] (5) Residues affecting chain orientation: Gly, Pro;

[0156] (6) Aromatic: Trp, Tyr, Phe.

[0157] The present invention provides, as another embodiment, a polynucleotide encoding an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof. In one embodiment, the polynucleotide may comprise or be composed of any one base sequence selected from the group consisting of SEQ ID NOs 121 to 130.

[0158] In the present invention, the term "polynucleotide" is also referred to as "nucleic acid" and refers to a polymer of nucleotides of any length. Specifically, the polynucleotide may be DNA or RNA.

[0159] In the present invention, if the polynucleotides encode the same polypeptide, one or more bases may be modified by substitution, deletion, insertion, or a combination thereof. When preparing polynucleotide sequences by chemical synthesis, synthesis methods widely known in the art may be used, for example, the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl, 37:73-127, 1988), and methods such as triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on solid supports may be used.

[0160] In addition, in the present invention, the polynucleotide may comprise or be composed of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with any one nucleotide sequence selected from the group consisting of SEQ ID NOs 121 to 130. Furthermore, it is obvious to a person skilled in the art that the polynucleotide according to the present invention may be provided with codon optimization as necessary.

[0161] The above polynucleotide may additionally include a signal sequence or a lead sequence.

[0162] As another embodiment, the present invention provides an expression vector loaded with a polynucleotide encoding an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof.

[0163] As used herein, the term "vector" refers to a substance for carrying or expressing a nucleic acid sequence comprising a nucleic acid sequence encoding the anti-STAT3 single-domain antibody variant described herein.

[0164] Specifically, the vector comprises linear nucleic acids, plasmids, phages, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, and baculoviruses. Additionally, the plasmid may include a selection marker, such as an antibiotic resistance gene, and the host cell containing the plasmid may be cultured under selective conditions.

[0165] More specifically, the vector may be plasmid DNA, phage DNA, etc., and may be a commercially developed plasmid (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (e.g., pUB110, pTP5, etc.), a yeast-derived plasmid (e.g., YEp13, YEp24, YCp50, etc.), a phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, gt10, gt11, AZAP, etc.), an animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (baculovirus, etc.). Since the protein expression levels and modifications of the above vector vary depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0166] In addition, the vector of the present invention may be fused with other sequences to facilitate the purification of antibodies expressed therefrom. The fused sequences may include, for example, Flag (IBI, USA), glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), 6xHis (Qiagen, USA), Halo, Myc, V5, HA, Strep-tag, T7 tag, NanoLuc, SUMO, MBP, Protein A, Protein G, or Fc fragment.

[0167] In addition, when the protein expressed by the vector of the present invention contains Fc, the expressed antibody can be easily purified through a Protein A column, etc., without an additional sequence for purification.

[0168] As another embodiment of the present invention, a transgenic cell is provided into which an expression vector loaded with an anti-STAT3 single-domain antibody or a polynucleotide encoding an antigen-binding fragment thereof is introduced.

[0169] As used herein, the term "transformed cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The transformed cell may be produced by introducing the vector into a host cell and transforming it. Additionally, the anti-STAT3 single-domain antibody of the present invention or its antigen-binding fragment may be produced by expressing the polynucleotide contained in the vector.

[0170] The above transformation can be performed by various methods. As long as the anti-STAT3 single-domain antibody of the present invention or its antigen-binding fragment can be produced, it is not particularly limited. Specifically, the transformation method may include the CaCl2 precipitation method, the Hanahan method which increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation method, protoplasmic fusion method, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation method, PEG-mediated transformation method, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation method. Additionally, the target product may be delivered into the cell using viral particles by means of infection. Furthermore, the vector may be introduced into the host cell by means of gene bombardment, etc.

[0171] In addition, the host cell used for producing the above-mentioned transformed cell is not particularly limited as long as it is capable of producing the antibody of the present invention. Specifically, the host cell may include, but is not limited to, prokaryotic cells, eukaryotic cells, mammalian, plant, insect, fungal, or cellular cells. As an example of the prokaryotic cell, Escherichia coli may be used. In addition, as an example of the eukaryotic cell, yeast may be used. Furthermore, as the mammalian cell, CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC6 cells, HEK293 cells, or HEK293T cells may be used, but is not limited thereto, and any cell known to those skilled in the art that can be used as a mammalian host cell may be used.

[0172] In addition, to optimize the properties of the antibody as a therapeutic agent or for other purposes, glycosylation-related genes in the host cell can be manipulated through methods known to those skilled in the art to adjust the glycosylation pattern of the antibody (e.g., sialic acid, fucosylation, glycosylation).

[0173] As another embodiment of the present invention, a method for producing an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof is provided, comprising the step of culturing the transformed cells.

[0174] Specifically, a method for producing the antibody or a fragment thereof may include the step of culturing a transformed cell to produce an antibody and the step of obtaining the antibody produced from the culture medium.

[0175] The method of culturing the above-mentioned transformed cells can be carried out using methods widely known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the anti-STAT3 single-domain antibody of the present invention or its antigen-binding fragment. Specifically, the culture can be carried out continuously in a batch process or a fed batch or repeated fed batch process.

[0176] Additionally, the step of obtaining the antibody specific to STAT3 and its fragment from the culture medium may be carried out by a method known in the art. Specifically, the obtaining method is not particularly limited as long as it can obtain the produced antibody of the present invention or its fragment. Preferably, the obtaining method may be a method such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion).

[0177] fusion protein (bioPROTAC)

[0178] In the present invention, a total of six types of STAT3 bioPROTACs (A4.005, 1D6.005, 2D1.005, 3E5.005, 4E8.005, 5A5.005) were produced by fusing the above anti-STAT3 single-domain antibodies with an E3 ubiquitin ligase fragment. In addition, additional bioPROTACs (4E8_A8.005, 4E8_A9.005, 4E8_A10.005) were produced based on variants (4E8_A8, 4E8_A9, 4E8_A10) with enhanced binding affinity to STAT3, and it was confirmed that the STAT3 protein degradation efficiency of these variant-based bioPROTACs increased.

[0179] Accordingly, in another aspect, the present invention relates to a bioPROTAC comprising an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof.

[0180] In one embodiment, the present invention provides a fusion protein comprising an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof; and a fragment of an E3 ubiquitin ligase or a variant thereof.

[0181] The anti-STAT3 single-domain antibody or its antigen-binding fragment is as described above.

[0182] The term "bioPROTAC" means a polypeptide comprising a target binding domain and an E3 ubiquitin ligase (or a fragment or variant thereof), and unless otherwise noted, it is also used to mean a polynucleotide encoding the same, a vector containing the same, a transformed cell containing the same, and a pharmaceutical composition. In the present invention, bioPROTAC may be used interchangeably with a fusion protein, a polynucleotide encoding the fusion protein, a vector containing the same, and a transformed cell containing the same.

[0183] The term "ubiquitin (UB)" refers to a protein composed of 76 amino acids that is present in almost all eukaryotic cells. Ubiquitin has the characteristic of covalently bonding to other proteins through an ATP-dependent reaction, which is called ubiquitination.

[0184] The above ubiquitin is covalently bonded to a target protein (hereinafter interchangeably with substrate protein) through 1) activation performed by a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), 2) conjugation, and 3) ligation. The target protein ubiquitinated (poly-Ub) as described above is degraded by a proteasome.

[0185] The term "E3 ubiquitin ligase" refers to an enzyme in the final step of ubiquitination that binds ubiquitin to a substrate. E3 ligase binds to a target protein and E2-Ub, thereby inducing the ubiquitination (poly-Ub) of the target protein by positioning the target protein close to E2-Ub. In the present invention, the E3 ubiquitin ligase can be used interchangeably as E3 ligase, E3 ubiquitin ligase, E3 ubiquitin binding enzyme, or E3.

[0186] The term "fragment" may refer to a specific substrate recognition domain, a ubiquitin transfer domain, or two of these domains within the E3 ubiquitin ligase complex.

[0187] In the present invention, the E3 ubiquitin ligase may be any one selected from the group consisting of βTrCP (beta-ransducin repeat-containing protein), SKP2 (S-phase kinase-associated protein 2), VHL (von hippel-lindau), SPOP (speckle-type BTB-POZ), SOCS2 (suppressor of cytokine signaling 2), CHIP (carboxy terminus of Hsp70-binding protein), DDB2 (damage DNA binding protein 2), CRBN (cereblon), ASB1 (ankyrin repeat and SOCS box protein 1), TRIM21 (tripartite motif-containing protein 21), RNF4 (RING finger protein), UBE2D1 (ubiquitin conjugating enzyme E2 D1), UBE2D4, and ZNRF1 (zinc and ring finger 1).

[0188] In one embodiment, the E3 ubiquitin ligase may be SPOP. In another embodiment, the E3 ubiquitin ligase fragment may be the SPOP 167-374 fragment of the entire SPOP (Uniprot ID: 043791) (amino acids 167-374, see SEQ ID No. 100 and FIG. 28, hereinafter used interchangeably with '005').

[0189] In one embodiment, a variant of βTrCP is βTrCP of full-length (Uniprot ID: Q9Y297) 2-263 (Amino acid 2-263, SEQ ID NO. 141) fragment or βTrCP 190-228 (Amino acids 190–228, SEQ ID NO. 142) may be a fragment. In one embodiment, the SKP2 variant is SKP2 of the full SKP2 length (Uniprot ID: Q13309).2-176 (Amino acid 2-176, SEQ ID NO. 143) may be a fragment. In one embodiment, the variant of VHL is VHL of the full length of VHL (Uniprot ID: P40337). 152-213 (Amino acids 152–213, SEQ ID NO. 144) may be a fragment. In one embodiment, the variant of SOCS2 is SOCS2 of the full length (Uniprot ID: 014508). 143-198 It may be a fragment (amino acids 143-198, SEQ ID 145). In one embodiment, the variant of CHIP is the CHIP of the full length of CHIP (Uniprot ID: Q9UNE). 128-303 It may be a fragment (amino acids 128–303, SEQ ID NO. 146). In one embodiment, the variant of DDB2 is DDB2 of the full length of DDB2 (Uniprot ID: Q92466). 2-114 (Amino acid at positions 2-114, SEQ ID NO. 147) may be a fragment. In one embodiment, the variant of CRBN is CRBN of the full length of CRBN (Uniprot ID: Q96SW2). 2-320 (Amino acid at positions 2-320, SEQ ID NO. 148) may be a fragment. In one embodiment, the variant of ASB1 is ASB1 of the full length of ASB1 (Uniprot ID: Q9Y576). 266-335 (Amino acids 266–335, SEQ ID 149) may be a fragment. In one embodiment, the variant of TRIM21 is TRIM21 of the full length of TRIM21 (Uniprot ID: P19474). 2-85 (Amino acid 2-85, SEQ ID NO. 150) may be a fragment. In one embodiment, the variant of RNF4 is RNF4 of the full length of RNF4 (Uniprot ID: P78317). 71-190 (Amino acid 71–190, SEQ ID NO. 151) may be a fragment. In one embodiment, the variant of UBE2D1 is UBE2D1 of the full length of UBE2D1 (Uniprot ID: P51668). 2-147(Amino acid 2-147, SEQ ID 152) may be a fragment. In one embodiment, the variant of UBE2D4 is UBE2D4 of the full length of UBE2D4 (Uniprot ID: Q9Y2X8). 2-147 (Amino acid 2-147, SEQ ID NO. 153) may be a fragment. In one embodiment, the variant of ZNRF1 is ZNRF1 of the full length of ZNRF1 (Uniprot ID: Q8ND2). 138-227 (Amino acid 138-227, SEQ ID No. 154) may be a fragment. Specific details regarding this may be found in International Publication No. 2025 / 105856, which is incorporated herein by reference.

[0190] In the fusion protein of the present invention, the anti-STAT3 single-domain antibody or its antigen-binding fragment may be located at the N'-terminus or C'-terminus of a fragment of E3 ubiquitin ligase or a variant thereof.

[0191] In one embodiment, the anti-STAT3 single-domain antibody or its antigen-binding fragment may be directly linked to a fragment of E3 ubiquitin ligase or a variant thereof.

[0192] In another embodiment, the fusion protein may include a linker. In this case, an anti-STAT3 single-domain antibody or its antigen-binding fragment may be combined with a fragment of E3 ubiquitin ligase or a variant thereof by the linker.

[0193] The above linker may be a peptide linker. In the present invention, the anti-STAT3 single-domain antibody of the fusion protein or its antigen-binding fragment, and the fragment of E3 ubiquitin ligase or a variant thereof may be connected through the linker.

[0194] The above peptide linker may consist of 1 to 30 consecutive amino acids, 2 to 20 consecutive amino acids, or 2 to 10 amino acids. In one embodiment, the above peptide linker may be (GS)n (where n is an integer from 1 to 10). In this case, n in (GS)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The above peptide linker may use, without special limitation, sequence linkers known in the art such as GS, GGGGS, (GGGGS)2, (GGGS)3, ASTKGP, ASTKGPSVFPLAP, TR, etc., which impart structural flexibility without being cleaved by proteolytic enzymes, but are not limited thereto. In one embodiment, the linker may be a GS linker.

[0195] In the present invention, the fusion protein may additionally include a sequence for purification (e.g., a tag protein sequence).

[0196] In one embodiment, the fusion protein may comprise or be composed of any one of the amino acid sequences of SEQ ID NOs 90 to 92 and SEQ ID NOs 4 to 10; and the amino acid sequence of SEQ ID NO. 100. The fusion protein may comprise or be composed of any one amino acid sequence selected from the group consisting of SEQ ID NOs 131 to 139. The fusion protein may comprise an amino acid sequence in which some amino acid residues in any one of the amino acid sequences selected from the group consisting of SEQ ID NOs 131 to 139 are conserved amino acid substituted. In some embodiments, the fusion protein comprises a GS peptide linker, but is not limited thereto. In the present invention, the fusion protein may be formed by directly combining two or more polypeptides or by combining them via different peptide linkers.

[0197] In another embodiment, the present invention provides a polynucleotide encoding a fusion protein comprising the anti-STAT3 single-domain antibody or its antigen-binding fragment; and a fragment of E3 ubiquitin ligase or a variant thereof.

[0198] In one embodiment, the polynucleotide encoding the fusion protein may comprise or be composed of any one of the base sequences of SEQ ID NO. 121 to SEQ ID NO. 130 and the base sequence of SEQ ID NO. 140.

[0199] The above polynucleotide may include or be composed of any one of the nucleotide sequences of SEQ ID NOs 101 to 106 and SEQ ID NOs 110 to 112.

[0200] The above polynucleotide may comprise or be composed of a base sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with any one base sequence selected from the group consisting of each of SEQ ID NOs 101 to 106 and SEQ ID NOs 110 to 112.

[0201] The above polynucleotide may be modified by substitution, deletion, insertion, or a combination thereof, of one or more bases. When preparing the nucleotide sequence by chemical synthesis, synthesis methods widely known in the art, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl, 37:73-127, 1988), may be used.

[0202] The above polynucleotide may be DNA, mRNA, plasmid DNA, etc., and the above polynucleotide or a vector containing (loaded with) it may be delivered into a cell by a cell-permeable functional nanocarrier to exhibit the same target protein degradation efficacy as a fusion protein.

[0203] The above polynucleotide may be mRNA. In some embodiments, the mRNA may be linear mRNA. Linear mRNA may include an open reading frame (ORF) encoding a protein and may additionally include a 5' cap structure and / or a poly(A) tail at the 3' end. In other embodiments, the mRNA may be circular mRNA. Circular mRNA has a ring structure in which both ends of the open reading frame (ORF) encoding a protein are connected. Due to these structural features, it has higher resistance to RNase, is more stable than linear mRNA, and allows for longer-term protein expression. Additionally, translation of circular mRNA may be initiated through a cap-independent mechanism, such as an internal ribosome entry site (IRES).

[0204] In another embodiment, the present invention provides an expression vector loaded with a polynucleotide encoding a fusion protein comprising the anti-STAT3 single-domain antibody or its antigen-binding fragment; and a fragment of E3 ubiquitin ligase or a variant thereof.

[0205] In one embodiment, the polynucleotide encoding the fusion protein may comprise or be composed of any one of the nucleotide sequences selected from SEQ ID NOs 101 to 106 and SEQ ID NOs 110 to 112.

[0206] In another embodiment, the present invention provides a transgenic cell into which an expression vector loaded with a polynucleotide encoding a fusion protein comprising the anti-STAT3 single-domain antibody or its antigen-binding fragment; and a fragment of E3 ubiquitin ligase or a variant thereof has been introduced.

[0207] In another embodiment, the present invention provides a method for producing said fusion protein comprising: the step of culturing said transformed cells; and the step of obtaining a fusion protein comprising an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof, and a fragment of E3 ubiquitin ligase or a variant thereof from a culture medium of said cells.

[0208] In the polynucleotide encoding a fusion protein, an expression vector loaded with said polynucleotide, a transformed cell into which said expression vector has been introduced, and a method for producing a fusion protein, reference may be made to the contents described in the paragraph 'anti-STAT3 single-domain antibody or antigen-binding fragment thereof' so as not to be mutually contradictory.

[0209] use

[0210] In this invention, STAT3 bioPROTAC not only demonstrated the effect of restoring the expression of key skin barrier proteins under atopic induction conditions, but also confirmed that atopic dermatitis can be effectively treated in an animal model. Furthermore, it was confirmed that it effectively inhibited cancer cell proliferation in various anticancer cell lines, as well as effectively inhibited tumor growth in a liver cancer animal model.

[0211] Accordingly, in another aspect, the present invention relates to the use of a fusion protein comprising an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof.

[0212] In one embodiment, the present invention provides a composition comprising, as an active ingredient, the fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector has been introduced.

[0213] The fusion protein, the polynucleotide encoding the fusion protein, and the vector containing the polynucleotide are the same as described above.

[0214] The above composition may be a pharmaceutical composition, and in one embodiment, may be a pharmaceutical composition for the prevention or treatment of atopic dermatitis or cancer.

[0215] The above composition may be a food composition, and in one embodiment, may be for the prevention or improvement of atopic dermatitis or cancer.

[0216] The above composition may be a cosmetic composition, and in one embodiment, may be for the prevention or improvement of atopic dermatitis or cancer.

[0217] The above composition may be a quasi-drug composition, and in one embodiment, may be for the prevention or improvement of atopic dermatitis or cancer.

[0218] In another embodiment, the present invention provides a method for preventing or treating atopic dermatitis, comprising the step of administering to a subject in need the above composition or, as an active ingredient of the above composition, a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector has been introduced.

[0219] In another embodiment, the present invention provides a use for preventing or treating atopic dermatitis in a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector has been introduced, as the composition or as an active ingredient of the composition.

[0220] In another embodiment, the present invention provides the use of a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector is introduced, as an active ingredient of the composition or the composition for manufacturing a drug for the prevention or treatment of atopic dermatitis.

[0221] In another embodiment, the present invention provides a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector is introduced, as an active ingredient of the composition or the composition for the prevention or treatment of atopic dermatitis.

[0222] In another embodiment, the present invention provides a method for preventing or treating cancer, comprising the step of administering to a subject requiring the above composition or, as an active ingredient of the above composition, a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector has been introduced.

[0223] In another embodiment, the present invention provides a use for preventing or treating cancer in a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector has been introduced, as the composition or as an active ingredient of the composition.

[0224] In another embodiment, the present invention provides the use of a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector is introduced, as an active ingredient of the composition or the composition for manufacturing a drug for the prevention or treatment of cancer.

[0225] In another embodiment, the present invention provides a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector is introduced, as an active ingredient of the composition or the composition for the prevention or treatment of cancer.

[0226] In one embodiment, the fusion protein may be formulated in the form of a purified recombinant protein and introduced into a cell. As a means of formulation, lipid nanoparticles (LNP) containing ionized lipids, liposomes, polymer nanoparticles (e.g., PLGA, PEI), exosomes / vesicles, or cell-permeable peptide fusions (TAT, etc.) may be used. The formulation may be designed to promote endosome escape and may add a target-directed ligand as needed.

[0227] In another embodiment, the polynucleotide or a vector containing (loaded with) the same may be expressed in vivo using an mRNA formulation (including LNP), plasmid DNA (non-viral), or viral vector (AAV, lentivirus, etc.). The polynucleotide may be DNA, mRNA, plasmid DNA, etc., and the polynucleotide or a vector containing (loaded with) the same may be delivered into cells by a cell-permeable functional nanocarrier to exhibit the same target protein degradation efficacy as the fusion protein. The mRNA may include a cap structure, a 5′′UTR, and a poly(A) tail, and may undergo processes such as codon optimization, base modification, and removal of unnecessary dsRNA as needed.

[0228] The term "atopic dermatitis" or "atopic dermatitis" refers to a chronic or recurrent inflammatory skin disease accompanied by pruritus, which is a general term for a condition exhibiting eczematous lesions (e.g., erythema, papules, exudation / erosion, scaling, lichenification, etc.) due to impaired skin barrier function and abnormal immune regulation.

[0229] The term "cancer" is a collective term for diseases caused by cells that possess aggressive characteristics—dividing and proliferating beyond normal growth limits—invasive characteristics—infiltrating surrounding tissues—and metastatic characteristics—spreading to other parts of the body; it is used synonymously with "malignant tumor."

[0230] The above cancer may be any one selected from the group consisting of breast cancer, colorectal cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, glioma, ovarian cancer, cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0231] In one embodiment, the fusion protein, the polynucleotide encoding the fusion protein, or the vector loaded with the polynucleotide as the active ingredient of the composition or the composition may be administered alone without being used in combination with other anticancer agents.

[0232] In another embodiment, the composition or, as an active ingredient of the composition, a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector has been introduced may be administered in combination with one or more other agents effective as anticancer agents. The other agents include, but are not limited to, any agents capable of preventing or treating cancer, such as compounds, gene therapies, proteins (including antibodies), and immunotherapies. When the composition disclosed herein or, as an active ingredient of the composition, a fusion protein, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, or a transformed cell into which the vector has been introduced is administered together with other agents, they may be formulated as a single composition for simultaneous delivery, or may be formulated individually into two or more compositions (e.g., a kit). Each component may be administered to the subject at a different time than when the other components are administered. In certain embodiments, each administration may be given non-simultaneously (e.g., individually or sequentially) at multiple intervals over a given period. In addition, individual components may be administered to the subject via the same or different routes.

[0233] In some embodiments, a fusion protein, a polynucleotide encoding said fusion protein, a vector loaded with said polynucleotide, or a transformed cell into which said vector has been introduced may be administered in combination with a compound as a composition disclosed herein or as an active ingredient of said composition. In one embodiment, the compound may be a multi-target tyrosine kinase inhibitor and may be, for example, one or more selected from sorafenib, regorafenib, lenvatinib, cabozantinib, sunitinib, pazopanib, vandetanib, axitinib, nintedanib, tivozanib, apatinib, ponatinib, ripretinib, foretinib, anlotinib, and donafenib, but is not limited thereto.

[0234] In some embodiments, the composition disclosed herein or as an active ingredient of said composition, a fusion protein, a polynucleotide encoding said fusion protein, a vector loaded with said polynucleotide, or a transformed cell into which said vector has been introduced may be administered in combination with a cytotoxic agent / chemotherapy agent. said cytotoxic agent / chemotherapy agent may be one or more selected from cisplatin, carboplatin, oxaliplatin, doxorubicin, epirubicin, paclitaxel, docetaxel, irinotecan, topotecan, gemcitabine, 5-fluorouracil (5-FU), capecitabine, cyclophosphamide, ifosfamide, methotrexate, bleomycin, vincristine, vinblastine, and vinorelbine, but is not limited thereto.

[0235] In some embodiments, the composition disclosed herein or the fusion protein, the polynucleotide encoding the fusion protein, the vector loaded with the polynucleotide, or the transformed cell into which the vector is introduced as an active ingredient of the composition may be administered in combination with an immunotherapeutic agent. Since the immunotherapeutic agent has a novel mechanism of killing cancer cells by activating the body's immune cells, it has the advantage of being widely applicable to most cancers even without specific gene mutations. Furthermore, in that the immunotherapeutic agent treats cancer by strengthening the patient's own immune system, it has fewer side effects and brings about effects that improve the patient's quality of life and significantly extend survival time. The immunotherapeutic agent that can be formulated as a single composition or individual composition with the composition of the present invention or the fusion protein, the polynucleotide encoding the fusion protein, the vector loaded with the polynucleotide, or the transformed cell into which the vector is introduced as an active ingredient of the composition may include an immune checkpoint inhibitor and may be manufactured by a known method or be a commercially available product. Examples of immunotherapies include, but are not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG-3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-4-1BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-VISTA, anti-NKG2A, anti-BTLA, anti-PVRIG, anti-CD73, anti-B7-H3, anti-IDO-1, anti-CD200, and anti-TIGIT antibodies.In some embodiments, the immunotherapeutic agent may comprise one or more selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG-3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-4-1BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-VISTA, anti-NKG2A, anti-BTLA, anti-PVRIG, anti-CD73, anti-B7-H3, anti-IDO-1, anti-CD200, and anti-TIGIT antibodies. In other embodiments, the immunotherapeutic agent may comprise one or more selected from the group consisting of anti-PD-1 and anti-PD-L1 antibodies.

[0236] In some embodiments, a method for preventing or treating cancer may be provided, comprising the step of administering one or more other agents effective as said anticancer agents sequentially, simultaneously, or in reverse order with the composition disclosed herein or said composition as an active ingredient of said composition, said fusion protein, said polynucleotide encoding said fusion protein, said polynucleotide loaded with said polynucleotide, or said vector introduced into a transformed cell.

[0237] The term "treatment" generally means achieving desired pharmacological and / or physiological effects. These effects are therapeutic in that they partially or completely cure a disease and / or other unwanted or undesirable conditions. Desirable therapeutic effects include, but are not limited to, the prevention of the onset or recurrence of the disease, improvement of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. Preferably, "treatment" may mean medical intervention for an already manifested disease or disorder.

[0238] The term "prevention" means obtaining the intended preventive pharmacological and / or physiological effects in the sense of partially or completely preventing a disease or its symptoms.

[0239] In the present invention, the pharmaceutical composition may be administered in a therapeutically effective amount. A "therapeutically effective amount" or "pharmaceuticalally effective amount" refers to an amount of a compound or composition effective in preventing or treating a target disease, which is sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause adverse effects. The level of the effective amount may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. In one embodiment, the therapeutically effective amount refers to an amount of drug effective in treating the disease.

[0240] The term "administration" means providing an active ingredient to an individual to achieve a preventive or therapeutic purpose.

[0241] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. A pharmaceutically acceptable adjuvant (buffer, dispersant) may also be included in the pharmaceutical composition.

[0242] The pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not possess toxicity beyond tolerable limits for the subject of application (prescription).

[0243] The pharmaceutical composition may be administered systemically via parenteral administration. Parenteral administration may be administered intranasally, intranasally, orally, intravenously, intramuscularly, intra-arterly, intramedullary, intradurally, intracardiaclysmically, transdermally, subcutaneously, intradermally, intraperitoneally, intestinally, topically, sublingually, or rectally, but is not limited thereto.

[0244] When a pharmaceutical composition comprises a polynucleotide or a vector containing (loaded with) the same as an active ingredient, the nucleic acid may be used in combination with various delivery vehicles, such as lipid nanoparticles (LNPs), liposomes, or vesicles, which are known to effectively deliver polynucleotides into cells, but are not limited thereto.

[0245] The pharmaceutical composition may be prepared in, for example, powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposome suspension, polyflex, emulsion, lipid nanoparticles (LNP) having RNA on their surface or encapsulated therein, or any other suitable form that can be administered to humans or mammals requiring treatment.

[0246] When a pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of an injectable, transdermal, nasal inhalant, or suppository according to methods known in the art with a suitable carrier. When formulated as an injectable, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof; preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or isotonic solutions such as 5% dextrose may be used. Regarding the formulation of pharmaceutical compositions, the art is well known, and specifically, reference may be made to the literature [Remington's Pharmaceutical Sciences (19th ed, 1995)]. The said literature is considered to be part of this specification.

[0247] The term "quasi-drug" refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, which have a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are defined as items excluding those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans or animals, as well as products that have a mild effect on the human body or do not act directly on it.

[0248] The quasi-drug composition of the present invention may be used for the purpose of preventing or improving atopic dermatitis, and is not particularly limited in its formulation, and may be a transdermal formulation such as a lotion, ointment, gel, cream, patch, or spray.

[0249] In addition, for each formulation, the quasi-drug composition may arbitrarily select and combine other ingredients according to the formulation or purpose of use of other quasi-drugs. The amount of the active ingredient can be appropriately determined according to the purpose of use (inhibition or alleviation). For example, it may include conventional adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0250] The cosmetic composition of the present invention may be used for the purpose of preventing or improving atopic dermatitis, and may have, for example, a formulation of a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a pack, a mask pack, a mask sheet, a gel, or a skin-adhesive type cosmetic.

[0251] In the present invention, the food may be a health functional food. The health functional food composition of the present invention may be used as a health functional food, a food additive, or a dietary supplement. When the composition of the present invention is used as a food additive, it may be appropriately used according to conventional methods, such as by adding it as is or by mixing it with other foods or food ingredients. In addition, the mixing amount of the health functional food composition may be appropriately changed according to the purpose of use (prevention, health, or therapeutic treatment).

[0252] The term "subject" is used interchangeably with "subject," "individual," and "patient," and may be a mammal requiring prevention or treatment of a disease or illness, e.g., primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In one embodiment, the subject to which the pharmaceutical composition may be applied (prescribed) may be a mammal, preferably a human.

[0253] To the extent that there is no mutual contradiction in this specification, descriptions relating to a pharmaceutical composition may also be interpreted as descriptions relating to an active ingredient of said composition.

[0254] In addition, in all cases described herein as embodiments in the form of a pharmaceutical composition, it should be understood that other similar forms described as pharmaceutical use forms, Swiss-type pharmaceutical use forms and / or treatment method forms for using the same are also provided.

[0255]

[0256] Examples

[0257] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0258] Example 1. Discovery of Anti-STAT3 Single-Domain Antibody (sdAb)

[0259] Signal transducer and activator of transcription 3 (STAT3 (Gly127-Arg688), UniProt: P40763, SEQ No. 1) (Sinobiological, Cat no. 10034-H07E), STAT1 (UniProt: P42224, SEQ No. 2) (Novusbio, Cat no. NBP3-18204), and STAT6 (UniProt: P42226, SEQ No. 3) (Sinobiological, Cat no. 13190-H08B) antigens were purchased and used for the discovery of STAT3-specific antibodies.

[0260] [Table 2]

[0261]

[0262] Anti-STAT3 sdAbs were selected from a single domain antibody (sdAb) naive phage display library using STAT3 (SEQ No. 1), STAT1 (SEQ No. 2), and STAT6 (SEQ No. 3) as antigens, respectively, by Genscript Probio. As a result of analyzing the sequences of anti-STAT3 sdAbs obtained through the selection of anti-STAT3 sdAbs that bind to STAT3 but not to STAT1 and STAT6 via 4 round panning, a total of 5 types were finally derived: 1D6 (SEQ No. 4), 2D1 (SEQ No. 5), 3E5 (SEQ No. 6), 4E8 (SEQ No. 7), and 5A5 (SEQ No. 8).

[0263] Selected clones, VHH13 clone (SBT-100, SEQ ID NO. 9, see US9,695,234) and A4 clone (VHH13 binding affinity enhancement variant clone, SEQ ID NO. 10) were inserted into pYD5, a yeast surface expression vector constructed with reference to the paper (Wang Zet et al., "A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities". Protein Eng Des Sel. (2005) 18:337-343, doi:10.1093 / protein / gzi036).

[0264] [Table 3]

[0265]

[0266] [Table 4]

[0267]

[0268] [Table 5]

[0269]

[0270]

[0271] [Table 6]

[0272]

[0273]

[0274] For reference, Table 5 shows the FR and CDR sequences of single-domain antibodies based on Kabat numbering, and Table 6 shows the FR and CDR sequences of single-domain antibodies based on IMGT numbering (see Fig. 27). Each of the above clones was cultured at 30°C for about 16 hours in SD-CAA medium (20 g glucose (SIGMA, Cat no. G7528), 14.7 g sodium citrate (SIGMA, Cat no. C8532), 4.3 g citric acid monohydrate (SIGMA, Cat no. C0706), 6.7 g yeast nitrogen base (BD Difco, Cat no. 291940), 5 g bacto casamino acid (BD Difco, Cat no. 223050), 100 µg / mL kanamycin (Biosesang, Cat no. KC1001-025-02) / 1 L distilled water). The above culture solution is added to SG-CAA medium via OD 600 After diluting to a value of 1 and culturing for 18 hours at 30°C, anti-STAT3 sdAb was induced to be expressed on the surface of the yeast.

[0275] Cultured yeast 0.5×10 7Primary staining was performed by mixing 10 nM biotinylated STAT3 or 100 nM biotinylated off-target antigens (STAT1, STAT6, and EGFRviii proteins (Acrobiosystems, Cat no. EGI-H52H4)) and a 1:500 ratio anti-V5 mouse antibody (Invitrogen, Cat no. R960-25) (diluted in wash buffer (PBS containing 0.1% BSA) at a 1:500 ratio) and reacting at room temperature for 30 minutes. Then, Streptavidin, R-Phycoerythrin Conjugate (SAPE) (Invitrogen, Cat no. SA10044), and anti-mouse IgG(H+L)-FITC antibody (Invitrogen, Cat no. 11-4011-85), diluted in wash buffer at a 1:100 ratio, were added and incubated in the dark at 4°C for 20 minutes (secondary Staining). The binding affinity of anti-STAT3 sdAb to STAT3 protein was confirmed by analyzing the above reaction results using a flow cytometer (SONY, SH800S).

[0276] As a result, as shown in Figure 1, it was confirmed that the anti-STAT3 sdAbs prepared as described above specifically bind only to STAT3.

[0277]

[0278] Example 2. Discovery of variants with enhanced binding affinity of anti-STAT3 single-domain antibody

[0279] Example 2.1. Construction of a library of anti-STAT3 single-domain antibody 4E8

[0280] To enhance the binding affinity of the single domain antibody (sdAb) clone 4E8 selected in Example 1 above, a library was constructed and selection was performed. The gene encoding the anti-STAT3 single domain antibody 4E8 was inserted into the yeast surface expression vector pYD5, and the corresponding plasmid was divided into four regions (H1 (SEQ No. 71), H2 (SEQ No. 55), H3A (NALRLI, SEQ No. 155), H3B (LPRYDY, SEQ No. 156)) containing the CDR, part of the CDR, or part of the CDR and surrounding amino acids. Primers were synthesized (IDT Technology) to induce random mutations in those regions, and PCR was performed using them.

[0281] To create a random library of the four regions mentioned above, two pairs of forward and reverse primers were used per region, and two PCR products (fragment1, fragment2) were produced through 1st PCR amplification using High-Fidelity DNA Polymerase (Thermo Scientific, F530L). Then, 2nd PCR (overlap extension PCR) was performed on the two fragments to obtain PCR products with random sequences in each region.

[0282] 5 μg of the final synthesized PCR product and 1 μg of pYD5 vector were placed in an electro-cuvette and electroporated into prepared EBY100 yeast (ATCC, Cat no. MYA-4941) competent cells to obtain approximately 0.5 × 10⁻⁶ 7 - 10×10 7 We built libraries with diversity for each.

[0283] Example 2.2. Analysis of a variant monoclone with enhanced binding affinity

[0284] The library constructed in Example 2.1 was induced to be expressed on the surface of yeast using the method of Example 1. Cultured yeast 4×10 7 Primary staining was performed by mixing 100 nM biotinylated STAT3 and a 1:500 ratio anti-V5 mouse antibody (Invitrogen, Cat no. R960-25) (diluted in wash buffer (PBS containing 0.1% BSA) at a 1:500 ratio) and reacting at room temperature for 30 minutes. Then, Streptavidin, R-Phycoerythrin Conjugate (SAPE) (Invitrogen, Cat no. SA10044), and anti-mouse IgG(H+L)-FITC antibody (Invitrogen, Cat no. 11-4011-85), diluted in wash buffer at a 1:100 ratio, were added and reacted in the dark at 4°C for 20 minutes (secondary staining). The above reaction mixture was analyzed using a flow cytometer (SONY, SH800S) to identify individuals within the top approximately 0.1% of yeast colonies with improved binding affinity to STAT3 compared to 4E8 The cells were isolated, and this process was repeated three times. After the last cycle, monoclones were expressed on the surface of the isolated yeast population using the same method, and after staining and analysis, clones with high binding affinity were selected.

[0285] [Table 7]

[0286]

[0287] [Table 8]

[0288]

[0289] Three selected anti-STAT3 single-domain antibody clones (4E8_A8, 4E8_A9, and 4E8_A10) were expressed and stained on the surface of yeast in the same manner as in Example 1, and then analyzed using a flow cytometer (SONY, SH800S) to confirm that the binding affinity to STAT3 protein was enhanced compared to 4E8 (Fig. 2).

[0290] Example 2.3. Confirmation of STAT3-specific binding affinity (NanoBRET assay)

[0291] To determine whether anti-STAT3 single-domain antibodies specifically bind to the STAT3 protein within actual cells, the STAT3-specific binding affinity of two anti-STAT3 single-domain antibodies (4E8 and 4E8_A8) was measured using a NanoBRET™® substrate (Promega, Cat no. N1661) according to the manufacturer's method.

[0292] Specifically, the expression vector for the STAT3 protein (pHTC-STAT3) was constructed by loading polynucleotides encoding STAT3 and Halo tags sequentially at the N-terminus into the pHTC vector (Promega, Cat no. G7711). In addition, the expression vectors for each of the two single-domain antibody clones (pNLF1-N-4E8, pNLF1-N-4E8_A8) were constructed by loading polynucleotides encoding NanoLuc tags and the two single-domain antibody clones sequentially at the N-terminus into the pNLF1 vector (Promega, Cat no. N1351), respectively.

[0293] [Table 9]

[0294]

[0295] [Table 10]

[0296]

[0297]

[0298] 0.5 ng of either the pNLF1-N-4E8 or pNLF1-N-4E8_A8 vector was added, and the pHTC-STAT3 vector was prepared by serially diluting it in 1 / 2 increments starting from 50 ng to a final concentration of 1.56 ng, and the two vectors were mixed in a ratio of 1:100 to 1:3.13. Subsequently, human embryonic kidney cell lines (HEK293T cells) (ATCC) were transduced using Lipofectamine 3000 (Invitrogen, Cat no. L3000001), and pNLF1-N-MDM2 and the pHTC-p53 expression vector (Promega, Cat no. G9801) were used as positive controls. 24 hours after transduction, 0.1 μL of HaloTag 618 (Promega, Cat no. G9801), a ligand that binds to the Halo tag, was added to HEK293T cells per well and cultured for an additional 6 hours. Subsequently, luminescence was induced by adding NanoBRET™Nano-Glo® substrate (Promega, Cat no. N1661) and measured using a microplate reader (BioTek, Synergy HTX).

[0299] As a result, as shown in Figure 3, it was confirmed that both types of anti-STAT3 single-domain antibodies specifically bind to the STAT3 protein within the cell.

[0300] Example 2.4. Confirmation of STAT3-specific binding affinity (FACS assay)

[0301] To confirm the binding affinity of the anti-STAT3 single-domain antibodies 4E8 and 4E8_A8 to the STAT3 protein, antigen binding affinity at different concentrations was measured using a flow cytometer. 4E8 and 4E8_A8 were induced to be expressed on the surface of yeast using the method of Example 1. Cultured yeast 0.5 × 10⁶ 7Primary staining was performed by mixing a mouse anti-V5 antibody at a 1:500 ratio with 19 concentrations of biotinylated STAT3 sequentially diluted by half from 0 nM to 400 nM and reacting at room temperature for 30 minutes. Secondary staining was performed by diluting Streptavidin, R-Phycoerythrin Conjugate (SAPE), and anti-mouse IgG(H+L)-FITC antibodies at a 1:100 ratio in wash buffer and reacting in the dark at 4°C for 20 minutes. The binding patterns of the reaction mixtures at different concentrations of biotinylated STAT3 in the final clones were measured using a flow cytometer (SONY, SH800S), and the results were analyzed using the flow-jo program.

[0302] As a result, as shown in Figure 4, both the anti-STAT3 single-domain antibodies 4E8 and 4E8_A8 specifically bound to the STAT3 protein. In particular, 4E8_A8 showed a rapid increase in binding signal at lower concentrations and a higher binding strength at the same concentration, demonstrating superior binding affinity and efficacy compared to 4E8.

[0303]

[0304] Example 3. Production of anti-STAT3 single-domain antibody and Fc fusion protein and confirmation of STAT3-specific binding

[0305] Example 3.1. Preparation of Anti-STAT3 Single-Domain Antibody and Fc Fusion Protein

[0306] Anti-STAT3 single-domain antibody 4E8 and anti-STAT3 single-domain antibody 4E8_A8, which have enhanced binding affinity with anti-STAT3 single-domain antibody 4E8, were loaded onto pAD15 (refer to Korean Patent Publication No. 10-2017-0053511) in a form conjugated with human Fc, expressed in Expi-CHO-S cells, and then purified into the form of sdAb-Fc using a Protein A affinity column (MabSelect SuRe (Pre-packed, Hiscreen), Cytiva, Cat no. 17-5474-15).

[0307] [Table 11]

[0308]

[0309] Example 3.2. Confirmation of STAT3-specific binding affinity (ELISA assay)

[0310] STAT3-specific binding of the purified 4E8-Fc and 4E8_A8-Fc from Example 3.1 was evaluated through ELISA analysis.

[0311] To this end, STAT3 protein or off-target antigens (STAT1, STAT6, and IRAK4 (Sinobiological., Cat no. 10735-H07B)) were mixed with 1X PBS to a concentration of 1 μg / mL, added to a 96-well microplate (Corning® Half Area Clear Flat Bottom Polystyrene High Bind Microplate; Corning, Cat no. 3690), and coated for approximately 16 hours. After washing with 0.05% PBS-T using a Microplate Washer (Biotek, 450TSRS-SN), the plates were blocked with 0.05% S-PBST (3% Skim milk) for 1 hour. Subsequently, the portion of the plate coated with the off-target antigen was reacted with 1 μM concentration of 4E8-Fc or 4E8_A8-Fc at room temperature for 2 hours, and the portion coated with STAT3 protein was reacted with 7 concentrations of 4E8-Fc or 4E8_A8-Fc, sequentially diluted 1 / 10 from 0 nM to 1 μM, at room temperature for 2 hours. After washing with 0.05% PBS-T, anti-Fc HRP (Invitrogen, Cat no. A18817) was diluted in 0.05% S-PBST (3% Skim milk) at a ratio of 1:5000 and reacted for 1 hour. Finally, the binding and non-specific binding patterns at different STAT3 concentrations were confirmed using a microplate reader (Biotek, EPOCH2NSC-SN).

[0312] As a result, as shown in Figure 5, both 4E8-Fc and 4E8_A8-Fc specifically bound to STAT3 in a concentration-dependent manner, and in particular, 4E8_A8-Fc showed a much stronger binding affinity to STAT3.

[0313] Example 3.3. STAT3 Binding Affinity Analysis (BLI assay)

[0314] The binding affinity of 4E8-Fc and 4E8_A8-Fc purified in Example 3.1 was analyzed. Binding affinity for STAT3 protein was determined according to the manufacturer's method using Octet®Fc Capture (AHC) Biosensors (Sartorius, Cat no. 18-5060) in Biolayer interference (BLI) (Sartorius, Octet R8 system). Specifically, after confirming the baseline, 4E8-Fc and 4E8_A8-Fc were captured in the AHC biosensor through the loading step. STAT3 protein was used as an analyte in the association step by sequentially diluting it in Octet®Buffer 10X (Sartorius, 18-1105) to eight concentrations ranging from 0 nM to 500 nM in 1 / 2 increments. The dissociation rate constant (kd) and binding rate constant (ka) of each antibody were analyzed using Octet Analysis Studio 13.0 software, and the equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0315] As a result, as shown in Figure 6, it was confirmed that both 4E8-Fc and 4E8_A8-Fc bind to STAT3 in a concentration-dependent manner. In particular, 4E8_A8-Fc showed a significantly lower equilibrium dissociation constant (KD) compared to 4E8-Fc, with a larger binding rate constant (ka) and a smaller dissociation rate constant (kd). This implies that 4E8_A8-Fc has a stronger and more stable binding affinity for STAT3.

[0316]

[0317] Example 4. Production of STAT3 bioPROTAC and Verification of Resolution

[0318] Example 4.1. Preparation of STAT3 bioPROTAC

[0319] To confirm the STAT3 protein-specific degradation ability of bioPROTAC containing each of the five selected single-domain antibodies and E3 ubiquitin ligase, fusion proteins containing each of the five selected single-domain antibodies (A4, 1D6, 2D1, 3E5, 4E8, and 5A5) and an E3 ubiquitin ligase fragment were constructed.

[0320] The above fusion protein comprises an anti-STAT3 sdAb (A4, 1D6, 2D1, 3E5, 4E8, or 5A5)-GS(linker)-E3 ubiquitin ligase fragment and was constructed in a form containing a Flag-tag at the N-terminus of the anti-STAT3 single-domain antibody. The polynucleotides encoding the above fusion protein were each loaded into a pCMV6 (Origene, Cat no. PS100001) vector for mammalian cell expression to construct six types of STAT3 bioPROTAC expression vectors. Hereinafter, the fusion proteins containing the above anti-STAT3 single-domain antibody and the E3 ubiquitin ligase fragment as essential components are described interchangeably with A4.005, 1D6.005, 2D1.005, 3E5.005, 4E8.005, and 5A5.005, respectively. As one example, A4.005, 1D6.005, 2D1.005, 3E5.005, 4E8.005, and 5A5.005 were fabricated to additionally include a Flag-tag and linker sequence (in the form of 'Flag Tag - TR linker - sdAb - GS linker - E3 fragment').

[0321] In addition, a polynucleotide encoding full-length STAT3 containing a V5-tag at the N-terminus was loaded into the pCMV3 (Sinobiological, Cat no. HG10034-NM, Myc-tag replaced with V5-tag) vector to construct a STAT3 expression vector.

[0322] [Table 12]

[0323]

[0324] [Table 13]

[0325]

[0326] [Table 14]

[0327]

[0328] [Table 15]

[0329]

[0330]

[0331] [Table 16]

[0332]

[0333] [Table 17]

[0334]

[0335] Example 4.2. Confirmation of STAT3 resolution ability of STAT3 bioPROTAC

[0336] To confirm the STAT3-specific degradation ability of the six types of bioPROTACs prepared in Example 4.1, each bioPROTAC expression vector and STAT3 expression vector were mixed into human embryonic kidney cell lines (HEK293; Human embryonic kidney 293 cells) (ATCC) at a ratio of 20:1 (specifically, 2 μg of bioPROTAC expression vector and 0.1 μg of STAT3 expression vector) and transduced using Lipofectamine 3000 (Invitrogen, Cat. No. L3000001). HEK293 cell lines were cultured in DMEM medium (Gibco, Cat. No. 11995065) supplemented with 10% FBS (Gibco, Cat. No. 26140-079) and 1% penicillin-streptomycin (Gibco, Cat. No. 15140-122) at 37°C, 5% CO₂, and >95% humidity.

[0337] 24 hours after transduction, each cell was washed twice with cold PBS buffer (Gibco, Cat no. 10010-023) and treated with cell lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS) containing a protein and phosphatase inhibitor cocktail (Thermofisher, Cat no. 1861284) to obtain cell extracts. After heating the obtained cell extracts at 95°C for 10 minutes, the protein content was quantified using a BCA kit (ThermoFisher, Cat no. 23227), and equal amounts of protein from each sample were electrophoresed on a 4-12% SDS-PAGE gel (Invitrogen, Cat no. NW04125BOX). Subsequently, the sample was transferred to a PVDF membrane (Invitrogen, Cat no. IB24002) and blocked for 60 minutes at room temperature using a blocking buffer [1X buffer (1X0.1% Tween 20 (Bio-rad, Cat no. 1706531)) containing 5% skim milk (BD, Cat no. 232100)]. Next, the primary antibody was added and the reaction was carried out at 4°C for 16 hours. As the primary antibody, anti-V5 antibody (Invitrogen, Cat no. 46-1157, 1:5000), anti-Flag antibody (Sigma, Cat no. F1804, 1:2500), or anti-β-actin antibody (Sigma, Cat no. A5441, 1:10000) were each diluted in the blocking buffer and used. After the primary antibody reaction, The membrane was washed three times for 10 minutes each in 1X buffer solution, treated with the secondary antibody, and reacted at room temperature for 1 hour. At this time, the secondary antibody used was an anti-mouse antibody (CST, Cat no. 7076) conjugated with horseradish peroxidase (HRP) diluted in blocking buffer solution. After the secondary antibody reaction, the membrane was washed three times for 10 minutes each with 1X buffer solution at room temperature.

[0338] The expression level of each protein was confirmed using Imager (Invitrogen, iBright, CL1500) after treatment with ECL (Cytiva, Cat no. RPN2232) solution, and the amount of V5 (STAT3) protein relative to β-actin was quantified and displayed as a graph (Graphpad prism software).

[0339] As a result, as shown in Figure 7a, it was confirmed that 4 out of 6 types of bioPROTACs effectively degraded STAT3 protein, and in particular, A4.005, 2D1.005, and 4E8.005 showed STAT3 protein degradation ability of more than 70% compared to the control group (Mock).

[0340] Four types of STAT3 bioPROTACs (A4.005, 1D6.005, 2D1.005, 4E8.005) that showed significant STAT3 degradation in the results of Fig. 7a were selected and cloned into a Linearized pIVT vector (TAKARA, Cat no. 6143) according to the manufacturer's method using the TAKARA Cloning Kit for mRNA Template (TAKARA, Cat no. 6143). Using the above vector as a template, mRNAs encoding the four types of STAT3 bioPROTACs were prepared using the Takara IVTpro™T7 mRNA Synthesis Kit (TAKARA, Cat no. 6144), CleanCap Reagent AG (TriLink, Cat no. N7113-10), and N1-Methyl-Pseudouridine-5'-Triphosphate (TriLink, Cat no. N1081-10).

[0341] The synthesized STAT3 bioPROTAC mRNA was transduced into human primary neonatal skin keratinocytes (HEKn cells; Normal human epidermal keratinocyte) (ATCC, Cat no. PCS-200-010) using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015).

[0342] HEKn cell lines were cultured in Dermal Cell Basal Medium (ATCC, Cat. No. PCS-200-030) supplemented with Keratinocyte Growth Kit (ATCC, Cat. No. PCS-200-040) and 1% Penicillin-Streptomycin-Amphotericin B Solution (ATCC, Cat. No. PCS-999-002) under conditions of 37°C, 5% CO₂, and >95% humidity. A negative control group treated only with Lipofectamine Messenger MAX (MM only) was included, and each bioPROTAC mRNA was treated at concentrations of 0.15, 1.5, and 15 nM. At this time, 1.5 mM calcium chloride (Sigma, Cat. No. 21115-100 ml) was added, and cells were harvested after 24 hours. STAT3 protein expression analysis was performed according to the previously described method, and anti-STAT3 (CST, 9139, 1:2500), anti-Flag (Sigma, Cat. No. F1804, 1:2500), and anti-β-actin (Sigma, A5441, 1:10000) were diluted in blocking buffer and used as primary antibodies. As a result of quantifying STAT3 protein expression levels relative to β-actin and graphing them using GraphPad Prism software, all four types of STAT3 bioPROTACs exhibited concentration-dependent STAT3 degradation activity, among which 4E8.005 showed the most superior STAT3 degradation ability (Fig. 7b).

[0343]

[0344] Example 5. Production of STAT3 bioPROTAC containing a variant with enhanced binding affinity of anti-STAT3 single-domain antibody and confirmation of STAT3 resolution

[0345] Example 5.1. Production of STAT3 bioPROTAC containing a binding affinity-enhancing variant of an anti-STAT3 single-domain antibody

[0346] To confirm the STAT3 protein-specific degradation ability of bioPROTAC containing a single domain antibody (4E8) or a binding affinity-enhancing variant (4E8_A8, 4E8_A9 and 4E8_A10) and an E3 ubiquitin ligase, a fusion protein was constructed using the method of Example 4.1.

[0347] The polynucleotides encoding the above fusion proteins were each loaded into the pCMV6 (Origene, Cat no. PS100001) vector for mammalian cell expression. Hereinafter, fusion proteins containing the above anti-STAT3 single-domain antibody and the E3 ubiquitin ligase fragment as essential components are described in combination with 4E8.005, 4E8_A8.005, 4E8_A9.005, or 4E8_A10.005. As one example, 4E8.005, 4E8_A8.005, 4E8_A9.005, or 4E8_A10.005 were constructed to additionally include a Flag-tag and a linker sequence (in the form of 'Flag Tag - TR linker - sdAb - GS linker - E3 fragment').

[0348] [Table 18]

[0349]

[0350] [Table 19]

[0351]

[0352] [Table 20]

[0353]

[0354] Meanwhile, polynucleotides encoding fusion proteins 4E8.005, 4E8_A8.005, 4E8_A9.005, or 4E8_A10.005 were each cloned into a Linearized pIVT vector (TAKARA, Cat no. 6143), and mRNAs encoding 4E8.005, 4E8_A8.005, 4E8_A9.005, or 4E8_A10.005 were each prepared using the vector as a template according to the method of Example 4.1.

[0355] Example 5.2. Confirmation of STAT3 resolution ability of STAT3 bioPROTAC containing a variant with enhanced binding affinity of anti-STAT3 single-domain antibody

[0356] The 4E8.005, 4E8_A8.005, 4E8_A9.005, or 4E8_A10.005 expression vectors prepared in Example 4.1 or 5.1 and the STAT3 expression vector prepared in Example 4.1 were transduced into HEK293 cell lines at a ratio of 20:1 using Lipofectamine 3000 (Invitrogen, Cat. No. L3000001) for 24 hours. The experimental method was the same as in Example 4.2.

[0357] Anti-V5 antibody (Invitrogen, Cat no. 46-1157, 1:5000), anti-Flag antibody (Sigma, Cat no. F1804, 1:2500), or anti-α-tubulin antibody (GeneTex, Cat no. GTX628802, 1:3000) were used as primary antibodies. As a result of quantifying the amount of V5 (STAT3) protein relative to α-tubulin and plotting it on a graph (Graphpad prism software), it was confirmed that STAT3 degradation by 4E8_A8.005 was superior to that of 4E8.005 (Fig. 8a).

[0358] In addition, four types of linear mRNA (0.1, 8 nM) encoding STAT3 bioPROTAC produced in Example 4.1 or Example 5.1 (4E8.005, 4E8_A8.005, 4E8_A9.005, or 4E8_A10.005) were transduced into human cervical cancer cell lines (SiHa cells) (ATCC) for 16 hours using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015).

[0359] SiHa cell lines were cultured in EMEM (Eagle's Minimum Essential Media; ATCC, Cat no. 30-2003) supplemented with 10% FBS (Gibco, 26140-079) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37°C, 5% CO₂, and >95% humidity.

[0360] Protein expression analysis was performed in the same manner as in Example 4.2, where the primary antibodies used were anti-STAT3 (CST, 9139, 1:2500), anti-Flag antibody (Sigma, Cat no. F1804, 1:2500), or anti-β-actin (Sigma, A5441, 1:10000), each diluted in blocking buffer. As a result of quantifying the amount of STAT3 protein relative to β-actin and plotting it on a graph (Graphpad prism software), it was confirmed that the STAT3 resolution ability of all three variants (4E8_A8.005, 4E8_A9.005, or 4E8_A10.005) bioPROTACs was improved compared to 4E8.005, and among them, 4E8_A8.005 was found to exhibit the best STAT3 resolution ability (Fig. 8b).

[0361]

[0362] Example 6. Confirmation of STAT3 degradation mechanism in STAT3 bioPROTAC

[0363] Example 6.1. Confirmation of Ubiquitin-Proteasome-Dependent STAT3 Degradation Ability

[0364] We confirmed whether the degradation of STAT3 protein induced by three types of STAT3 bioPROTACs (A4.005, 4E8.005, 4E8_A8.005) occurs through the intracellular ubiquitin-proteasome system (UPS).

[0365] Specifically, human primary normal human epidermal keratinocytes (HEKn cells) were pretreated for 30 minutes with the ubiquitin E1 activator MLN7243 (MedChemExpress, Cat no. HY-100487) or the proteasome inhibitor MG132 (Sigma, Cat no. 474790) at concentrations of 1 μM and 10 μM, respectively, and then transduced with 1 nM of mRNA encoding A4.005, 4E8.005, or 4E8_A8.005 using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015) and cultured for 4 hours. At this time, a group treated with 0.5 μM of SD-36 (MCE, Cat no. HY-129602), a STAT3 compound PROTAC, was included as a positive control, and a group treated only with Lipofectamine-Messengermax (MM only) was included as a negative control. Protein expression analysis was performed in the same manner as in Example 4.2, and the primary antibodies used were anti-STAT3 (CST, 9139, 1:2500), anti-Flag (Sigma, Cat no. F1804, 1:2500), anti-ubiquitin (SCBT, sc-8017, 1:1000), or anti-β-actin (Sigma, A5441, 1:10000), each diluted in blocking buffer.

[0366] As a result, as shown in Fig. 9, it was confirmed that the degradation of STAT3 by A4.005, 4E8.005, or 4E8_A8.005 was significantly reduced by MLN7243 or MG132. Therefore, it was found that the degradation of STAT3 by the STAT3 bioPROTAC of the present invention is induced in a ubiquitin-proteasome system-dependent manner.

[0367] Example 6.2. Confirmation of the Mechanism of Action of STAT3 BioPROTAC

[0368] To confirm the mechanism of action of the STAT3 bioPROTAC of the present invention, mCherry, VHH GFP For the expression of 4.005, 4E8_A8, 4E8_A8.005, or 4E8_A8.005_L2, the polynucleotides encoding each were cloned into a Linearized pIVT vector (TAKARA, Cat no. 6146), and mCherry, VHH GFP Linear mRNAs encoding 4.005, 4E8_A8, 4E8_A8.005, or 4E8_A8.005_L2, respectively, were prepared according to the method of Example 4.1.

[0369] [Table 21]

[0370]

[0371] [Table 22]

[0372]

[0373]

[0374] Recombinant proteins of human interleukin-4 (Peprotech, Cat. No. 200-04), interleukin-13 (Peprotech, Cat. No. 200-13), interleukin-22 (Peprotech, Cat. No. 200-22), and interleukin-31 (Peprotech, Cat. No. 200-31) were each treated to HEKn cell lines at a concentration of 10 ng / mL. After 15 minutes, the synthesized linear mRNA (1 nM) was transduced into the HEKn cell lines using Lipofectamine MessengerMAX (Invitrogen, Cat. No. LMRNA015). After 24 hours, STAT3 protein expression was compared and analyzed using the same method as in Example 4.2. For protein detection, anti-STAT3 (CST, Cat. No. 9139, 1:2500), anti-phosphorylated STAT3 (Y705) (CST, Cat. No. 9138, 1:1000), anti-VHH (GenScript, Cat. No. A02016, 1:5000), or anti-β-actin (Sigma, Cat. No. A5441, 1:10000) were each diluted in blocking buffer and used as primary antibodies.

[0375] As a result, as shown in Figure 10, significant STAT3 degradation was confirmed in the experimental group transduced with 4E8_A8.005 mRNA, whereas STAT3 degradation was not clearly induced in the other experimental groups. Therefore, it was confirmed that the normal function of the anti-STAT3 single-domain antibody portion and the E3 ubiquitin ligase fragment portion of STAT3 bioPROTAC is essential for STAT3 degradation.

[0376]

[0377] Example 7. Confirmation of STAT3 selective degradation ability of STAT3 bioPROTAC

[0378] Example 7.1. Detection of changes in STAT family proteins

[0379] STAT3 protein belongs to the STAT family and has high amino acid sequence similarity with STAT1, STAT5, and STAT6. Therefore, we investigated whether STAT3 bioPROTAC selectively degrades only STAT3 among the STAT family proteins.

[0380] Linear mRNA (1 nM) of mCherry, 005, 4E8.005, and 4E8_A8.005, prepared according to the same method as in Example 4.2, were each transduced into HEKn cell lines using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015), and STAT3 compound PROTAC SD-36 (MCE, Cat no. HY-129602) (1 μM) was treated as a control. After 4 hours, STAT1, STAT3, STAT5, and STAT6 protein expression analysis was performed in the same manner as in Example 4.2, and anti-STAT1 (CST, 9172, 1:1000), anti-STAT3 (CST, 9139, 1:2500), anti-STAT5 (CST, 94205, 1:1000), anti-STAT6 (CST, 9362, 1:1000), or anti-β-actin (Sigma, A5441, 1:10000) were used as primary antibodies.

[0381] As a result, as shown in Figure 11, it was confirmed that both types of STAT3 bioPROTAC (4E8.005, 4E8_A8.005) selectively degraded only STAT3 without affecting other STAT family proteins such as STAT1, STAT5, and STAT6.

[0382] Example 7.2. Quantitative proteomic analysis

[0383] Quantitative proteomic analysis was performed to confirm the selective resolution of STAT3 by two types of STAT3 bioPROTACs (4E8.005, 4E8_A8.005).

[0384] Linear mRNAs of 005, 4E8.005, and 4E8_A8.005 were transduced into HEKn cell lines using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015). Four hours after transduction, each cell was washed once with PBS buffer (Gibco, Cat no. 10010-023), detached using Trypsin-EDTA for Primary Cells (ATCC, Cat no. PCS-999-003), and the reaction was terminated using an equal amount of Trypsin Neutralizing Solution (ATCC, Cat no. PCS-999-004). The cell pellet separated by centrifugation was washed once with PBS buffer (Gibco, Cat no. 10010-023) and centrifuged again to obtain the cell pellet. Each protein sample was labeled using TMTpro reagent (Thermo Fisher Scientific), and the labeled samples were pooled and fractionated via high-pH reversed-phase fractionation. Subsequently, LC-MS / MS analysis was performed using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) and a nanoLC system. The acquired MS / MS data were mapped to the Uniprot human database using SAGE software, and relative protein expression levels were calculated based on reporter ion intensity. Differentially expressed proteins (DEPs) were analyzed using log₂(fold change) and -log₂(fold change). 10 Analysis was performed based on (p-value), and visualized using a volcano plot.

[0385] As a result, as shown in Figure 12, in the differential expression protein analysis between the 005 only group compared to 4E8.005 and the 005 only group compared to 4E8_A8.005, it was confirmed that STAT3 was selectively reduced in the STAT3 bioPROTAC-treated groups in both cases. On the other hand, no significant changes were observed in most proteins, including STAT family proteins other than STAT3. Therefore, it was confirmed that both types of STAT3 bioPROTAC selectively degrade STAT3 among intracellular proteins.

[0386]

[0387] Example 8. Comparison of STAT3 degradation ability between two STAT3 bioPROTACs and a STAT3 PROTAC compound

[0388] The STAT3 degradation ability of STAT3 bioPROTAC was compared and analyzed with SD-36, a compound-based STAT3 PROTAC.

[0389] Specifically, HEKn cells were cultured as in Example 4.2, and the human keratinocyte cell line HaCaT (AddexBio, Cat. No. T0020001) was cultured in DMEM medium (Gibco, Cat. No. 11995065) supplemented with 10% FBS (Gibco, Cat. No. 26140-079) and 1% penicillin-streptomycin (Gibco, Cat. No. 15140-122) under conditions of 37°C, 5% CO₂, and >95% humidity. For both cell lines, 16 hours prior to transduction, each cell was seeded into a 12-well plate at a density of 1.0 × 10⁵ cells / well and allowed to reach 70-80% confluence the following day.

[0390] For each cell line, 4E8.005 or 4E8_A8.005 mRNA was treated with serial dilutions starting from 5 nM in 1 / 3 increments to a final concentration of 0.25 pM, and transduction was performed using Lipofectamine MessengerMAX (Invitrogen, Cat. No. LMRNA015) according to the manufacturer's instructions. As a control, STAT3 PROTAC SD-36 was treated with serial dilutions starting from 5 μM in 1 / 3 increments to a final concentration of 0.25 nM.

[0391] Protein expression analysis was performed in the same manner as in Example 4.2, and the degradation rate was calculated as 100 minus relative intensity (%). Concentration-response curves were fitted using a 4-parameter logistic (Top / Bottom / Hill slope free) model with GraphPad Prism to DC 50 The (50% decomposition concentration) and Dmax (maximum decomposition rate) were calculated.

[0392] As a result, as shown in Figure 13, it was confirmed that both types of STAT3 bioPROTACs exhibited superior STAT3 resolution compared to SD-36.

[0393]

[0394] Example 9. Treatment with STAT3 bioPROTAC under atopic induction conditions

[0395] Example 9.1. Restorative efficacy of skin barrier proteins

[0396] To evaluate the therapeutic efficacy of STAT3 bioPROTAC under conditions inducing atopic dermatitis, skin barrier-related gene expression was analyzed.

[0397] Specifically, after culturing HEKn cells according to the manufacturer's instructions, 2.4 × 10⁴ cells were placed in a 6-well plate 16 hours prior to transduction. 5Cells were seeded into wells and allowed to reach 70-80% confluence the following day. For atopic induction, 1.5 mM CaCl₂ was added to the medium, and HEKn cells were treated with human recombinant interleukin-4, interleukin-13, interleukin-22, and interleukin-31 at a concentration of 5 ng / mL each.

[0398] Fifteen minutes after inducing atopy using interleukin-4, interleukin-13, interleukin-22, and interleukin-31, 4E8_A8.005 mRNA was transduced at a concentration of 0.33 or 1 nM using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015) according to the manufacturer's instructions. For the control group, the STAT3 compound-based PROTAC KT-333 (MCE, Cat no. HY-156730), the JAK1 inhibitor Upadacitinib (Selleckchem, Cat no. S8162), and the IL-4Ra neutralizing antibody Dupilumab (Selleckchem, Cat no. A2038) were treated at a concentration of 1 or 10 nM, respectively.

[0399] After 72 hours, cells were washed twice with cold PBS buffer (Gibco, Cat. No. 10010-023), and total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Cat no. 74136). Subsequently, cDNA was synthesized using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, 1809105), and quantitative PCR (qPCR) was performed using the primers listed in Table 23. The analysis results compared the relative expression levels of skin barrier-related genes (Filaggrin, Involucrin) based on GAPDH expression.

[0400] [Table 23]

[0401]

[0402] As a result, as shown in Figure 14, the reduced skin barrier-related gene expression under atopic induction conditions was restored by treatment with 4E8_A8.005 and a comparative drug. In particular, the highest recovery efficacy was observed in the 4E8_A8.005 treatment group, confirming that STAT3 bioPROTAC has superior therapeutic efficacy in atopic skin diseases compared to competing drugs.

[0403] Example 9.2. Efficacy of skin barrier protein recovery under various atopic dermatitis-induced conditions

[0404] The efficacy of STAT3 bioPROTAC in restoring skin barrier-related gene expression was compared with dupilumab, an IL-4Ra neutralizing antibody, under various atopic dermatitis induction conditions.

[0405] The experiment was performed in the same manner as in Example 9.1, with only the atopic induction conditions differing, and (i) recombinant human IL-4 and IL-13 (10 ng / mL each), and (ii) recombinant human IL-4, IL-13, IL-22, and IL-31 (10 ng / mL each) were administered. STAT3 bioPROTAC was administered starting from 3 nM and serially diluted in 1 / 3 increments down to 0.45 pM, and dupilumab (Dupilumab, Selleckchem, Cat no. A2038) was administered starting from 300 nM and serially diluted in 1 / 3 increments down to 45 pM.

[0406] As a result, as shown in Figure 15, STAT3 bioPROTAC more effectively restored the reduced expression of Filaggrin and Loricrin compared to dupilumab in both atopic induction conditions, thereby confirming the superior atopic therapeutic efficacy of STAT3 bioPROTAC.

[0407]

[0408] Example 10. Confirmation of STAT3 resolution ability of STAT3 bioPROTAC composed of linear or circular mRNA form

[0409] The STAT3 resolution of linear or circular STAT3 bioPROTAC (4E8_A8.005) was verified.

[0410] Human primary neonatal epidermal keratinocytes (HEKn) and human primary adult epidermal keratinocytes (HEKa) (ATCC, Cat no. PCS-200-011) were cultured according to the manufacturer's instructions. 16 hours before transduction, each cell was seeded into a 6-well plate at a density of 2.4 × 10⁵ cells / well to achieve 70-80% confluence at the time of transduction.

[0411] The linear mRNA of 4E8_A8.005 was synthesized via T7 in vitro transcription, and all UTPs were replaced with N1-methyl-pseudouridine-TP. Subsequently, after DNase treatment, Cap-1 was conferred via Vaccinia capping / 2′, and a poly(A) tail (approx. 100-120 nt) was added. The dsRNA of the synthesis product was removed using a cellulose spin column or RP-HPLC. The circular mRNA of 4E8_A8.005 was obtained by submitting a production request to GenScript, providing its amino acid sequence information.

[0412] Linear or circular mRNA was treated with serial dilutions starting from 3 nM in 1 / 3 increments up to 5.65 fM. Transduction was performed using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015) according to the manufacturer's instructions. As a control, STAT3 PROTAC SD-36 (MedChemExpress, HY-129602) was treated with serial dilutions starting from 3 μM in 1 / 3 increments up to 5.65 pM.

[0413] Protein expression analysis was performed 16 or 72 hours after treatment in the same manner as in Example 4.2. The degradation rate was calculated as 100 minus relative intensity (%), and the concentration-response curve was fitted using a 4-parameter logistic model (Top / Bottom / Hill slopes free) with GraphPad Prism to DC 50 (=50% decomposition concentration) and Dmax (maximum decomposition rate) were derived.

[0414] As a result, as shown in Figure 16, STAT3 degradation increased in a concentration-dependent manner in both linear and circular mRNA treatment groups, and both conditions showed superior STAT3 degradation efficacy compared to SD-36, a compound-based PROTAC.

[0415]

[0416] Example 11. Comparison of gene expression of skin barrier proteins by linear or circular mRNA forms of STAT3 bioPROTAC and a competitor drug (Dupilumab) under atopy-induced conditions

[0417] The skin barrier-related gene expression recovery effect of linear or circular STAT3 bioPROTAC (4E8_A8.005) was compared with dupilumab, a competing drug and IL-4Rα neutralizing antibody.

[0418] Human primary neonatal epidermal keratinocytes (HEKn) were cultured according to the manufacturer's instructions. 16 hours prior to transduction, each cell was seeded into a 6-well plate at a density of 2.4 × 10⁵ cells / well to achieve 70-80% confluence at the time of transduction.

[0419] Atopy induction was performed by adding 1.5 mM CaCl₂ to the medium and then stimulating for 15 minutes with recombinant human IL-4, IL-13, IL-22, and IL-31 (R&D Systems, see Example 6.2) at a final concentration of 10 ng / mL each. Subsequently, linear or circular mRNA of 4E8_A8.005 was treated by serially diluting from 3 nM in 1 / 3 increments to a final concentration of 0.45 pM, and transduction was performed using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015) according to the manufacturer's instructions. As a control, dupilumab was treated by serially diluting from 300 nM in 1 / 3 increments to a final concentration of 45 pM.

[0420] After 72 hours, total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized. The expression of skin barrier-related genes (Filaggrin, Loricrin) was analyzed by quantitative PCR (qPCR) using primers listed in Table 23, and the relative expression levels were calculated using GAPDH as an intrinsic control gene.

[0421] As a result, as shown in Figure 17, both linear and circular STAT3 bioPROTACs showed superior expression recovery effects of skin barrier-related genes (Filaggrin, Loricrin) compared to dupilumab under conditions of atopic dermatitis induction. In particular, it was confirmed that the circular mRNA-based STAT3 bioPROTAC exhibited higher efficacy than the linear form.

[0422]

[0423] Example 12. Comparison of the efficacy of restoring skin barrier proteins by STAT3 bioPROTAC in linear or circular mRNA form and a competitor drug (Upadacitinib) under atopic induction conditions.

[0424] Using the same experimental method as in Example 11, the effect of STAT3 bioPROTAC in linear or circular mRNA form on the restoration of skin barrier-related gene (Filaggrin, Loricrin) expression under conditions of atopic dermatitis induction was compared with that of the JAK1 inhibitor upadacitinib. Upadacitinib (Selleckchem, Cat no. S8162) was administered starting at 300 nM and serially diluted in 1 / 3 increments up to a final concentration of 45 pM.

[0425] As a result, as shown in Figure 18, it was confirmed that the reduced gene expression of Filaggrin and Loricrin under atopic induction conditions was restored in a concentration-dependent manner by treatment with linear and circular STAT3 bioPROTACs. In particular, both forms of STAT3 bioPROTACs showed superior recovery efficacy compared to upadacitinib.

[0426]

[0427] Example 13. Confirmation of mouse STAT3 resolution ability of two STAT3 bioPROTAC types

[0428] The resolution ability of STAT3 bioPROTAC against mouse STAT3 was verified.

[0429] Specifically, mouse neuroblastoma cell line Neuro-2a (ATCC, CCL-131) was cultured in EMEM (ATCC, Cat no. 30-2003) supplemented with 10% FBS and 1% penicillin-streptomycin under conditions of 37°C, 5% CO₂, and >95% humidity. Sixteen hours prior to transduction, the cells were seeded into 12-well plates at a density of 1.0 × 10⁵ cells / well to achieve a cell density of 70-80% confluence at the time of transduction.

[0430] Subsequently, 4E8.005 mRNA and 4E8_A8.005 mRNA were treated with serial dilutions starting from 3 nM and increasing by 1 / 3 to a final concentration of 51 fM. Transfection was performed using Lipofectamine Messenger MAX (Invitrogen, Cat no. LMRNA015) according to the manufacturer's instructions. Cells were harvested 16 hours after treatment, and protein expression was analyzed using the same method as in Example 4.2.

[0431] STAT3 protein expression levels under each condition were normalized as a percentage (%) relative to the vehicle, and concentration-response curves were fitted using a 4-parameter logistic (4PL) model with GraphPad Prism to DC 50 The (50% decomposition concentration) and Dmax (maximum decomposition rate) were calculated.

[0432] As a result, as shown in Figure 19, it was confirmed that both 4E8.005 and 4E8_A8.005 mRNAs induce concentration-dependent degradation of mouse STAT3 protein.

[0433]

[0434] Example 14. Confirmation of therapeutic efficacy of STAT3 bioPROTAC in an atopic animal model

[0435] Example 14.1. Comparison of the therapeutic efficacy of STAT3 bioPROTAC and a competitor drug (Dupilumab) for atopic dermatitis in an animal model induced by MC903

[0436] To evaluate the therapeutic efficacy of STAT3 bioPROTAC against atopic skin diseases, an atopic dermatitis-induced mouse model using MC903 was used.

[0437] Specifically, 7-week-old female B-hIL4 / hIL4RA mice (Biocytogen Jiangsu Co., Ltd.) had their backs shaved 3 days prior to the start of the experiment, and were then randomly assigned to groups of 5 mice each. The atopic dermatitis induction group was treated with MC903 (vitamin D3 analog, Calcipotriol, Tocris Bioscience, Cat no. 2700) at a concentration of 25 μM daily for 14 days, while the normal control group was treated with the same amount of ethanol.

[0438] The experimental group was administered lipid nanoparticles (SM102, GenScript) loaded with 4E8_A8.005 mRNA at a dose of 1 mg / kg twice a week for a total of 5 times via intradermal (id) or subcutaneous (sc). The control group was administered human immunoglobulin G4 (hIgG4, Selleck.cn, Cat no. A2052) at a concentration of 25 mg / kg according to the same schedule, and the comparative experimental group was administered dupilumab (Dupilumab, Selleckchem, Cat no. A2038) subcutaneously at a concentration of 25 mg / kg.

[0439] The improvement of symptoms in the affected areas of atopic dermatitis was analyzed through visual observation and sensory evaluation. For the sensory evaluation, on the 14th day, the severity of erythema, edema, erosion, and desquamation was assigned on a scale from 0 (none) to 3 (severe), respectively, to calculate a total score (out of 12 points), and the individual scores were averaged and compared.

[0440] As a result, as shown in Figure 20a, the STAT3 bioPROTAC (4E8_A8.005) subcutaneous administration group showed a significant decrease in sensory evaluation scores compared to the hIgG4 control group and showed a greater improvement in dermatitis symptoms than the dupilumab comparison group.

[0441] In addition, blood was collected 24 hours after the last drug administration, serum was separated, and serum IgE concentrations were measured using ELISA (Biolegend, Cat no. 432404) according to the manufacturer's instructions. Subsequently, CCL17 (TARC) and IL-22 concentrations were measured using the ultra-sensitive electrochemiluminescence assay with the U-PLEX Custom Biomarker Group 1 (mouse) Assay kit (MSD, Cat no. K15069M-1) according to the manufacturer's instructions. As a result, as shown in Figures 20b-d, the STAT3 bioPROTAC (4E8_A8.005) administration group showed significantly reduced serum IgE, CCL17, and IL-22 concentrations compared to the hIgG4 control group, and demonstrated an inhibitory effect equivalent to or better than that of the dupilumab treatment group.

[0442] At the same time as blood collection, a 1×1 cm dorsal skin tissue was excised, fixed in 10% neutral formalin for 24–48 hours, paraffin embedded, 5 μm sections prepared, and H&E stained. Epidermal thickness was measured in 5 randomized fields per mouse using ImageJ and the mean values ​​were calculated. The epidermal proliferation index was assigned based on the number of epidermal layers as follows: 0.5 points (2–4 layers), 1.0 points (4–6 layers), 1.5 points (6–8 layers), and 2.0 points (8–10 layers). As shown in Figures 20e and 20f, the STAT3 bioPROTAC administration group (4E8_A8.005) showed a decrease in both epidermal thickness and epidermal proliferation index compared to the hIgG4 control group, and demonstrated a greater improvement effect than the dupilumab treatment group.

[0443] Statistical analysis was performed using one-way ANOVA followed by Tukey correction, with significance levels set at *p<0.05 and **p<0.01.

[0444] Example 14.2. Comparison of the therapeutic efficacy of STAT3 bioPROTAC and a competitor drug (Dupilumab) for atopic dermatitis treatment via histology analysis in an atopic dermatitis-induced mouse model using MC903

[0445] The skin tissue obtained in Example 14.1 was stained with hematoxylin-eosin (H&E) and observed under a microscope.

[0446] As a result, as shown in Figure 21, inhibition of epidermal proliferation, alleviation of hyperkeratosis, and reduction of dyskeratosis were confirmed in the group administered STAT3 bioPROTAC (4E8_A8.005) subcutaneously compared to the hIgG4 control group. In addition, it was confirmed that these improvement effects were superior to those of the competing drug dupilumab.

[0447] Thus, it was confirmed that the STAT3 bioPROTAC according to the present invention exhibits superior therapeutic efficacy, equivalent to or greater than that of the competing drug dupilumab, in an animal model of atopic dermatitis.

[0448]

[0449] Example 15. Confirmation of STAT3 degradation ability of two STAT3 bioPROTACs in cancer cell lines

[0450] The efficacy of STAT3 protein degradation according to the concentration of STAT3 bioPROTAC was evaluated in SiHa cells, a human cervical cancer cell line.

[0451] Specifically, SiHa cells (ATCC, Cat no. HTB-35) were cultured in Eagle's Minimum Essential Media (EMEM, ATCC, Cat no. 30-2003) supplemented with 10% FBS (Gibco, 26140-079) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37°C, 5% CO₂, and >95% humidity. 16 hours prior to transduction, cells were seeded into 12-well plates at a density of 0.6 × 10⁵ cells / well and the experiment was conducted at approximately 70-80% confluence.

[0452] STAT3 bioPROTACs 4E8.005 mRNA or 4E8_A8.005 mRNA were transduced using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015) according to the manufacturer's protocol, serially diluted from 5 nM in 1 / 3 increments up to a final concentration of 0.25 pM. As a control group, the compound-based STAT3 PROTAC SD-36 (MCE, Cat no. HY-129602) was treated serially from 5 μM in 1 / 3 increments up to a final concentration of 0.25 nM.

[0453] Sixteen hours after treatment, cells were washed twice with cold PBS, and protein expression was analyzed using the same method as in Example 4.2. The STAT3 degradation rate was calculated as 100 minus relative signal intensity (%), and the concentration-response curve was fitted using a 4-parameter logistic model (Top / Bottom / Hill slope free) in GraphPad Prism to DC 50 The (50% decomposition concentration) and Dmax (maximum decomposition rate) were calculated.

[0454] As a result, as shown in Figure 22, it was confirmed that both types of STAT3 bioPROTACs induced STAT3 protein degradation in proportion to the treatment concentration, and that they exhibited significantly superior STAT3 degradation efficacy compared to SD-36, a compound-based STAT3 PROTAC.

[0455]

[0456] Example 16. Confirmation of anticancer efficacy of STAT3 bioPROTAC

[0457] Example 16.1. Confirmation of the cancer cell growth inhibitory ability of STAT3 bioPROTAC in various cancer types

[0458] The inhibitory effect of STAT3 bioPROTAC treatment on cancer cell growth in various human cancer cell lines was evaluated.

[0459] Specifically, human cervical cancer cell line (SiHa), human non-small cell lung cancer cell line (SW900) (KCLB, Cat no. 30089) and human laryngeal squamous cell carcinoma cell line (FaDu) (KCLB, Cat no. 30043) were cultured as in Example 15.

[0460] Human non-small cell lung cancer cell line (NCI-H1730) (KCLB, Cat no. 91703) was cultured in RPMI 1640 medium (Gibco, Cat no. A1049101) supplemented with 10% FBS (Gibco, 26140-079) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37°C, 5% CO₂, and >95% humidity.

[0461] Sixteen hours prior to transduction, 5.5×10³ SiHa, 7.5×10³ NCI-H1730, 7.5×10³ SW900, and 7.5×10³ FaDu cells / well (100 μL) were seeded into 96-well plates, respectively. Subsequently, 4E8_A8.005 mRNA was transduced using Lipofectamine MessengerMAX (Invitrogen, LMRNA015) according to the manufacturer's instructions at concentrations of 0.33 nM for SiHa and NCI-H1730 cell lines and 0.89 nM for SW900 and FaDu cell lines. Immediately after drug treatment, the plates were mounted on an Incucyte (Sartorius), and continuous imaging was performed for a total of 72 hours at 3-hour intervals using a phase-contrast channel, a 10× objective lens, and three fields per well under conditions of 37°C and 5% CO₂. The acquired images were segmented using a single phase mask in the Basic Analyzer of Incucyte software to generate confluence (%) time series, and statistical analysis was performed using an unpaired t-test with Welch correction applied, expressed as mean ± standard error (SEM) (*p<0.05, **p<0.01).

[0462] As a result, as shown in Figure 23, it was confirmed that cell growth was significantly inhibited in the 4E8_A8.005 mRNA treatment group compared to the mCherry control group in all four human cancer cell lines.

[0463] Example 16.2. Confirmation of concentration-dependent in vitro anticancer efficacy of STAT3 bioPROTAC

[0464] The anticancer efficacy of STAT3 bioPROTAC treatment was evaluated in human blood cancer cell lines.

[0465] Specifically, human blood cancer cell lines (SUP-M2) (DSMZ, Cat no. ACC509) were cultured in RPMI-1640 medium (Gibco, Cat no. A1049101) supplemented with 10% heat-inactivated FBS (Gibco, Cat no. 26140-079) and 1% penicillin-streptomycin (Gibco, Cat no. 15140-122) at 37°C, 5% CO₂, and >95% humidity. Subsequently, the cells were seeded into 96-well plates at a density of 3.0 × 10⁴ cells / well.

[0466] For the experimental group, 4E8_A8.005 mRNA, a STAT3 bioPROTAC, was treated at concentrations of 0.33, 1.0, and 3.0 nM, and transduction was performed using Lipofectamine Messenger MAX (Invitrogen, Cat no. LMRNA015) according to the manufacturer's instructions. As a negative control, mCherry mRNA was treated at the same concentration. For the comparative experimental group, the STAT3 PROTAC KT-333 (MedChemExpress, Cat no. HY-156730A) was treated at concentrations of 3, 30, and 300 nM, or STAT3 siRNA (Origene, Cat no. SR321907-B) was treated at a concentration of 20 nM, respectively. As a positive control, oxaliplatin (Sigma, Cat no. O9512) was treated at a concentration of 100 μM.

[0467] Cell viability was measured 72 hours after drug treatment using CellTiter-Glo®2.0 (Promega, Cat no. G9242) according to the manufacturer's instructions.

[0468] As a result, as shown in Figure 24, a concentration-dependent reduction in cancer cell survival was confirmed in the STAT3 bioPROTAC-treated group, and it showed superior anticancer efficacy compared to KT-333.

[0469]

[0470] Example 17. Confirmation of STAT3 resolution ability by STAT3 bioPROTAC in linear or circular mRNA forms in various cancer cell lines

[0471] The efficacy of STAT3 protein degradation following treatment with linear or circular mRNA forms of STAT3 bioPROTAC was evaluated in various human cancer cell lines.

[0472] Specifically, human cervical cancer cell lines (SiHa) were cultured as in Example 15. Human liver cancer cell lines (Huh-7) (KCLB, Cat no. 60104) were cultured in RPMI 1640 medium (Gibco, Cat no. A1049101) supplemented with 10% FBS (Gibco, Cat no. 26140-079) and 1% penicillin-streptomycin (Gibco, Cat no. 15140-122) at 37°C and 5% CO₂. Cells were seeded into 12-well plates at a density of 5.0 × 10⁴ cells / well.

[0473] Linear or circular 4E8_A8.005 mRNA prepared in the same manner as in Example 11 was treated by serial dilution from 3 nM in 1 / 3 increments up to 50.8 fM, and transduction was performed using Lipofectamine Messenger MAX (Invitrogen, Cat no. LMRNA015) according to the manufacturer's instructions. STAT3 PROTAC SD-36 (MCE, Cat no. HY-129602) was used as the comparative experimental group, and after dissolution in DMSO, it was treated by serial dilution from 8 μM in 1 / 3 increments up to 0.14 nM. For the vehicle control group, an equal volume of Lipofectamine Messenger MAX or DMSO was added.

[0474] Cells were harvested 16 hours after treatment, and the STAT3 signal for each condition was normalized to a percentage relative to the vehicle using the same method as in Example 4.2. Subsequently, the concentration-response curves were fitted using a 4-parameter logistic (4PL) model in GraphPad Prism to DC 50 and the Dmax value was calculated.

[0475] As a result, as shown in Figure 25, concentration-dependent STAT3 degradation was confirmed in the STAT3 bioPROTAC treatment group in linear or circular mRNA form, and it was confirmed to exhibit stronger degradation ability compared to the existing STAT3 PROTAC, SD-36.

[0476]

[0477] Example 18. Confirmation of anticancer efficacy of STAT3 bioPROTAC in a liver cancer animal model

[0478] To verify the anticancer activity of STAT3 bioPROTAC according to the present invention, tumor suppression efficacy was evaluated using a human liver cancer cell line MHCC-97H transplant animal model.

[0479] Specifically, 5.0 × 10⁶ MHCC-97H cells were injected subcutaneously into the right flank of 6–8 week old female BALB / c nude mice to induce tumor formation. When the average tumor size reached approximately 80–100 mm³, mice were randomly assigned 5 to each group. The administration groups were set as follows:

[0480] (1) vehicle(saline) control group

[0481] (2) sorafenib (20 mg / kg, Shanghai Bide Pharmatech Co., Ltd) monotherapy group

[0482] (3) STAT3 bioPROTAC monotherapy group (0.1 mg / kg, 0.3 mg / kg)

[0483] (4) sorafenib (20 mg / kg) and STAT3 bioPROTAC (0.1 mg / kg, 0.3 mg / kg) combination therapy group

[0484] STAT3 bioPROTAC was prepared by formulating 4E8_A8.005 mRNA into lipid nanoparticles (LNP, SM102; GenScript, particle size approx. 80-100 nm, PDI <0.2) and homogenized by inverting several times immediately before administration. LNP was injected intratumorally (it) once a week for a total of four times, and sorafenib was prepared by dissolving it in a Cremophor EL / ethanol (50:50) solution to a concentration of 2 mg / ml and administered orally daily.

[0485] Tumor volume was calculated according to the following mathematical formula 1 based on the long axis and short axis measured twice a week, and the body weight of each individual was also recorded.

[0486] <Mathematical Formula 1>

[0487] Tumor volume (mm³) = 0.5 × long axis × short axis 2

[0488] All animal experiments were performed under randomization and blinding conditions.

[0489] As a result, as shown in Figure 26, tumor growth inhibition was confirmed in the STAT3 bioPROTAC monotherapy group, and particularly, a more potent anticancer effect was observed in the STAT3 bioPROTAC combination therapy group compared to the sorafenib monotherapy group.

[0490]

[0491] Although the present invention has been described above with specific details such as specific components, limited embodiments, and drawings, this is provided only to aid in a more comprehensive understanding of the invention, and the invention is not limited to the above embodiments, and a person skilled in the art to which the invention belongs can make various modifications and variations from this description.

[0492] Accordingly, the scope of the present invention should not be limited to the embodiments described above, and all modifications equivalent to or equivalent to the claims set forth below, as well as the claims described below, shall be considered to fall within the scope of the concept of the present invention.

Claims

1. HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 50, SEQ ID NO 12, SEQ ID NO 19, SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 40, and SEQ ID NO 46; HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 14, SEQ ID NO 21, SEQ ID NO 28, SEQ ID NO 36, SEQ ID NO 42, and SEQ ID NO 47; and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 44, SEQ ID NO 48, and SEQ ID NO 51; comprising, Anti-STAT3 single-domain antibody or its antigen-binding fragment.

2. In claim 1, the anti-STAT3 single-domain antibody or its antigen-binding fragment is (i) HCDR1 having the amino acid sequence of SEQ ID NO. 50, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 16; (ii) HCDR1 comprising the amino acid sequence of SEQ ID NO. 12, HCDR2 comprising the amino acid sequence of SEQ ID NO. 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 16; (iii) HCDR1 having the amino acid sequence of SEQ ID NO. 19, HCDR2 having the amino acid sequence of SEQ ID NO. 21, and HCDR3 having the amino acid sequence of SEQ ID NO. 23; (iv) HCDR1 having the amino acid sequence of SEQ ID NO. 26, HCDR2 having the amino acid sequence of SEQ ID NO. 28, and HCDR3 having the amino acid sequence of SEQ ID NO. 30; (v) HCDR1 having the amino acid sequence of SEQ ID NO. 34, HCDR2 having the amino acid sequence of SEQ ID NO. 36, and HCDR3 having the amino acid sequence of SEQ ID NO. 38; (vi) HCDR1 comprising the amino acid sequence of SEQ ID NO. 40, HCDR2 comprising the amino acid sequence of SEQ ID NO. 42, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 44; (vii) HCDR1 comprising the amino acid sequence of SEQ ID NO. 46, HCDR2 comprising the amino acid sequence of SEQ ID NO. 47, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 48; (viii) HCDR1 having the amino acid sequence of SEQ ID NO. 12, HCDR2 having the amino acid sequence of SEQ ID NO. 14, and HCDR3 having the amino acid sequence of SEQ ID NO. 51; or (ix) HCDR1 comprising the amino acid sequence of SEQ ID NO. 50, HCDR2 comprising the amino acid sequence of SEQ ID NO. 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 51; Anti-STAT3 single-domain antibody or its antigen-binding fragment.

3. In Paragraph 1, The above anti-STAT3 single-domain antibody or its antigen-binding fragment is A sequence comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 90 to 92 and SEQ ID NOs 4 to 10, Anti-STAT3 single-domain antibody or its antigen-binding fragment.

4. HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 88, SEQ ID NO 53, SEQ ID NO 59, SEQ ID NO 65, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 79 and SEQ ID NO 84; HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 55, SEQ ID NO 61, SEQ ID NO 67, SEQ ID NO 75, SEQ ID NO 81 and SEQ ID NO 86; and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO 57, SEQ ID NO 63, SEQ ID NO 69, SEQ ID NO 77, SEQ ID NO 83, SEQ ID NO 87 and SEQ ID NO 89; comprising, Anti-STAT3 single-domain antibody or its antigen-binding fragment.

5. In paragraph 4, the anti-STAT3 single-domain antibody or its antigen-binding fragment is (i) HCDR1 comprising the amino acid sequence of SEQ ID NO. 88, HCDR2 comprising the amino acid sequence of SEQ ID NO. 55, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 57; (ii) HCDR1 having the amino acid sequence of SEQ ID NO. 53, HCDR2 having the amino acid sequence of SEQ ID NO. 55; and HCDR3 having the amino acid sequence of SEQ ID NO. 57; (iii) HCDR1 having the amino acid sequence of SEQ ID NO. 59, HCDR2 having the amino acid sequence of SEQ ID NO. 61; and HCDR3 having the amino acid sequence of SEQ ID NO. 63; (iv) HCDR1 having the amino acid sequence of SEQ ID NO. 65, HCDR2 having the amino acid sequence of SEQ ID NO. 67; and HCDR3 having the amino acid sequence of SEQ ID NO. 69; (v) HCDR1 having the amino acid sequence of SEQ ID NO. 71, HCDR2 having the amino acid sequence of SEQ ID NO. 55; and HCDR3 having the amino acid sequence of SEQ ID NO. 57; (vi) HCDR1 having the amino acid sequence of SEQ ID NO. 73, HCDR2 having the amino acid sequence of SEQ ID NO. 75; and HCDR3 having the amino acid sequence of SEQ ID NO. 77; (vii) HCDR1 comprising the amino acid sequence of SEQ ID NO. 79, HCDR2 comprising the amino acid sequence of SEQ ID NO. 81; and HCDR3 comprising the amino acid sequence of SEQ ID NO. 83; (viii) HCDR1 having the amino acid sequence of SEQ ID NO. 84, HCDR2 having the amino acid sequence of SEQ ID NO. 86; and HCDR3 having the amino acid sequence of SEQ ID NO. 87; (ix) HCDR1 comprising the amino acid sequence of SEQ ID NO. 71, HCDR2 comprising the amino acid sequence of SEQ ID NO. 55; and HCDR3 comprising the amino acid sequence of SEQ ID NO. 89; or (x) HCDR1 comprising the amino acid sequence of SEQ ID NO. 88, HCDR2 comprising the amino acid sequence of SEQ ID NO. 55; and HCDR3 comprising the amino acid sequence of SEQ ID NO. 89; Anti-STAT3 single-domain antibody or its antigen-binding fragment.

6. A polynucleotide encoding an anti-STAT3 single-domain antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof.

7. An expression vector loaded with the polynucleotide of claim 6.

8. Transformed cells into which the expression vector of claim 7 has been introduced. 9.i) a step of culturing the transformed cells of claim 8; and ii) a step of obtaining an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof from a culture medium in which the transformed cells were cultured; comprising Method for preparing an anti-STAT3 single-domain antibody or an antigen-binding fragment thereof.

10. An anti-STAT3 single-domain antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof; and a fragment of an E3 ubiquitin ligase or a variant thereof Fusion protein.

11. In paragraph 10, the fragment of the E3 ubiquitin ligase or a variant thereof is a fragment of SPOP (speckle-type BTB-POZ) or a variant thereof, Fusion protein.

12. In claim 11, the fragment of the SPOP (speckle-type BTB-POZ) or a variant thereof comprises the amino acid sequence of SEQ ID NO. 100, Fusion protein.

13. In paragraph 10, the fusion protein comprises a linker, Fusion protein.

14. In paragraph 10, the anti-STAT3 single-domain antibody or its antigen-binding fragment is located at the N-terminus or C-terminus of a fragment of E3 ubiquitin ligase or a variant thereof, Fusion protein.

15. In claim 10, the fusion protein comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs 90 to 92 and SEQ ID NOs 4 to 10; and the amino acid sequence of SEQ ID NO.

100. Fusion protein.

16. A polynucleotide encoding the fusion protein of paragraph 15.

17. An expression vector loaded with the polynucleotide of claim 16.

18. Transformed cell into which the expression vector of paragraph 17 has been introduced. 19.i) A step of culturing the transformed cells of paragraph 18; and ii) a step of obtaining a fusion protein from a culture medium in which the transformed cells are cultured; comprising Method for manufacturing a fusion protein.

20. A pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17.

21. A quasi-drug composition for preventing or improving atopic dermatitis comprising a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17.

22. A cosmetic composition for preventing or improving atopic dermatitis comprising a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17.

23. A food composition for preventing or improving atopic dermatitis comprising a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17.

24. A method for preventing or treating atopic dermatitis comprising the step of administering to a subject requiring the fusion protein of any one of claims 10 to 15; the polynucleotide of claim 16; or the expression vector of claim 17.

25. Use of a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17 for the manufacture of a drug for the prevention or treatment of atopic dermatitis.

26. A fusion protein of any one of claims 10 to 15 for the prevention or treatment of atopic dermatitis; a polynucleotide of claim 16; or an expression vector of claim 17.

27. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17.

28. A pharmaceutical composition for the prevention or treatment of cancer according to claim 27, wherein the cancer is any one selected from the group consisting of breast cancer, colorectal cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, glioma, ovarian cancer, cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

29. A pharmaceutical composition for the prevention or treatment of cancer, wherein, in paragraph 27, the pharmaceutical composition is administered in combination with one or more other preparations effective as anticancer agents.

30. In Paragraph 29, The above preparation is (i) sorafenib, regorafenib, lenvatinib, cabozantinib, sunitinib, pazopanib, vandetanib, axitinib, nintedanib, tivozanib, apatinib, ponatinib, ripretinib, foretinib, anlotinib, or donafenib; (ii) cisplatin, carboplatin, oxaliplatin, doxorubicin, epirubicin, paclitaxel, docetaxel, irinotecan, topotecan, gemcitabine, 5-fluorouracil (5-FU), capecitabine, cyclophosphamide, ifosfamide, methotrexate, bleomycin, vincristine, vinblastine or vinorelbine; or (iii) antibodies that are anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG-3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-4-1BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-VISTA, anti-NKG2A, anti-BTLA, anti-PVRIG, anti-CD73, anti-B7-H3, anti-IDO-1, anti-CD200, or anti-TIGIT Pharmaceutical composition for the prevention or treatment of cancer.

31. A method for preventing or treating cancer comprising the step of administering a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17 to a subject requiring the same.

32. Use of a fusion protein of any one of claims 10 to 15; a polynucleotide of claim 16; or an expression vector of claim 17 for the manufacture of a drug for the prevention or treatment of cancer.

33. A fusion protein of any one of claims 10 to 15 for the purpose of preventing or treating cancer; a polynucleotide of claim 16; or an expression vector of claim 17.

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