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 SOX2 via E3 ubiquitin ligase or an antigen-binding fragment thereof, a fusion protein for enhancing proteolysis using same, and a use thereof. The fusion protein comprising the anti-SOX2 single domain antibody and the E3 ubiquitin ligase fragment of the present invention specifically binds to SOX2 to degrade SOX2. SOX2 degradation by the fusion protein promotes growth inhibition and apoptosis of cancer cell lines. In addition, when used in combination with an anticancer agent, the fusion protein inhibited the growth of cancer cells that exhibit drug resistance. The fusion protein inhibits tumor growth in a tumor mouse model. Therefore, the fusion protein of the present invention can be advantageously used for preventing or treating cancer.

Description

Single domain antibody specifically binding to SOX2, fusion protein comprising same, and use thereof

[0001] The present invention relates to a single domain antibody or antigen-binding fragment thereof that specifically binds to SOX2 by E3 ubiquitin ligase, a fusion protein using the antibody or antigen-binding fragment thereof for enhancing protein degradation, and a use of the fusion protein. Specifically, the present invention relates to the use of a fusion protein comprising an anti-SOX2 single domain antibody targeting SOX2 or an antigen-binding fragment thereof and an E3 ubiquitin ligase fragment or a variant thereof for preventing or treating a SOX2-mediated disease.

[0002] To date, new drugs have been developed based on the principle of inhibiting the active site or ligand binding site of disease-causing proteins. Of the approximately 20,000 human proteins, only about 3% are known to be FDA-approved drug targets. Approximately 3,100 proteins (16%), representing the majority of disease-causing proteins, are considered inapplicable to current drug development technologies. Furthermore, existing active site-directed drugs often suffer from issues related to drug resistance.

[0003] Proteolysis-targeting chimeras (PROTACs) are double-stranded molecules that bind to disease-causing proteins and induce proteolysis via the proteasome. PROTACs utilize the ubiquitin-proteasome system (UPS), the cell's endogenous protein degradation mechanism, enabling the induction of proteolysis of proteins previously inaccessible to conventional technologies. Therefore, they are attracting attention as a new paradigm for overcoming the limitations of traditional drug development.

[0004] Protacs are composed of two protein-binding molecules, each of which binds to an E3 ubiquitin ligase and a target protein. Through this binding, Protacs bring the target protein close to the E3 ligase, thereby inducing ubiquitination. Ubiquitination involves three steps: activation, conjugation, and ligation, carried out by a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). As a result of this cascade, ubiquitin is covalently bound to the target protein, and the ubiquitinated protein is degraded by the proteasome.

[0005] Protac technology was first introduced in 2001 and is currently receiving significant attention, with active development underway. Most protac drugs have been developed based on small-molecule compounds. While known protac drugs are highly useful, improvements are needed to address concerns about the inherent low solubility of small-molecule compounds, reduced intracellular penetration, and safety issues associated with reduced proteolytic efficiency. Therefore, there is a growing need for protac drugs that address these issues.

[0006] Accordingly, the inventors of the present invention conducted research to develop a protease that specifically targets SOX2 (SRY-box transcription factor 2) and induces degradation of SOX2, and as a result, they confirmed that a fusion protein comprising a novel anti-SOX2 single domain antibody and a fragment of E3 ubiquitin ligase specifically targets SOX2 and promotes its degradation, thereby completing the present invention.

[0007] In order to achieve the above object, one aspect of the present invention provides an anti-SOX2 single domain antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising: HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 63, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 87, and SEQ ID NO: 94; HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 89; and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO: 96.

[0008] Another aspect of the present invention provides a polynucleotide encoding the antibody or an antigen-binding fragment thereof, an expression vector loaded with the polynucleotide, and a transformed cell into which the expression vector has been introduced.

[0009] Another aspect of the present invention provides a method for producing an anti-SOX2 single domain antibody or antigen-binding fragment thereof, comprising the steps of culturing the transformed cell and obtaining the antibody or antigen-binding fragment thereof from the culture medium.

[0010] Another aspect of the present invention provides a fusion protein comprising the antibody or an antigen-binding fragment thereof; and a fragment of an E3 ubiquitin ligase or a variant thereof.

[0011] Another aspect of the present invention provides a polynucleotide encoding the fusion protein, an expression vector loaded with the polynucleotide, and a transformed cell into which the expression vector has been introduced.

[0012] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the fusion protein as an active ingredient.

[0013] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject the fusion protein, a polynucleotide encoding the fusion protein, or a vector loaded with the polynucleotide.

[0014] Another aspect of the present invention provides a use of the fusion protein, a polynucleotide encoding the fusion protein, or a vector loaded with the polynucleotide for preventing or treating cancer.

[0015] The fusion protein comprising an anti-SOX2 single-domain antibody and an E3 ubiquitin ligase fragment of the present invention specifically binds to SOX2 and degrades SOX2. The degradation of SOX2 by the fusion protein inhibited the growth of cancer cell lines and promoted apoptosis. Furthermore, when the fusion protein was used in combination with an anticancer drug, the growth of cancer cells exhibiting anticancer drug resistance was inhibited. Furthermore, the fusion protein inhibited tumor growth in a tumor mouse model. Therefore, the fusion protein of the present invention can be usefully used for the prevention or treatment of cancer.

[0016] FIG. 1 is a drawing showing the amino acid sequences of 13 anti-huSOX2 VHH antibody clones as one specific example of the present invention.

[0017] Figures 2a and 2b are graphs showing the results of flow cytometry analysis to confirm the binding ability of anti-huSOX2 VHH antibody clones to huSOX2 protein.

[0018] Figure 3 is a graph showing the results of confirming the binding ability of an anti-huSOX2 VHH antibody clone to the huSOX2 protein at the cellular level.

[0019] FIG. 4 is a drawing (a) showing the results of confirming the SOX2 protein-specific degradation ability by a fusion protein comprising S14 (anti-huSOX2 VHH antibody clone), which is one specific example of the present invention, and an E3 ubiquitin ligase fragment, using Western blot, and a graph (b) showing the quantitative results.

[0020] FIGS. 5A to 5C are graphs showing the results of confirming the cell growth inhibition and apoptosis induction effects on cancer cells of mRNA encoding a fusion protein including S14 and E3 ubiquitin ligase fragments, which is one specific example of the present invention.

[0021] Figure 6 is a graph showing the results of confirming the cell growth inhibition effect of cancer cells according to concentration-dependent treatment of mRNA encoding a fusion protein including S14 and E3 ubiquitin ligase fragments, which is one specific example of the present invention.

[0022] Figures 7a and 7b show the tamoxifen-resistant cancer cell line (MCF7-Tam R ) are drawings and graphs showing the results of confirming the expression level of SOX2 (Fig. 7a) and the effect of inducing cell growth and apoptosis by combined use of mRNA encoding a fusion protein including S14 and E3 ubiquitin ligase fragments, which is one specific example of the present invention, and tamoxifen (Fig. 7b).

[0023] Figures 8a and 8b show doxorubicin-resistant cancer cell lines (H69-Dox R ) are drawings and graphs showing the results of confirming the expression level of SOX2 (Fig. 8a) and the effect of inducing cell growth and apoptosis by combined use of mRNA encoding a fusion protein including S14 and E3 ubiquitin ligase fragments, which is one specific example of the present invention, and doxorubicin (Fig. 8b).

[0024] FIG. 9 is a drawing (a) showing the results of confirming the SOX2 protein-specific degradation ability by an expression vector expressing a fusion protein including 3A2 (anti-huSOX2 VHH antibody clone), which is one specific example of the present invention, and an E3 ubiquitin ligase fragment, and a graph (b) showing the results quantitatively.

[0025] Figure 10 is a graph showing the results of confirming the SOX2 protein degradation ability by treatment of mRNA encoding a fusion protein including 3A2 (anti-huSOX2 VHH antibody clone), which is an example of the present invention, and an E3 ubiquitin ligase fragment in human cancer cell lines (lung cancer, liver cancer).

[0026] FIG. 11 is a drawing showing the results of confirming whether the SOX2 degradation mechanism is ubiquitin-proteasome dependent using an expression vector expressing a fusion protein including 3A2 (anti-huSOX2 VHH antibody clone), which is one specific example of the present invention, and an E3 ubiquitin ligase fragment.

[0027] Figure 12 is a graph showing the results of confirming the effect of inhibiting SOX2 expression and cancer cell growth after treatment with mRNA encoding a fusion protein containing SOX2 siRNA or 3A2 and E3 ubiquitin ligase fragments in a human non-small cell lung cancer cell line (A549 cells).

[0028] Figures 13a and 13b are graphs showing the results of confirming the cell growth inhibition effect by treatment of mRNA encoding a fusion protein including 3A2 (anti-huSOX2 VHH antibody clone), which is an example of the present invention, and an E3 ubiquitin ligase fragment, in human cancer cell lines (lung cancer, pancreatic cancer, cervical cancer, liver cancer).

[0029] Figure 14 shows each anticancer drug-resistant cancer cell line (MCF7-Tam R , H69-Dox R) is a graph showing the results of confirming the cell growth inhibition effect by the combined use of mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is one specific example of the present invention, and an anticancer agent (tamoxifen or doxorubicin). LipoMM: Treatment with only Lipofectamine-MessengerMax reagent.

[0030] Figure 15 is a graph showing the results of confirming the tumor growth inhibition effect and body weight change by treatment with mRNA encoding a fusion protein including S14 and E3 ubiquitin ligase fragments, which is one specific example of the present invention, in a human cancer cell line (A549 cell, BxPC cell) transplant tumor mouse model.

[0031] Figure 16 is a graph showing the results of confirming the cell growth inhibition effect and the apoptosis induction effect by treatment of mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is an example of the present invention, in a human non-small cell lung cancer cell line (A549 cells).

[0032] Figure 17 is a drawing showing the results of confirming the expression of the fusion protein (degraders) and the change in SOX2 protein expression over time after treating a human kidney cell line (HEK293 cell) with mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is an example of the present invention.

[0033] Figure 18 is a graph showing the results of confirming changes in the expression of SOX2 protein according to treatment with different concentrations of mRNA or SOX2 siRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is an example of the present invention, in a human non-small cell lung cancer cell line (SW900 cells).

[0034] Figure 19 is a graph showing the results of confirming the change in expression of SOX2 by treatment with different concentrations of mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is an example of the present invention, in human non-small cell lung cancer cell lines (A549 cells, SW900 cells).

[0035] Figure 20 is a graph showing the results of confirming the cell growth inhibition effect by treatment with different concentrations of mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is an example of the present invention, in human non-small cell lung cancer cell lines (A549 cells, SW900 cells).

[0036] Figure 21a is a diagram showing the results of comparing SOX2 protein expression by treatment with mRNA encoding a 3A2 single domain antibody (binder only), an E3 ubiquitin ligase fragment (E3 only), or a fusion protein comprising 3A2 and an E3 ubiquitin ligase fragment, which is an embodiment of the present invention, in human non-small cell lung cancer cell lines (A549 cells, SW900 cells). LipoMM: Treatment with Lipofectamine-MessengerMax reagent only.

[0037] Figure 21b is a graph showing the results of confirming the cell growth inhibition effect by treatment with mRNA encoding a fusion protein comprising a 3A2 single domain antibody (binder only), an E3 ubiquitin ligase fragment (E3 only), or a specific example of the present invention, 3A2 and E3 ubiquitin ligase fragments, in human non-small cell lung cancer cell lines (A549 cells, SW900 cells). LipoMM: Treatment with Lipofectamine-MessengerMax reagent only.

[0038] FIG. 22 is a drawing showing the results of confirming changes in the expression of SOX2 protein in tumor tissues by treatment with mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is one specific example of the present invention, in a tumor mouse model transplanted with a human cancer cell line (A549 cells), and a graph showing the results quantified.

[0039] Figure 23 is a drawing showing the results of confirming the change in expression of the fusion protein (degrader) and SOX2 protein over time after treatment of mRNA encoding the fusion protein comprising 3A2 and E3 ubiquitin ligase fragments, which is an example of the present invention, in a human non-small cell lung cancer cell line (SW900 cells), and a graph showing the quantification of the results. MM only or LipoMM: Treatment with Lipofectamine-MessengerMax reagent only.

[0040] Figures 24a and 24b are graphs showing the expression of the fusion protein (degrader) and SOX2 protein over time after treatment of mRNA encoding the fusion protein comprising 3A2 and E3 ubiquitin ligase fragments, which is an embodiment of the present invention, in a human non-small cell lung cancer cell line (SW900 cells).

[0041] Figure 25 shows each anticancer drug-resistant cancer cell line (MCF7-Tam R Cells, H69-Dox R This graph shows the results of confirming the cell growth inhibition effect by combining mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is one specific example of the present invention, and an anticancer agent (tamoxifen or doxorubicin) in cells. LipoMM: Treatment with only Lipofectamine-MessengerMax reagent.

[0042] Figure 26 shows a doxorubicin-resistant cancer cell line (H69-Dox RThis graph shows the results of confirming the cell growth inhibition effect by the combined use of mRNA encoding a fusion protein including 3A2 and E3 ubiquitin ligase fragments, which is one specific example of the present invention, and doxorubicin in resistant cancer cells (H69 cells) or non-resistant cancer cells. LipoMM: Treatment with Lipofectamine-MessengerMax reagent only.

[0043] Figure 27 shows a tamoxifen-resistant cancer cell line (MCF7-Tam R This graph shows the results of confirming the cell growth inhibition effect by the combined use of mRNA encoding a fusion protein comprising a 3A2 and E3 ubiquitin ligase fragment, which is one specific example of the present invention, and tamoxifen in resistant cancer cells (MCF7 cells) or non-resistant cancer cells (MCF7 cells). LipoMM: Treatment with Lipofectamine-MessengerMax reagent only.

[0044] Anti-SOX2 single domain antibody or antigen-binding fragment thereof

[0045] One aspect of the present invention provides an anti-SOX2 single domain antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising: HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 63, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 87, and SEQ ID NO: 94; HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 89; and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO: 96.

[0046] The term "SOX2 (SRY-box transcription factor 2)" used herein is known to be a transcription factor essential for maintaining the self-renewal or pluripotency of undifferentiated embryonic stem cells. Furthermore, SOX2 is overexpressed in various types of human cancer, and its overexpression has been reported to be correlated with a decreased survival rate in cancer patients. SOX2 promotes cancer cell proliferation, survival, invasion / metastasis, cancer stemness, and the development of drug resistance. Therefore, SOX2 has recently been attracting attention as a target for cancer treatment.

[0047] In the present invention, the SOX2 may include, without limitation, any mammalian SOX2, but preferably refers to human SOX2 or ape SOX2. In addition, the SOX2 protein in the present invention includes, but is not limited to, a natural type, a fragment or a mutant thereof. The "wild type" includes all proteins found in nature or nucleic acids encoding the same, and may be described interchangeably with the wild type. The natural type SOX2 protein generally refers to a polypeptide including the amino acid sequence of the natural type SOX2 protein, and the amino acid sequence of the natural type SOX2 protein generally refers to an amino acid sequence found in naturally occurring SOX2. Information on the SOX2 can be obtained from known databases such as GenBank of the National Institutes of Health in the United States. The SOX2 protein in the present invention may include, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0048] The above fragment refers to a truncated fragment of a native SOX2 protein at a portion of the N-terminus and / or C-terminus. Specifically, the SOX2 fragment may be one in which 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 amino acids are deleted consecutively from the 1st amino acid of the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 1. In one specific example, the SOX2 fragment may include the amino acid sequence of SEQ ID NO: 4.

[0049] As used herein, the term “anti-SOX2 single domain antibody” refers to an antibody molecule that specifically binds to SOX2 having a single variable region.

[0050] The above "single domain antibody (sdAb)" generally refers to an antibody having antigen binding activity that contains only one heavy chain variable region (VH). sdAbs derived from heavy chains are mainly used, but single variable region fragments derived from light chains have also been reported to specifically bind to antigens. In the present invention, the single domain antibody may be an antibody that does not contain a light chain and contains a single chain FR1-VHHCDR1-FR2-VHHCDR2-FR3-VHHCDR3-FR4 in the order of N-terminus to C-terminus. The single domain antibody is used interchangeably with heavy chain antibody, nanobody VHH, nanobody or HCAb (heavy chain only antibody), and although HCAb does not have a light chain, it has a certain antigen binding mechanism part. 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 antibodies may include antigen-binding fragments of antibody molecules as well as complete antibody forms.

[0051] As used herein, the term “antigen-binding fragment” refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen, i.e., SOX2. 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, triabodies, and tetrabodies, Fab fragments, F(ab')2 fragments, Fd, scFv, domain antibodies, minibodies, sterol regulatory binding protein cleavage activating protein (scap), 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 region derivatives, and synthetic antibodies based on protein scaffolds having binding ability to SOX2.

[0052] It will be apparent to those skilled in the art that any fragment of an antibody according to the present invention will exhibit the same properties as the antibody according to the present invention, as long as the binding function to SOX2 is maintained.

[0053] As used herein, the term "heavy chain (HC)" refers to a full-length heavy chain and fragments thereof comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3.

[0054] As used herein, the term "variable" (V) indicates that a specific portion of the variable region differs significantly in sequence among antibodies. The variable region mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. Variability is concentrated in three segments called hypervariable regions (HVRs), or CDRs, in all heavy-chain variable regions. The more highly conserved portions of the variable region are called framework (FR) regions. The heavy-chain variable region has the following structures, from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0055] As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region (HVR) of an immunoglobulin heavy chain. Each heavy chain (HCDR1, HCDR2, and HCDR3) contains three CDRs. CDRs provide key contact residues for antibody binding to an antigen or epitope.

[0056] In the present invention, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising: HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 63, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 87, and SEQ ID NO: 94; HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 89; and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO: 96.

[0057] In one embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 56, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 58, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 60. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 5.

[0058] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 63, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 65, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 67. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 6.

[0059] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 69, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 67. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 7.

[0060] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 63, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 67. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 8.

[0061] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 73, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 74, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 67. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 9.

[0062] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 63, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 75, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 67. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

[0063] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 63, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 76, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 67. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 12.

[0064] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 82, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 85. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 13.

[0065] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 78, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 79, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 81. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 14.

[0066] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 87, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 89, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 91. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 15.

[0067] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 94, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 89, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 91. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 16.

[0068] In one specific embodiment, the anti-SOX2 single domain antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 94, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 89, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 96. The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 17.

[0069] Additionally, the heavy chain variable region of the antibody may include an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or about 100% identity with the amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 17.

[0070] Polynucleotide encoding an anti-SOX2 single domain antibody or antigen-binding fragment thereof

[0071] Another aspect of the present invention provides a polynucleotide encoding an anti-SOX2 single domain antibody or an antigen-binding fragment thereof. The polynucleotide may comprise any one base sequence selected from the group consisting of SEQ ID NO: 101 to SEQ ID NO: 113.

[0072] The above anti-SOX2 single domain antibody and antigen binding fragment are the same as described above.

[0073] As used herein, the term "polynucleotide," also referred to as "nucleic acid," refers to a polymer of nucleotides of any length. Specifically, the polynucleotide may be DNA or RNA.

[0074] The polynucleotide may be modified by one or more base substitutions, deletions, insertions, or a combination thereof, as long as it encodes the same polypeptide. When producing a polynucleotide sequence by chemical synthesis, synthetic methods well known in the art can be used, such as those described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and examples thereof include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on solid supports.

[0075] In addition, in the present invention, the polynucleotide may include or consist of a base sequence having about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, at least about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with the base sequence of SEQ ID NO: 101 to SEQ ID NO: 113.

[0076] The above polynucleotide may additionally comprise a signal sequence or a leader sequence.

[0077] A vector loaded with a polynucleotide encoding an anti-SOX2 single domain antibody or an antigen-binding fragment thereof

[0078] Another aspect of the present invention provides an expression vector loaded with a polynucleotide encoding an anti-SOX2 single domain antibody or an antigen-binding fragment thereof. The anti-SOX2 single domain antibody, antigen-binding fragment, and polynucleotide are the same as described above.

[0079] As used herein, the term "vector" means a material for carrying or expressing a nucleic acid sequence comprising a nucleic acid sequence encoding an anti-SOX2 single domain antibody variant described herein.

[0080] Specifically, the vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. Additionally, the plasmid may include a selectable marker, such as an antibiotic resistance gene, and the host cell harboring the plasmid may be cultured under selective conditions.

[0081] More specifically, the vector may be a 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, λgt10, λgt11, λZAP, etc.), an animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (baculovirus, etc.). The above vector can be used to express proteins in different amounts and in different forms depending on the host cell. Therefore, it is desirable to select and use the host cell most suitable for the purpose.

[0082] Additionally, the vector of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Examples of sequences to be fused include Flag (IBI, USA), glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), 6×His (Qiagen, USA), Halo, or Myc.

[0083] In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a Protein A column or the like without an additional sequence for purification.

[0084] Transformed cells expressing anti-SOX2 single domain antibody or antigen-binding fragment thereof

[0085] Another aspect of the present invention provides a transformed cell into which an expression vector loaded with a polynucleotide encoding the anti-SOX2 single domain antibody or an antigen-binding fragment thereof is introduced. The anti-SOX2 single domain antibody, antigen-binding fragment, polynucleotide, and expression vector are the same as those described above.

[0086] As used herein, the term "transformed cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. The transformed cell can be produced by introducing the vector into a host cell and transforming the cell. In addition, the polynucleotide contained in the vector can be expressed to produce the anti-SOX2 single-domain antibody of the present invention or an antigen-binding fragment thereof.

[0087] The above transformation can be performed by various methods. It is not particularly limited thereto, as long as it can produce the anti-SOX2 single domain antibody of the present invention or an antigen-binding fragment thereof. Specifically, the transformation method may be a CaCl2 precipitation method, a Hanahan method that increases efficiency by using a reducing substance called dimethyl sulfoxide (DMSO) in the CaCl2 precipitation method, electroporation, a calcium phosphate precipitation method, a protoplast fusion method, a stirring method using silicon carbide fibers, an Agrobacterium-mediated transformation method, a transformation method using PEG, a dextran sulfate, lipofectamine, and a drying / inhibition-mediated transformation method. In addition, a target object can be delivered into a cell using a virus particle by means of infection. In addition, a vector can be introduced into a host cell by gene bombardment, etc.

[0088] In addition, the host cell used for producing the transformed cell is not particularly limited as long as it can produce the antibody of the present invention. Specifically, the host cell may include, but is not limited to, prokaryotic cells, eukaryotic cells, mammals, plants, insects, fungi, or cells of cellular origin. An example of the prokaryotic cell may be Escherichia coli. In addition, an example of the eukaryotic cell may be yeast. In addition, the mammalian cell may be 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, PERC.6 cells, HEK293 cells, or HEK293T cells, but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art may be used.

[0089] Additionally, to optimize the therapeutic properties of the antibody or for other purposes, the glycosylation-related genes of the host cell can be manipulated using methods known to those skilled in the art to adjust the sugar chain pattern of the antibody (e.g., sialic acid, fucosylation, glycosylation).

[0090] Method for producing an anti-SOX2 single domain antibody or antigen-binding fragment thereof

[0091] Another aspect of the present invention provides a method for producing an anti-SOX2 single domain antibody or antigen-binding fragment thereof, comprising the step of culturing the transformed cell.

[0092] The anti-SOX2 single domain antibody, antigen-binding fragment, and transformed cells are the same as described above.

[0093] Specifically, the method for producing the antibody or fragment thereof may include a step of culturing a transformed cell to produce an antibody and a step of obtaining the produced antibody from the culture solution.

[0094] The method for culturing the above-mentioned transformed cells can be performed using methods widely known in the art. Specifically, the culturing is not particularly limited as long as it can express and produce the anti-SOX2 single-domain antibody or antigen-binding fragment thereof of the present invention. Specifically, the culturing can be continuously performed in a batch process or a fed batch or repeated fed batch process.

[0095] In addition, the step of obtaining the antibody and fragment thereof specific to SOX2 from the culture medium can be performed by a method known in the art. Specifically, the obtaining method is not particularly limited as long as it can obtain the antibody or fragment thereof of the present invention produced. Preferably, the obtaining method can be a method such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion).

[0096] Fusion protein comprising an anti-SOX2 single domain antibody or an antigen-binding fragment thereof and a fragment of E3 ubiquitin ligase or a variant thereof

[0097] Another aspect of the present invention provides a fusion protein comprising the anti-SOX2 single domain antibody or an antigen-binding fragment thereof; and a fragment of E3 ubiquitin ligase or a variant thereof.

[0098] The above anti-SOX2 single domain antibody and antigen binding fragment are the same as described above.

[0099] As used herein, the term "ubiquitin (UB)" refers to a 76-amino acid protein found in nearly all eukaryotic cells. Ubiquitin has the property of covalently linking to other proteins through an ATP-dependent reaction, a process called ubiquitination.

[0100] The above ubiquitin is covalently linked to a target protein (hereinafter, referred to as a substrate protein) through 1) activation performed by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), 2) conjugation, and 3) ligation. The target protein ubiquitinated (poly-Ub) as described above is degraded by the proteasome.

[0101] The term "E3 ubiquitin ligase" as used herein refers to an enzyme in the final step of ubiquitination that attaches ubiquitin to a substrate. E3 ligase binds to a target protein and E2-Ub, thereby inducing ubiquitination (poly-Ub) of the target protein by positioning the target protein close to E2-Ub. In the present invention, the E3 ubiquitin ligase may be used interchangeably as E3 ligase, E3 ubiquitin ligase, E3 ubiquitin conjugase, or E3.

[0102] As used herein, the term "fragment" may refer to a specific substrate recognition domain, a ubiquitin transfer domain, or two domains thereof within an E3 ubiquitin ligase complex.

[0103] Table 1 below shows the types of subunits of the E3 ubiquitin ligase complex and examples thereof according to the type of CRL.

[0104] Cullin protein adapter subunit exampleCul1Skp1F-box proteinSkp2, βFbw, Fbxo, FbxlCul2EloBCVHL-box proteinVHLCul3BTBBTB domain proteinKLHL, SPOPCul4ADDB1DCAF / H-box proteinDCAF, DDB2, CSA, CDT2, CRBNCul4BDDB1DCAF / H-box proteinDCAF, DDB2Cul5EloBCSOCS-box proteinSOCS1~SOCS7Cul7Skp1F-box proteinFbxw8

[0105] In the present invention, the fragment of the E3 ubiquitin ligase or a variant thereof may be any one selected from the group consisting of βTrCP (beta-transducin 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 ZNFR1 (zinc and ring finger 1) and specific domains thereof.

[0106] In one specific example, the variant of βTrCP is βTrCP full-length (Uniprot ID: Q9Y297) 2-263(amino acids 2-263, SEQ ID NO: 206) fragment or βTrCP 190-228 (amino acids 190-228, SEQ ID NO: 18) fragment. In one specific example, the variant of SKP2 is SKP2 of full-length SKP2 (Uniprot ID: Q13309). 2-176 (amino acids 2-176, SEQ ID NO: 19) fragment. In one specific example, the variant of VHL is a VHL fragment of full-length VHL (Uniprot ID: P40337). 152-213 (amino acids 152-213, SEQ ID NO: 20) fragment. In one specific example, the variant of SPOP is a fragment of SPOP full-length (Uniprot ID: O43791). 167-374 (amino acids 167-374, SEQ ID NO: 21) fragment. In one specific example, the variant of SOCS2 is a SOCS2 fragment of the full-length SOCS2 (Uniprot ID: 014508). 143-198 (amino acids 143-198, SEQ ID NO: 22) fragment. In one specific example, the variant of CHIP is a fragment of the full-length CHIP (Uniprot ID: Q9UNE). 128-303 (amino acids 128-303, SEQ ID NO: 23) fragment. In one specific example, the variant of DDB2 is a fragment of the full-length DDB2 (Uniprot ID: Q92466). 2-114 (amino acids 2-114, SEQ ID NO: 24) fragment. In one specific example, the variant of CRBN is a CRBN fragment of the full-length CRBN (Uniprot ID: Q96SW2). 2-320 (amino acids 2-320, SEQ ID NO: 25) fragment. In one specific example, the variant of ASB1 is a fragment of the full-length ASB1 (Uniprot ID: Q9Y576). 266-335 (amino acids 266-335, SEQ ID NO: 26) fragment. In one specific example, the variant of TRIM21 is a TRIM21 fragment of the full-length TRIM21 (Uniprot ID: P19474). 2-85(amino acids 2-85, SEQ ID NO: 27) fragment. In one specific example, the variant of RNF4 is a fragment of the full-length RNF4 (Uniprot ID: P78317). 71-190 (amino acids 71-190, SEQ ID NO: 28) fragment. In one specific example, the variant of UBE2D1 is a fragment of the full-length UBE2D1 (Uniprot ID: P51668). 2-147 (amino acid 2-147, SEQ ID NO: 29) fragment. In one specific example, the variant of UBE2D4 is a fragment of the full-length UBE2D4 (Uniprot ID: Q9Y2X8). 2-147 (amino acids 2-147, SEQ ID NO: 30) fragment. In one specific example, the variant of ZNFR1 is a fragment of the full-length ZNFR1 (Uniprot ID: Q8ND2). 138-227 (Amino acids 138-227, SEQ ID NO: 31) may be a fragment.

[0107] In one specific example, the fragment of the E3 ubiquitin ligase or a variant thereof may comprise any one amino acid sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 31 and SEQ ID NO: 206.

[0108] The variant of the fragment of the above E3 ubiquitin ligase refers to a form in which some of the amino acids of the fragment or a fragment thereof are substituted. In other words, the variant of the fragment refers to a peptide having a sequence different from that of the native fragment or a fragment thereof and having the function of binding to an adapter protein.

[0109] In one specific example, the variant of the fragment may have 5 to 18 amino acids substituted in the native βTrCP fragment.

[0110] More specifically, the βTrCP variant has the 8th, 10th, 11th, 16th, 18th, 28th, 33rd, 35th, 38th, 47th, 91st, 95th, 98th, 102nd, 105th, 110th, 114th, 115th, 120th, 122nd, 124th, 125th, 127th, 132nd, 144th, 146th, 148th, 159th, 170th, 172nd, 173rd, 183rd, 185th, 192nd, 197th, 198th, 202nd, 203rd, 204th, in the amino acid sequence of SEQ ID NO: 206. Any one amino acid selected from the group consisting of the 225th, 234th, 236th, 240th, 241st, 242nd, 250th, 251st, 260th, and combinations thereof may be substituted.

[0111] More specifically, the variant comprises L8, E10, K11, M16, S18, E28, I33, P35, N38, C47, S91, E95, K98, V102, F105, E110, V114, E115, L120, S122, M124, C125, Y127, I132, F144, T146, L148, N159, C170, A172, E173, T183, D185, L192, V197, R198, L202, W203, R204, N225, A234, Y236, I240, Q241, D242, W250, R251, I260 and Any one amino acid selected from the group consisting of these combinations may be substituted for another amino acid.

[0112] At this time, the "amino acid" introduced by the above substitution may be any one selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, and proline.

[0113] More specifically, the variants are L8P, E10V, K11E, M16L, S18T, E28G, I33T, P35A, N38D, C47S, S91T, E95G, K98R, V102A, F105L, E110G, V114A, E115G, L120P, S122P, M124V, C125R, Y127C, I132T, F144S, T146P, L148P, N159K, C170R, A172V, E173V, T183S, D185G, D185E, L192P, V197A, R198G, L202Q, L202R, in the amino acid sequence of SEQ ID NO: 206. It can be substituted with any one amino acid selected from the group consisting of W203R, R204G, N225Y, A234V, Y236C, I240M, Q241R, D242E, W250R, R251G, I260S, and combinations thereof. In this case, when D185 is substituted, it can be substituted with D185G or D185E. In addition, L202 can be substituted with L202Q or L202R.

[0114] The above βTrCP variant may comprise or consist of any one amino acid sequence selected from the group consisting of SEQ ID NO: 207 to SEQ ID NO: 214.

[0115] In one specific example, the variant fragment may have 7 to 14 amino acids substituted in the native SKP2 fragment.

[0116] More specifically, the SKP2 fragment variant may be one in which any one amino acid selected from the group consisting of the 2nd, 4th, 11th, 12th, 19th, 24th, 29th, 33rd, 44th, 59th, 78th, 98th, 102nd, 107th, 111th, 112th, 113th, 124th, 128th, 130th, 133rd, 135th, 137th, 138th, 139th, 148th, 156th, 161st, 176th, and combinations thereof in the amino acid sequence of SEQ ID NO: 19 is substituted.

[0117] More specifically, the variant may be one in which any one amino acid selected from the group consisting of H2, K4, D11, L12, S19, W24, T29, L33, E44, L59, D78, D98, D102, G107, C111, L112, C113, C124, Y128, L130, D133, S135, W137, Q138, T139, H148, L156, I161, E176 and combinations thereof in the amino acid sequence of SEQ ID NO: 19 is substituted with another amino acid. At this time, the amino acid introduced for the substitution is the same as described above.

[0118] More specifically, the variant may be substituted with an amino acid selected from the group consisting of H2R, K4R, D11G, L12P, S19G, W24R, T29A, L33P, E44K, L59P, D78G, D98V, D102N, G107R, C111R, L112P, L112R, C113R, C124S, Y128C, L130Q, D133E, S135P, W137R, Q138K, T139S, H148R, L156Q, L156R, I161A, I161V, E176G and combinations thereof in the amino acid sequence of SEQ ID NO: 19. In this case, when L112 is substituted, it may be substituted with L112P or L112R. When L156 is substituted, it can be substituted with L156Q or L156R. When I161 is substituted, it can be substituted with I161A or I161V.

[0119] The above SKP2 variant may comprise or consist of any one amino acid sequence selected from the group consisting of SEQ ID NO: 215 to SEQ ID NO: 221.

[0120] In the present invention, the fusion protein may include a linker. In this case, the linker may be a peptide linker. In the present invention, the anti-SOX2 single-domain antibody or antigen-binding fragment thereof of the fusion protein and the fragment or variant thereof of the E3 ubiquitin ligase may be linked via the linker.

[0121] Structure of fusion proteins

[0122] Specifically, the above fusion protein may be composed of the following structural formula (I) or (II).

[0123] N'-TB-(L)n-SR-C' (I)

[0124] N'-SR-(L)n-TB-C' (II)

[0125] At this time, in the structural formulas (I) and (II),

[0126] The above N' is the N-terminus of the fusion protein,

[0127] The above C' is the C-terminus of the fusion protein,

[0128] The above TB is an anti-SOX2 single domain antibody or an antigen-binding fragment thereof,

[0129] The above SR is a fragment of E3 ubiquitin ligase or a variant thereof,

[0130] The above L is a peptide linker,

[0131] The above n is 0 or 1.

[0132] At this time, the anti-SOX2 single domain antibody, antigen binding fragment and fragment of E3 ubiquitin ligase or variant thereof are the same as described above.

[0133] The peptide linker may be composed of 1 to 30 consecutive amino acids, or 2 to 20 consecutive amino acids, or 2 to 10 amino acids. In one specific example, the peptide linker may be (GS)n (wherein 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 peptide linker may use, without particular limitation, a sequence linker known in the art, such as GS, GGGGS, (GGGGS)2, (GGGS)3, ASTKGP, or ASTKGPSVFPLAP, which provides structural flexibility without being cleaved by proteolytic enzymes. In one specific example, the peptide linker may include an amino acid sequence of SEQ ID NO: 157 (amino acid sequence: GS) or SEQ ID NO: 238.

[0134] In one specific example, the fusion protein may comprise any one amino acid sequence selected from the group consisting of SEQ ID NO: 164 to SEQ ID NO: 184.

[0135] Polynucleotide encoding a fusion protein comprising an anti-SOX2 single domain antibody and a fragment of E3 ubiquitin ligase

[0136] Another aspect of the present invention provides a polynucleotide encoding a fusion protein comprising the anti-SOX2 single domain antibody or an antigen-binding fragment thereof; and a fragment of an E3 ubiquitin ligase or a variant thereof. Specifically, the polynucleotide encoding the fusion protein may comprise a base sequence of SEQ ID NO: 185 to SEQ ID NO: 205.

[0137] The above anti-SOX2 single domain antibody, antigen binding fragment, E3 ubiquitin ligase and fragment are the same as described above.

[0138] Additionally, the polynucleotide may include a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identity with the base sequence of each of SEQ ID NOs: 185 to 205.

[0139] The polynucleotide may be mutated by one or more base substitutions, deletions, insertions, or a combination thereof. When producing a nucleotide sequence by chemical synthesis, synthetic methods widely known in the art, such as those described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), may be used.

[0140] A vector loaded with a polynucleotide encoding a fusion protein

[0141] Another aspect of the present invention provides an expression vector loaded with a polynucleotide encoding a fusion protein comprising the anti-SOX2 single domain antibody or an antigen-binding fragment thereof; and a fragment of an E3 ubiquitin ligase or a variant thereof.

[0142] The above anti-SOX2 single domain antibody, antigen binding fragment, E3 ubiquitin ligase, fragment, fusion protein and polynucleotide are the same as described above.

[0143] In one specific example, the polynucleotide encoding the fusion protein may comprise a base sequence of SEQ ID NO: 185 to SEQ ID NO: 205.

[0144] Specifically, the vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. Additionally, the plasmid may include a selectable marker, such as an antibiotic resistance gene, and the host cell harboring the plasmid may be cultured under selective conditions.

[0145] Additionally, the vector of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Examples of sequences to be fused include Flag (IBI, USA), glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), 6×His (Qiagen, USA), Halo, or Myc.

[0146] Transformed cells expressing the fusion protein

[0147] Another aspect of the present invention provides a transformed cell into which an expression vector is introduced, wherein the expression vector comprises a polynucleotide encoding a fusion protein comprising the anti-SOX2 single domain antibody or an antigen-binding fragment thereof; and a fragment of an E3 ubiquitin ligase or a variant thereof.

[0148] The above anti-SOX2 single domain antibody, antigen-binding fragment, E3 ubiquitin ligase, fragment, variant, fusion protein, polynucleotide and expression vector are the same as described above.

[0149] The above-mentioned transformed cell can be produced by introducing a vector into a host cell and transforming it. In addition, the polynucleotide contained in the vector can be expressed to produce the fusion protein of the present invention.

[0150] The above transformation can be performed by various methods, such as CaCl2 precipitation, Hanahan method, electroporation, calcium phosphate precipitation, protoplast fusion, Agrobacterium-mediated transformation, PEG-based transformation, etc., and is not particularly limited thereto, as long as it can produce a fusion protein comprising the anti-SOX2 single domain antibody variant of the present invention and the variant of the E3 ubiquitin ligase fragment.

[0151] In addition, the host cell used for producing the above-mentioned transformed cell is not particularly limited as long as it can produce the anti-SOX2 single domain antibody variant of the present invention.

[0152] Method for producing fusion proteins

[0153] Another aspect of the present invention provides a method for producing the fusion protein, comprising: culturing the transformed cell; and obtaining a fusion protein comprising an anti-SOX2 single domain antibody or an antigen-binding fragment thereof and a fragment of E3 ubiquitin ligase or a variant thereof from the culture medium of the cell.

[0154] The method for culturing the above-mentioned transformed cells can be performed using methods widely known in the art. Specifically, the culturing can be performed continuously in a batch process or a fed batch or repeated fed batch process.

[0155] In addition, the step of recovering the fusion protein from the culture can be performed by a method known in the art. Specifically, the recovery method is not particularly limited as long as it can recover the produced fusion protein of the present invention. Preferably, the recovery method can be a method such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion).

[0156] A pharmaceutical composition comprising a fusion protein, a polynucleotide encoding the same, or a vector loaded with the polynucleotide

[0157] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, the fusion protein, a polynucleotide encoding the fusion protein, or a vector loaded with the polynucleotide. In this case, the polynucleotide may be DNA, mRNA, plasmid DNA, etc., and the polynucleotide or the vector containing (loaded with) the same may be delivered into cells by a cell-penetrating functional nanocarrier and exhibit the same target protein degradation efficacy as the fusion protein.

[0158] The above fusion protein, the polynucleotide encoding the fusion protein, and the vector containing the polynucleotide are the same as described above.

[0159] The term "cancer" as used herein refers to a disease caused by cells that have aggressive characteristics in which cells divide and proliferate while ignoring normal growth limits, invasive characteristics in which cells invade surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body, and is used with the same meaning as a malignant tumor.

[0160] The cancer may be any one selected from the group consisting of, but not limited to, breast cancer, colon 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.

[0161] As used herein, the term "treatment" may be used to encompass both therapeutic treatment and preventative treatment. In this context, prevention may be used to mean alleviating or reducing a pathological condition or disease in an individual. In one embodiment, the term "treatment" encompasses all applications or any form of medication for treating a disease in mammals, including humans. Furthermore, the term encompasses inhibiting or slowing the progression of a disease; restoring or repairing damaged or defective functions, thereby partially or completely alleviating a disease; or stimulating an inefficient process; or alleviating a serious disease.

[0162] As used herein, the term "treatment of cancer" means inhibiting or preventing the growth of cancer cells or tissues, and may also include reducing cancer growth and metastasis compared to when no treatment or treatment is performed, and reducing resistance to anticancer drugs to enhance the therapeutic effect. The term "cancer metastasis" refers to the process by which tumor (cancer) cells spread to distant parts of the body, and "resistance to anticancer drugs" or "anticancer drug resistance" refers to the absence of a therapeutic effect from the beginning of treatment when treating a cancer patient with an anticancer drug, or the loss of a cancer treatment effect during the initial treatment process even though the cancer treatment effect is lost during continued treatment. "Prevention" refers to any act of inhibiting the occurrence of cancer or delaying its onset by administering the pharmaceutical composition.

[0163] Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also influence efficacy. Therefore, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and enhanced efficacy, and can be measured by comparing parameters such as clearance rate and tumor treatment or improvement in test animals or human subjects.

[0164] Here, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the 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 a drug that is effective in treating a disease.

[0165] At this time, the pharmaceutical composition may further include 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. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0166] Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the carrier does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the tolerable level of the intended subject.

[0167] In the present invention, the pharmaceutical composition may be administered systemically via parenteral administration. Parenteral administration may include, but is not limited to, intranasal, intranasal, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intradermal, intraperitoneal, enteral, topical, sublingual, or rectal administration.

[0168] In the present invention, when the pharmaceutical composition comprises a polynucleotide or a vector containing (loaded with) the same as an active ingredient, the nucleic acid may be used together with various carriers such as lipid nanoparticles (LNPs), liposomes, or vesicles, which are known to effectively deliver polynucleotides into cells, but are not limited thereto.

[0169] The pharmaceutical composition may be prepared in the form of, for example, a powder, a tablet, a capsule, a liquid, an ointment, a cream, a gel, a hydrogel, an aerosol, a spray, a micellar solution, a transdermal patch, a liposomal suspension, a polyplex, an emulsion, a lipid nanoparticle (LNP) (having RNA on its surface or encapsulated therein) or any other suitable form that can be administered to a human or mammal in need of treatment.

[0170] In addition, when the pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. Methods for formulating pharmaceutical compositions are known in the art, and specific references can be made to literature [Remington's Pharmaceutical Sciences (19th ed., 1995)], etc. The above literature is considered to be a part of the present specification.

[0171] The preferred dosage of the above pharmaceutical composition may range from 0.01 ug / kg to 10 g / kg or from 0.01 mg / kg to 1 g / kg per day, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once a day or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0172] The subject to which the above pharmaceutical composition can be applied (prescribed) may be a mammal, preferably a human. The pharmaceutical composition of the present application may additionally contain, in addition to the active ingredient, any compound or natural extract known to have anticancer activity.

[0173] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject the fusion protein, a polynucleotide encoding the fusion protein, or a vector loaded with the polynucleotide.

[0174] Another aspect of the present invention provides a use of the fusion protein, a polynucleotide encoding the fusion protein, or a vector loaded with the polynucleotide for preventing or treating cancer.

[0175] The above fusion protein, the polynucleotide encoding the fusion protein, the vector loaded with the polynucleotide, administration, prevention and treatment are the same as described above.

[0176] At this time, the subject may be an subject suffering from a disease, etc. In addition, the subject may be a mammal, and preferably a human.

[0177] The above fusion protein or nucleic acid may be administered to a subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects. The optimal administration method, dosage, and frequency can be selected within an appropriate range by a person skilled in the art. In addition, the above fusion protein or nucleic acid may be administered in combination with other drugs or physiologically active substances known to have therapeutic effects against the disease to be treated, or may be formulated in the form of a combination preparation with other drugs.

[0178] Mutant fragment of E3 ubiquitin ligase

[0179] Another aspect of the present invention provides a fragment of βTrCP (beta-transducin repeat-containing protein) or a variant thereof. In addition, another aspect of the present invention provides a fragment of SKP2 (S-phase kinase-associated protein 2) or a variant thereof.

[0180] The above βTrCP (beta-transducin repeat-containing protein) fragment or variant thereof and SKP2 (S-phase kinase-associated protein 2) fragment or variant thereof are the same as described above.

[0181] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the scope of the present invention is not limited to these examples.

[0182] Example 1. Screening of anti-huSOX2 VHH antibodies

[0183] Human SRY-box transcription factor 2 (huSOX2, UniProt: P48431, SEQ ID NO: 1), CynoSOX2 (UniProt: A0A2K5UL35, SEQ ID NO: 2), huSOX3 (UniProt: P41225, SEQ ID NO: 3) and fragment 135-317 of huSOX2 (huSOX2 135-317 , and the antigen was prepared by attaching a 6×His-tag sequence to the C-terminus of each of the sequences (SEQ ID NO: 4) and requesting production from Biointron.

[0184] Specifically, the protein was expressed in 4 L cultured Escherichia coli (E. coli), purified using a His-tag, and harvested. Other antigens were purchased from the relevant vendors.

[0185] The discovery of nanobodies binding to huSOX2 was commissioned to Elpis Biotech, and four rounds of panning were conducted using the huSOX2 (Cusabio, Cat no. CSB-EP022426HU(M)) antigen. As a result, the huSOX2-specific VHH antibody S14 (SEQ ID NO: 5) was secured.

[0186] Panning was performed using the VHH phage library. huSOX2 produced by Biointron was placed in an immunotube. 135-317 Protein was added at a concentration of 20 ug / mL and adsorbed on the surface of the test tube for 1 hour. Then, 3% non-fat milk solution was added to the test tube and huSOX2 135-317 The surface was protected from adsorption. After emptying the test tube, 2×10 dispersed in 3% non-fat milk solution was placed therein. 11The antibody phage library of CFU was added and reacted with the antigen. Non-specifically bound phages were washed three times with 1× PBST (phosphate-buffered saline + Tween 20) buffer. The remaining antigen-specific phage antibodies were eluted with 100 mM triethylamine (Sigma, Cat no. 471283) solution. The eluted phages were neutralized with 1.0 M Tris-HCl (Biosesang, TR2016-050-75) buffer and then transfected into TG1 (Lucigen, Cat no. 60502-2) Escherichia coli to measure the titer. The phages were amplified in the same host before the next round of panning.

[0187] The amplified E. coli was suspended in 5 mL of SB medium (20 g Yeast extract (Duchfa, Cat no. T1333), 30 g Trypton (Duchfa, Cat no. T1332), 10 g MOPS (Duchefa, Cat no. M1502), and 100 ug / mL ampicillin (Biosesang, Cat no. AC1043-025-00) / 1 L distilled water). 50 uL of the suspension was inoculated into 20 mL of SB-ampicillin solution and cultured at 37°C. The absorbance (OD) of the culture was determined by 600 ) becomes 0.5, 10 10 PFU of M13KO7 helper phage (Invitrogen, Cat no. 18311-019) was added and cultured at 37°C with gentle shaking. After 1 hour, 70 μg / mL of kanamycin (Biosesang, Cat no. KC1001-025-02) was added and cultured overnight at 30°C with rapid shaking (220 rpm).

[0188] The following day, the culture solution was centrifuged, and the supernatant containing phage particles was transferred to a new 50 mL tube. 5 mL of PEG / NaCl solution was added and incubated on ice for 30 minutes. The precipitated phage was centrifuged and dissolved in 1× PBS buffer. The phage solution was used as a library, and the panning process was repeated to enrich antigen-specific clones.

[0189] Individual colonies after panning were incubated with 10 ug / mL off-target antigen, 20 ug / mL huSOX2, and huSOX2 135-317 A 96-well ELISA screening was performed for the protein. The sequences of clones that showed negative signals for off-target antigen proteins and positive signals for huSOX2 proteins (two types) were confirmed, resulting in nine clones (SEQ ID NOs: 6 to 14) with different sequences.

[0190] Anti-huSOX2 VHH antibodies were screened from a single domain antibody (sdAb) naive phage display library using huSOX2 (SEQ ID NO: 1), cynoSOX2 (SEQ ID NO: 2), and huSOX3 (SEQ ID NO: 3) produced by Biointron at the request of Genscript Probio. After three rounds of panning, 3F11 (SEQ ID NO: 15), 3A2 (SEQ ID NO: 16), and 3H9 (SEQ ID NO: 17) (a total of three) were selected as anti-huSOX2 VHH antibodies that bound to huSOX2 and cynoSOX2, but not to huSOX3.

[0191] As a result of analyzing the sequences of anti-huSOX2 VHH antibodies obtained through the antibody selection using the above three methods, 13 clones having the amino acid sequences described in Fig. 1 and Table 2 were finally derived.

[0192]

[0193]

[0194] Example 2. Confirmation of binding affinity of selected anti-huSOX2 VHH antibody clones

[0195] Nine of the 13 selected clones were selected and 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).

[0196] The above clones were cultured at 30°C for approximately 16 hours using 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 ug / mL kanamycin (Biosesang, Cat no. KC1001-025-02) / 1 L distilled water). The culture was transferred to SG-CAA medium at OD 600 =1, and was further cultured at 30°C for 18 hours to induce the expression of anti-huSOX2 VHH antibodies on the yeast surface.

[0197] 0.5×10 cultured yeast 7Primary staining was performed by mixing 2 uM of biotinylated huSOX2 (Biointron) or 2 uM of biotinylated off-target antigen (human recombinant SKP1 protein, Sinobiological, Cat no. 14161-H40E-B) and anti-V5 mouse antibody (Invitrogen, Cat no. R960-25) (1:500 diluted in wash buffer (PBS containing 0.1% BSA)) at a ratio of 1:500 and incubating at room temperature for 30 minutes.

[0198] 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 ratio of 1:100 were added and incubated in the dark at 4°C for 20 minutes (secondary staining). The reaction product was analyzed using a flow cytometer (SONY, SH800S) to confirm the binding affinity of the anti-huSOX2 VHH antibody to the huSOX2 protein.

[0199] As a result, as shown in Figures 2a and 2b, it was confirmed that the anti-huSOX2 antibody produced as described above specifically binds only to SOX2.

[0200] Example 3. Production and antigen binding assay of anti-huSOX2 VHH antibodies

[0201] The affinity of the antigen for anti-huSOX2 VHH antibodies (3 types; 3A2, 3F11, 3H9) produced by the method of Example 1 above was evaluated.

[0202] First, a single domain antibody (sdAb) conjugated with human Fc (SEQ ID NO: 151) after codon optimization was expressed in Expi-CHO-S cells, and then the sdAb-Fc was purified using a Protein A affinity column (Table 3). The binding affinity of each antibody for the huSOX2 protein was confirmed using a Surface Plasmon Resonance (SPR) biosensor (Biacore 8K).

[0203]

[0204] Specifically, the antibody was captured at approximately 100 RU on the Protein A sensor chip via the Fc capture method. The huSOX2 protein was diluted as an analyte and injected onto the surface of flows 1 and 2, followed by injection of running buffer as a dissociation step. The dissociation rate constant (kd) and association rate constant (ka) were analyzed using Biacore 8K evaluation software. In addition, the equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. The results of the analysis are shown in Table 4.

[0205] Antibody type 3F113A23H9 Antigen huSOX2 huSOX2 huSOX2 ka (1 / Ms) 1.75E+05 1.48E+05 2.13E+05 kd (1 / s) 5.41E-04 3.52E-04 2.26E-04 KD (M) 2.12E-09 2.39E-09 1.06E-09

[0206] Example 4. Confirmation of specific binding affinity of anti-huSOX2 VHH antibody and target protein

[0207] Among the single domain antibody clones selected in Example 1, five types were used to measure the specific binding affinity for huSOX2 protein using NanoBRET™Nano-Glo® substrate (Promega, Cat no. N1661).

[0208] Specifically, the expression vector for the huSOX2 protein was constructed by sequentially loading a polynucleotide encoding a Halo tag (SEQ ID NO: 154) at the N terminus and a polynucleotide encoding huSOX2 (SEQ ID NO: 97) into the pHTN vector (Promega, Cat no. G7721). In addition, the expression vectors for each of the five single-domain antibody clones were sequentially loaded with the NanoLuc tag (SEQ ID NO: 156) and the polynucleotides encoding the five single-domain antibody clones (SEQ ID NO: 101, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 113) at the N terminus into the pNLF1 vector (Promega, Cat no. N1351) to construct expression vectors (pNLF1-N-S14, pNLF1-N-2E8, pNLF1-N-3F11, pNLF1-N-3A2, pNLF1-N-3H9).

[0209] The expression vectors manufactured as above were combined in each ratio and transfected into human kidney cell line (HEK293T cells) using Lipofectamine 3000 (Lipofectamine 3000, Invitrogen, Cat no. L3000001). 24 hours after transfection, HEK293T cells were treated with HaloTag 618, a ligand that binds to the Halo tag, and cultured for an additional 6 hours. Afterwards, the cells were treated with Nano-Glo substrate to induce luminescence, and the luminescence value was measured using a microplate reader (BioTek, Synergy HTX). The results were graphed using Graphpad prism software.

[0210] As a result, as shown in Fig. 3, it was confirmed that all five antibody clones used specifically bind to the huSOX2 protein.

[0211] Example 5. Production of bioprotactic products containing anti-huSOX2 VHH antibodies

[0212] Example 5.1. Production of a fusion protein comprising S14 and E3 ubiquitin ligase fragments

[0213] To confirm the SOX2 protein-specific degradation ability of a fusion protein comprising the selected single-domain antibody S14 and E3 ubiquitin ligase, a fusion protein comprising an E3 ubiquitin ligase fragment and S14 was constructed. The amino acid sequences of the ubiquitin ligase fragments (11 types) used are shown in Table 5.

[0214] The above fusion protein was produced in a form including E3 ubiquitin ligase fragment (SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 27)-GS (SEQ ID NO: 157)-S14 (SEQ ID NO: 5) or S14 (SEQ ID NO: 5)-GS (SEQ ID NO: 157)-E3 ubiquitin ligase fragment (SEQ ID NO: 20 to SEQ ID NO: 23, SEQ ID NO: 26 or SEQ ID NO: 28), and a Flag-tag (SEQ ID NO: 162) was included at the N-terminus of the E3 ubiquitin ligase fragment or S14 located at the N-terminus. The polynucleotides (SEQ ID NO: 129 to SEQ ID NO: 139) encoding the above fusion protein were each loaded into a pCMV6 (Origene, Cat no. PS100001) vector for mammalian cell expression. The sequence of the above fusion protein is described in Table 6. Hereinafter, the fusion protein containing the S14 and E3 ubiquitin ligase fragments is described interchangeably with “S14-E3 ubiquitin ligase.”

[0215] An expression vector was constructed by loading a polynucleotide (SEQ ID NO: 128) encoding huSOX2.Myc (SEQ ID NO: 32) containing a Myc tag (SEQ ID NO: 158) at the C terminus into the vector.

[0216] Name: UniProt ID. Location: Sequence number: βTrCP 190-228　Q9Y297190-228DPAEAVLQEKALKFMNSSEREDCNNGEPPRKIIPEKNSLRQTYNSCARLCLNQETVCLASTAMKTENCVAKTKLANGTSSMIVPKQRKLSASYEKEKELCVKYFEQWSESDQVEFVEHLISQMCHYQHGHINSYLKPMLQRDFITALPARGLDHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLWKKLIERMVRTDSLWRGLAERRGWGQYLFKNKPPDGNAPPNSFYRALYPKIIQDIETIESNWRCGRHSLQRIHCR18SKP2 2-176 Q133092-176HRKHLQEIPDLSSNVATSFTWGWDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLGIFSCLCLPELLKVSGVCKRWYRLASDESLWQTLDLTGKNLHPDVTGRLLSQGVIAFRCPRSFMDQPLAE19VHL 152-213 P40337152-213TLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD20SPOP 167-374 043791167-374SVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQS21SOCS2 143-198 O14508143-198PRNGTVHLYLTKPLYTSAPSLQHLCRLTINKCTGAIWGLPLPTRLKDYLEEYKFQV22CHIP 128-303Q9UNE7128-303RLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKRDIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLAMKEVIDAFISENGWVEDY23DDB2 2-114 Q924662-114APKKRPETQKTSEIVLRPRNKRSRSPLELEPEAKKLCAKGSGPSRRCDSDCLWVGLAGPQILPPCRSIVRTLHQHKLGRASWPSVQQGLQQSFLHTLDSYRILQKAAPFDRRA24CRBN 2-320 Q96SW22-320AGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTS 25ASB1 266-335 Q9Y576266-335VKWESLGPESRGRRKVDPEALQVFKEARSVPRTLLCLCRVAVRRALGKHRLHLIPSLPLPDPIKKFLLHE 26TRIM21 2-85　 P194742-85ASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEARE 27RNF4 71-190P7831771-190DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI28

[0217]

[0218]

[0219]

[0220] Example 5.2. Construction of a fusion protein comprising 3A2 and E3 ubiquitin ligase fragments

[0221] In order to confirm the SOX2 protein-specific degradation ability of a fusion protein comprising the single-domain antibody 3A2 selected in Example 1 and the E3 ubiquitin ligase fragment, a fusion protein comprising the E3 ubiquitin ligase fragment and 3A2 was produced. The amino acid sequences of the ubiquitin ligase fragments (10 types) used are shown in Table 7.

[0222] The above fusion protein comprises 3A2 (SEQ ID NO: 16)-GS (SEQ ID NO: 157)-E3 ubiquitin ligase fragment (SEQ ID NO: 20 to SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 28 to SEQ ID NO: 31), or E3 ubiquitin ligase fragment (SEQ ID NO: 27)-GS (SEQ ID NO: 157)-3A2 (SEQ ID NO: 16), and is produced in a form in which a Flag-tag is included at the N-terminus of the 3A2 or E3 ubiquitin ligase fragment located at the N-terminus.

[0223] The polynucleotides encoding the above fusion proteins (SEQ ID NOs: 140 to 149) were each loaded into the pCMV6 (Origene, Cat no. PS100001) vector for mammalian cell expression. The sequences of the above fusion proteins are described in Table 8. Hereinafter, the fusion proteins containing the 3A2 and E3 ubiquitin ligase fragments are described interchangeably as "3A2-E3 ubiquitin ligase."

[0224] An expression vector was constructed by loading a polynucleotide (SEQ ID NO: 150) encoding huSOX2.V5 (SEQ ID NO: 54) containing a V5-tag (SEQ ID NO: 160) at the C-terminus into the vector.

[0225] Name: UniProt ID. Location: Sequence number: VHL 152-213 P40337152-213TLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD20SPOP 167-374 043791167-374SVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEEPEVFKEMMCFIYTGKAPNLD KMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQS21SOCS2 143-198 O14508143-198PRNGTVHLYLTKPLYTSAPSLQHLCRLTINKCTGAIWGLPLPTRLKDYLEEYKFQV22CHIP 128-303Q9UNE7128-303RLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKRDIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLAMKEVIDAFISENGWVEDY23ASB1 266-335 Q9Y576266-335VKWESLGPESRGRRKVDPEALQVFKEARSVPRTLLCLCRVAVRRALGKHRLHLIPSLPLPDPIKKFLLHE 26TRIM21 2-85　 P194742-85ASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEARE 27RNF4 71-190 P7831771-190DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI 28UBE2D1 2-147 P516682-147ALKRIQKELSDLQRDPPAHCSAGPVGDDLFHWQATIMGPPDSAYQGGVFFLTVHFPTDYPFKPPKIAFTTKIYHPNINSNGSICLDILRSQWSPALTVSKVLLSICSLLCDPNPDDPLVPDIAQIYKSDKEKYNRHAREWTQKYAM29UBE2D4 2-147 Q9Y2X82-147ALKRIQKELTDLQRDPPAQCSAGPVGDDLFHWQATIMGPNDSPYQGGVFFLTIHFPTDYPFKPPKVAFTTKIYHPNINSNGSICLDILRSQWSPALTVSKVLLSICSLLCDPNPDDPLVPEIAHTYKADREKYNRLAREWTQKYAM30ZNFR1 138-227Q8ND25138-227SSHSGFKCPICSKSVASDEMEMHFIMCLSKPRLSYNDDVLTKDAGECVICLEELLQGDTIARLPCLCIYHKSCIDSWEFEVNRSCPEHPAD31

[0226]

[0227]

[0228]

[0229] Example 6. Evaluation of SOX2 protein degradation ability of fusion proteins containing S14 and E3 ubiquitin ligase fragments

[0230] The SOX2 protein-specific degradation ability of the S14-E3 ubiquitin ligase fusion protein through binding of S14 and SOX2 was confirmed.

[0231] Specifically, the expression vectors of 11 kinds of S14-E3 ubiquitin ligase fusion proteins and the huSOX2.Myc protein expression vector produced by the method of Example 5.1 were each transfected into MDA-MB-231 cells, a human breast cancer cell line, using Lipofectamine 3000 (Lipofectamine 3000, Invitrogen, Cat no. L3000001). 24 hours after transfection, the cells were washed twice with cold PBS buffer (Gibco, Cat no. 10010-023) and treated with M-PER protein extract (M-PER Mammalian Protein Extraction Reagent, Thermofisher, Cat no. 78505) containing a protease inhibitor cocktail (Thermofisher, Cat no. 78440) to obtain cell extracts. Each of the above cell extracts was uniformly disrupted using an ultrasonicator, centrifuged, and the supernatant was obtained. The protein amount of each supernatant was quantified using a BCA kit (ThermoFisher, Cat no. 23227), and an equal amount of protein (30 ug) for each sample was electrophoresed on a 4-12% SDS PAGE gel (Invitrogen, Cat no. NW04125BOX).

[0232] The separated proteins were transferred to a PVDF membrane (Invitrogen, Cat no. IB24002), and the membrane was blocked with a blocking buffer solution [1× PBST buffer solution (1× PBS, 0.1% Tween 20) containing 5% skim milk (BD, Cat no. 232100)] for 60 minutes. After blocking, the membrane was treated with primary antibodies and reacted at 4°C for 16 hours. At this time, the primary antibodies were anti-c-Myc antibody (SantaCruz, Cat no. sc-40, 1:5000), anti-FLAG antibody (Sigma, Cat no. F1804, 1:2500), or anti-α-tubulin antibody (GeneTex, Cat no. GTX628802, 1:3000), each diluted in blocking buffer. After the primary antibody reaction, the membrane was washed three times for 10 minutes each in 1× PBST buffer. Then, the membrane was treated with each 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 to horseradish peroxidase (HRP) diluted in blocking buffer. After the secondary antibody reaction, the membrane was washed three times for 10 minutes each in 1× PBST buffer.

[0233] The expression level of each protein was confirmed using an Imager (Invitrogen, iBright, CL1500) after treatment with ECL (Cytiva, Cat no. RPN2232 or Thermofisher, Cat no. 34096) solution. In addition, the expression level of the protein was quantified as the amount of SOX2 protein relative to α-tubulin and presented in a graph (Graphpad prism software).

[0234] As a result, as shown in Fig. 4, it was confirmed that 9 out of 11 S14-E3 ubiquitin ligase fusion proteins degrade SOX2 protein. In particular, S14.010 and S14.012 exhibited SOX2 protein degradation ability of more than 70% compared to the control group (Mock).

[0235] Example 7. Cancer cell growth inhibition and apoptosis induction effects of a fusion protein comprising S14 and E3 ubiquitin ligase fragments

[0236] Example 7.1. Confirmation of cell growth inhibition and apoptosis induction effects by a fusion protein containing S14 and E3 ubiquitin ligase fragments in human cancer cell lines.

[0237] The cancer cell growth inhibition and apoptosis induction effects of the S14-E3 ubiquitin ligase fusion protein were confirmed.

[0238] First, each mRNA was produced by Genscript Probio using a polynucleotide encoding S14.010 (SEQ ID NO: 137) or a polynucleotide encoding S14.012 (SEQ ID NO: 139) as a template.

[0239] S14.010 mRNA or S14.012 mRNA, produced as described above, was transfected into human pancreatic cancer cell lines (BxPC3 cells) or human non-small cell lung cancer cell lines (NCI-H1703 cells, A549 cells) using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015). At this time, SOX2 siRNA (Origene, Cat no. SR321861-A) was used as a positive control, and siRNA (Santacruz, Cat no. sc-37007) was used as a control to confirm the apoptosis induction effect according to SOX2 protein expression in cancer cell lines.

[0240] Additionally, apoptosis was confirmed by treating each transduced cell with Caspase-3 / 7 Green Dye for Apoptosis (Satorius, Cat no. 4440), and then analyzing images taken at 3-hour intervals using the phase channel and fluorescence channel using Incucyte (Satorius) equipment (using Incucyte software).

[0241] As a result, as shown in Figures 5a to 5c, it was confirmed that cell death increased in all three cancer cell lines in the SOX2 siRNA treatment group, S14.010 mRNA treatment group, and S14.012 mRNA treatment group compared to the control group. In particular, it was confirmed that cell death and cell growth inhibition occurred more significantly in the S14.010 mRNA treatment group and the S14.012 mRNA treatment group, respectively.

[0242] Example 7.2. Confirmation of the cell growth inhibition effect of a fusion protein containing S14 and E3 ubiquitin ligase fragments in human cancer cell lines.

[0243] The growth inhibition effect of cancer cells was confirmed according to the concentration of S14-E3 ubiquitin ligase fusion protein.

[0244] Specifically, S14.010 mRNA and S14.012 mRNA produced by the method of Example 7.1 were serially diluted by half each, and then transduced into human non-small cell lung cancer cell lines (A549 cells, NCI-H1703 cells) and human cervical cancer cell lines (SiHa cells) using Lipofectamine-MessengerMax. The degree of cell growth for each cell was confirmed by taking images through the phase channel at 3-hour intervals using Incucyte (Satorius) equipment in the same manner as Example 7.1, and analyzing the images using Incucyte software.

[0245] As a result, as shown in Fig. 6, the cell growth inhibition effect was confirmed for all cancer cell lines in the S14.010 mRNA treatment group and the S14.012 mRNA treatment group, respectively.

[0246] Example 7.3. Effect of combined use of a fusion protein containing S14 and E3 ubiquitin ligase fragments and an anticancer drug on cell growth inhibition and apoptosis in anticancer drug-resistant human cancer cell lines.

[0247] It has been reported that the SOX2 protein is involved in cancer cell acquisition of anticancer drug resistance. The anticancer activity of the fusion protein of the present invention against anticancer drug-resistant cancer cells was confirmed.

[0248] Example 7.3.1. Confirmation of the combined effect of fusion proteins containing S14 and E3 ubiquitin ligase fragments and tamoxifen in tamoxifen-resistant cancer cell lines.

[0249] First, to confirm the correlation between the anticancer drug resistance and SOX2 protein, MCF-7 cells (human breast cancer cells) and MCF7-Tam cells showing tamoxifen resistance R The intracellular SOX2 expression level was confirmed using the same method as in Example 6. At this time, the primary antibody used was an anti-SOX2 antibody (CST, Cat no. 3579, 1:1000).

[0250] As a result, as shown in Fig. 7a, compared to MCF7 cells, MCF7-Tam R It was confirmed that SOX2 protein was expressed approximately 6 times more in the cells.

[0251] Next, the fusion protein of the present invention, S14.010 or S14.012, is MCF7-Tam R We confirmed whether the therapeutic effect of tamoxifen could be enhanced by inducing SOX2 protein degradation in each cell.

[0252] Specifically, Lipofectamine-MessengerMax was serially diluted by 1 / 2 each of S14.010 mRNA and S14.012 mRNA produced by the method of Example 7.1 above, and MCF7-Tam R Cells were transduced. After transduction, each cell was treated with Caspase-3 / 7 Green Dye for Apoptosis (Satorius, Cat no. 4440) and cultured for an additional 24 hours, followed by treatment with tamoxifen (20 uM). Apoptosis was confirmed using Incucyte (Satorius) equipment in the same manner as in Example 7.1. The degree of cell growth and apoptosis signal at the last imaging point were analyzed using Incucyte software, and the results were plotted using Graphpad prism software.

[0253] As a result, as shown in Fig. 7b, MCF7-Tam R Compared to the tamoxifen-only treatment group, the combined treatment group of tamoxifen and S14.010 mRNA or S14.012 mRNA showed an increased effect on inducing apoptosis and inhibiting cell growth in cancer cells.

[0254] Example 7.3.2. Confirmation of the combined effect of doxorubicin and a fusion protein containing S14 and E3 ubiquitin ligase fragments in doxorubicin-resistant human cancer cell lines.

[0255] H69 cells (human non-small cell lung cancer cell line) and doxorubicin-resistant H69-DOX R The expression levels of SOX2 protein in cells were compared.

[0256] Specifically, each of the above cells was washed twice with PBS buffer, and then each cell was treated with cell lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS) containing a protease inhibitor cocktail (Thermofisher, 78440) to obtain a cell extract. The cell extract was uniformly mixed using a vortex and heated at 95°C for 10 minutes. The protein amount of the cell extract was quantified using a BCA kit (ThermoFisher, 23227), and the protein sample was prepared by mixing with 10X sample buffer (Proteinsimple, Cat no. 042-195), 400 mM DTT solution, and Fluorescent 5X Master Mix (Proteinsimple, Cat no. PS-ST01EZ) and heating at 95°C for 5 minutes.

[0257] SOX2 was detected using a Fluorescence Separation Module (Proteinsimple, Cat no. SM-FL004) with an anti-SOX2 antibody (CST, Cat no. 3579) and an antibody anti-rabbit secondary detection module (Protein simple, Cat no. DM-001). The detection results were quantified using a Protein Normalization Module (Proteinsimple, Cat no. DM-PN02). Detection was performed using a JESS (Proteinsimple) device, and analysis was performed using Gaussian Fit in Compass for SW software.

[0258] As a result, as shown in Fig. 8a, compared to H69 cells, H69-DOX R It was confirmed that the expression of SOX2 protein in the cells increased by approximately 2-fold.

[0259] Next, the fusion protein of the present invention, S14.010 or S14.012, is H69-DOX RIt was confirmed whether the therapeutic effect of doxorubicin could be enhanced by inducing SOX2 protein degradation in each cell. At this time, the experiment was conducted in the same manner as in Example 7.3.1, and doxorubicin was treated at a concentration of 10 uM.

[0260] As a result, as shown in Fig. 8b, H69-DOX R Compared to the doxorubicin-only treatment group, the combined treatment group with doxorubicin and S14.010 mRNA or S14.012 mRNA showed increased apoptosis induction and cell growth inhibition effects in cancer cells.

[0261] Example 8. Evaluation of SOX2 protein degradation ability of fusion proteins containing 3A2 and E3 ubiquitin ligase fragments

[0262] Example 8.1. Confirmation of SOX2 protein degradation ability of fusion proteins containing 3A2 and E3 ubiquitin ligase fragments.

[0263] The SOX2 protein-specific degradation ability of the 3A2-E3 ubiquitin ligase fusion protein through binding of 3A2 and SOX2 was confirmed.

[0264] The expression vectors of 10 kinds of 3A2-E3 ubiquitin ligase fusion proteins produced by the method of Example 5.2 and the huSOX2.V5 protein expression vector were each transduced into human kidney cell line (HEK-293 cells) using Lipofectamine 3000 (Lipofectamine 3000, Invitrogen, Cat no. L3000001). SOX2 protein expression in the transduced cells was confirmed by the same method as Example 6. At this time, anti-V5 antibody (Invitrogen, Cat no. 46-1157), anti-Flag antibody (Sigma, cat no. F1804), Fluorescence Separation Module (Proteinsimple, Cat no. SM-FL004), and anti-mouse-HRP antibody (Proteinsimple, Cat no. 042-206) were additionally used.

[0265] As a result, as shown in Fig. 9, it was confirmed that six out of ten 3A2-E3 ubiquitin ligase fusion proteins degrade SOX2 protein. In particular, the 3A2.005 treatment group, the 3A2.007 treatment group, the 3A2.011 treatment group, and the 3A2.012 treatment group each exhibited a SOX2 degradation ability of approximately 70% or more compared to the control group (Mock).

[0266] Example 8.2. Evaluation of SOX2 protein degradation capacity according to concentration of fusion protein containing 3A2 and E3 ubiquitin ligase fragments in human cancer cell lines.

[0267] Intracellular SOX2 protein degradation was determined in human cancer cell lines according to the concentration of fusion proteins containing 3A2 and E3 ubiquitin ligase fragments.

[0268] First, the polynucleotide encoding 3A2.005 (SEQ ID NO: 141) or the polynucleotide encoding 3A2.012 (SEQ ID NO: 146) was cloned into the 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 vector as a template, 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) were used to produce mRNAs encoding 3A2.005 and 3A2.012, respectively.

[0269] The above 3A2.005 mRNA and 3A2.012 mRNA were transfected into human non-small cell lung cancer cell lines (NSCLC) (A549 cells, NCI-H1703 cells, HCC827 cells, SK-MES-1 cells, SW900 cells) and human hepatoma cell lines (HCC) (Hep3B cells) using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015), respectively. The expression of SOX2 protein in the transfected cells was confirmed in the same manner as in Example 7.3.2.

[0270] As a result, as shown in Fig. 10, it was confirmed that the 3A2.005 mRNA treatment group or the 3A2.012 mRNA treatment group increased SOX2 degradation in a concentration-dependent manner in all cancer cell lines. The 50% concentration of the maximum degradation of SOX2 protein for each cancer cell line (DC 50 , nM) are listed in Table 9.

[0271] cancer cell line DC 50(nM)3A2.0053A2.012NSCLCA5490.040.02NCI-H17030.030.05HCC8270.010.05SK-MES-10.030.13SW9000.120.57HCCHep3B0.050.03

[0272] Example 8.3. Confirmation of ubiquitin-proteasome-dependent SOX2 protein degradation by a fusion protein containing 3A2 and E3 ubiquitin ligase fragments.

[0273] We confirmed that the degradation of SOX2 protein induced by the 3A2-E3 ubiquitin ligase fusion protein occurs through the intracellular ubiquitin-proteasome system (UPS).

[0274] Specifically, the 3A2.005 expression vector, 3A2.012 expression vector, and huSOX2.V5 expression vector prepared by the method of Example 5.2 were transfected into human kidney cell line (HEK-293 cells) using Lipofectamine 3000 (Lipofectamine 3000, Invitrogen, Cat no. L3000001). 18 hours after transfection, the cells were treated with MLN7243, an ubiquitin E1 activating enzyme inhibitor, or MG132, a proteasome inhibitor, at concentrations of 1 uM and 20 uM, respectively, and incubated for an additional 6 hours. At this time, DMSO was treated as a control group. The amount of SOX2 protein expression in the cells treated as described above was confirmed by the same method as in Example 7.3.2. At this time, anti-V5 antibody (Invitrogen, Cat no. 46-1157) and anti-Flag antibody (Sigma, cat no. F1804) were used.

[0275] As a result, as shown in Fig. 11, compared to the control group, the SOX2 protein degradation effect was significantly reduced by the expression of 3A2.005 or 3A2.012 in the MLN7243 treatment group and the MG132 treatment group, respectively. Therefore, it was confirmed that the degradation of SOX2 protein by the expression of the fusion protein containing the 3A2 and E3 ubiquitin ligase fragments was induced in a ubiquitin-proteasome dependent manner.

[0276] Example 9. Cell growth inhibition effect of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments on cancer cells

[0277] Example 9.1. Comparison of cell growth inhibition effects of fusion proteins containing SOX2 siRNA and 3A2 and E3 ubiquitin ligase fragments on cancer cells

[0278] SOX2 siRNA (Origene, Cat no. SR321861-A) and 3A2.005 mRNA or 3A2.012 mRNA were transfected into human non-small cell lung cancer cell lines (A549 cells) using Lipofectamine-RNAiMAX (Lipofectamine RNAiMAX, Invitrogen, Cat no. 13778150) and Lipofectamine-MessengerMAX (Lipofectamine MessengerMAX, Invitrogen, Cat no. LMRNA015), and the degree of cell growth inhibition was confirmed. At this time, the 3A2.005 mRNA and 3A2.012 mRNA were prepared and used in the same manner as in Example 8.2. In addition, the detection of the intracellular SOX2 protein expression level and the degree of cancer cell growth inhibition were confirmed in the same manner as in Example 7.3.2 and Example 7.2.

[0279] As a result, as shown in Fig. 12, it was confirmed that the SOX2 protein degradation and cancer cell growth inhibition effects were increased in the 3A2.005 mRNA treatment group and the 3A2.012 mRNA treatment group, respectively, compared to the SOX2 siRNA treatment group.

[0280] Example 9.2. Confirmation of the efficacy of a fusion protein containing 3A2 and E3 ubiquitin ligases according to concentration in human cancer cell lines.

[0281] The growth inhibition effect of cancer cells according to the concentration of 3A2-E3 ubiquitin ligase fusion protein treatment in human cancer cell lines was confirmed.

[0282] Specifically, 3A2.005 mRNA and 3A2.012 mRNA were serially diluted and transduced into human non-small cell lung cancer cell lines (A549 cells, NCI-H1703 cells, Calu-1 cells, HCC15 cells, SW900 cells, SK-MES-1 cells), human pancreatic cancer cell lines (BxPC3 cells, CFPAC-1 cells), human cervical cancer cell lines (SiHa cells), or human hepatoma cell lines (HepG2 cells) using Lipofectamine-MessengerMax. The degree of cell growth of the transduced cells was imaged using the phase channel at 3-hour intervals with an Incucyte (Satorius) device, and then analyzed using Incucyte software at the last imaging time point. The 3A2.005 mRNA and 3A2.012 mRNA were prepared and used in the same manner as in Example 8.2.

[0283] As a result, as shown in Figures 13a and 13b, the 3A2.005 mRNA treatment group and the 3A2.012 mRNA treatment group showed cell growth inhibition effects on all cancer cell lines. The maximum cell proliferation of the cancer cell lines was 50% inhibitory concentration GI 50 (nM) values ​​are listed in Table 10.

[0284] Cancer cell line GI 50(nM)3A2.0053A2.012NSCLCA5490.431.16NCI-H17030.110.15Calu-10.511.26HCC150.330.84S W9000.140.45SK-MES-10.450.57PancreaticcancerCFPAC-1<0.010.94BxPC30.450.48Cervical cancerSiHa0.190.25HCCHepG20.430.93

[0285] Example 9.3. Effect of combined use of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments and an anticancer drug on cell growth inhibition and apoptosis induction in anticancer drug-resistant human cancer cell lines.

[0286] Tamoxifen-resistant breast cancer cell line MCF7-Tam R Cells and doxorubicin-resistant lung cancer cell line H69-Dox R The combined effect was confirmed by transducing cells with tamoxifen or doxorubicin and 3A2.005 mRNA or 3A2.012 mRNA at 1 nM each using the same method as in Example 7.2. At this time, tamoxifen was serially diluted from 100 uM to 2 / 3 and treated at each concentration, and doxorubicin was serially diluted from 150 uM to 1 / 2 and treated at each concentration.

[0287] As a result, as shown in Fig. 14, MCF7-Tam R Compared to the tamoxifen-only treatment group, the combined treatment group with tamoxifen and 3A2.005 mRNA or 3A2.012 mRNA showed higher apoptosis induction and cell growth inhibition effects. In addition, H69-Dox R In cells, the apoptosis induction and cell growth inhibition effects were higher in the doxorubicin and 3A2.005 mRNA or 3A2.012 mRNA combination treatment group compared to the doxorubicin treatment group alone.

[0288] Example 10. Evaluation of the tumor growth inhibitory effect of a fusion protein containing S14 and E3 ubiquitin ligase fragments in a human cancer cell line transplant tumor mouse model.

[0289] To confirm the tumor suppression efficacy of the S14-E3 ubiquitin ligase fusion protein, a mouse tumor model was created by transplanting a non-small cell lung cancer cell line (A549 cells) or a pancreatic cancer cell line (BxPC3 cells).

[0290] Specifically, 5.0×10 6- to 8-week-old female M-NSG mice (Shanghai Model Organisms Center, Inc.) were injected 6 cells (A549 cells) or 1.0×10 7 Cells (BxPC3 cells) were transplanted subcutaneously. Afterwards, the tumor size of each individual was approximately 150 mm 3 Upon reaching the target, the mice were randomly assigned to groups of five per group. Lipid nanoparticles containing S14.012 mRNA were then administered intravenously at a concentration of 2 mg / kg, once a week, for a total of three doses. PBS was administered intravenously to the control group.

[0291] Tumor volume was calculated using the following <Mathematical Formula 1> based on values ​​measured twice a week. The body weight of each individual was also measured at this time.

[0292] <Mathematical Formula 1>

[0293] Tumor volume (㎣) = 0.5 × (long axis) × (short axis) 2

[0294] As a result, as shown in Fig. 15, the S14.012 mRNA administration group exhibited a tumor growth inhibitory effect in both mouse tumor models implanted with A549 cells or BxPC3 cells. At this time, no significant change in body weight was observed in the S14.012 mRNA administration group compared to the control group.

[0295] Example 11. Confirmation of the cancer cell growth inhibition and apoptosis induction effects of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments in human non-small cell lung cancer cell lines.

[0296] 3A2.005 mRNA or 3A2.012 mRNA was transfected into human non-small cell lung cancer cell lines (A549 cells) using Lipofectamine MessengerMAX (Invitrogen, Cat no. LMRNA015). At this time, SOX2 siRNA (Origene, Cat no. SR321861-A) and doxorubicin were used as positive controls, and control siRNA (Santacruz, Cat no. sc-37007) was used as a control to confirm the effect of inducing cell death according to the regulation of SOX2 protein expression in cancer cell lines. The 3A2.005 mRNA or 3A2.012 mRNA was prepared and used in the same manner as in Example 8.2.

[0297] Additionally, apoptosis was confirmed by treating each transduced cell with Caspase-3 / 7 Green Dye for Apoptosis (Satorius, Cat no. 4440), and then analyzing images taken at 3-hour intervals using the phase channel and fluorescence channel using Incucyte (Satorius) equipment (using Incucyte software).

[0298] As a result, as shown in Fig. 16, it was confirmed that cell growth was inhibited and cell death increased in the SOX2 siRNA treatment group, doxorubicin, 3A2.005 mRNA treatment group, and 3A2.012 mRNA treatment group compared to the control group in the cancer cell lines. In particular, cell growth inhibition and cell death in the 3A2.005 mRNA treatment group and the 3A2.012 mRNA treatment group were confirmed to be at a similar level to the positive control group, doxorubicin.

[0299] Example 12. Expression of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments and evaluation of the persistence of SOX2 protein degradation.

[0300] The expression of the 3A2-E3 ubiquitin ligase fusion protein in cells and the duration of the fusion protein's ability to degrade SOX2 proteins were confirmed.

[0301] Specifically, the expression vectors of the 3A2.005 fusion protein or the 3A2.012 fusion protein produced by the method of Example 5.2 were transfected into human kidney cell lines (HEK-293 cells) together with the huSOX2.V5 protein expression vector using Lipofectamine 3000 (Invitrogen, Cat no. L3000001). Then, cell extracts were obtained at 24-hour intervals from 72 hours to 168 hours. The amount of SOX2 protein in the cell extracts was confirmed by the same method as in Example 7.3.2. At this time, SOX2 was detected using the primary antibody anti-V5 antibody (Invitrogen, Cat no. 46-1157), and the 3A2-E3 ubiquitin ligase fusion protein was detected using anti-Flag antibody (Sigma, cat no. F1804). The secondary antibody used was anti-mouse-HRP antibody (Proteinsimple, Cat no. 042-206).

[0302] As a result, as shown in Fig. 17, it was confirmed that the expression of the 3A2.005 and 3A2.012 fusion proteins and the SOX2 protein degradation effect of the fusion proteins continued from 72 hours to 168 hours.

[0303] Example 13. Comparison of SOX2 protein degradation according to treatment concentration of fusion proteins containing 3A2 and E3 ubiquitin ligase fragments and SOX2 siRNA

[0304] SOX2 siRNA (Origene, Cat no. SR321861-A) or 3A2.012 mRNA was transfected into human non-small cell lung cancer cell lines (SW900 cells) using Lipofectamine-RNAiMAX (Lipofectamine RNAiMAX, Invitrogen, Cat no. 13778150) or Lipofectamine-MessengerMax, respectively, and the amount of intracellular SOX2 protein was detected using the same method as in Example 7.3.2. The results were expressed as a graph using Graphpad prism software. 3A2.012 mRNA was prepared and used using the same method as in Example 8.2.

[0305] As a result, as shown in Fig. 18, it was confirmed that the SOX2 protein degradation ability was significantly increased in a concentration-dependent manner in the 3A2.012 mRNA treatment group compared to the SOX2 siRNA treatment group.

[0306] Example 14. Confirmation of SOX2 protein degradation ability according to treatment concentration of fusion protein containing 3A2 and E3 ubiquitin ligase fragments in human cancer cell lines.

[0307] The intracellular SOX2 protein degradation capacity was determined according to the treatment concentration of 3A2-E3 ubiquitin ligase fusion protein in human cancer cell lines.

[0308] 3A2.005 mRNA and 3A2.007 mRNA were serially diluted at each concentration and transduced into human non-small cell lung cancer cell lines (NSCLC) (A549 cells, SW900 cells) using Lipofectamine-MessengerMax, respectively. At this time, 3A2.007 mRNA was produced using the polynucleotide (SEQ ID NO: 143) encoding 3A2.007 and used in the same manner as in Example 8.2. In addition, 3A2.005 mRNA was also produced using the same manner as in Example 8.2 and used. After 24 hours, each cell extract was obtained, and the amount of SOX2 protein was confirmed in the same manner as in Example 7.3.2.

[0309] As a result, as shown in Fig. 19, it was confirmed that the 3A2.005 mRNA treatment group and the 3A2.007 mRNA treatment group increased SOX2 degradation in a concentration-dependent manner in all cancer cell lines, respectively. The 50% concentration of the maximum degradation of SOX2 protein for each cancer cell line (DC 50 , nM) are listed in Table 11.

[0310] cancer cell line DC 50 (nM)3A2.0053A2.007NSCLCA5490.100.03SW9000.290.18

[0311] Example 15. Evaluation of cell growth inhibition efficacy according to concentration of fusion protein containing 3A2 and E3 ubiquitin ligase fragments in human cancer cell lines

[0312] The cell growth inhibition efficacy of 3A2-E3 ubiquitin ligase fusion protein was confirmed according to the concentration in human cancer cell lines.

[0313] 3A2.005 mRNA or 3A2.007 mRNA was serially diluted at each concentration and transfected into human non-small cell lung cancer cell lines (NSCLC) (A549 cells, SW900 cells) using Lipofectamine-MessengerMax, respectively. Images taken at 3-hour intervals using the phase channel with Incucyte (Satorius) equipment were analyzed (using Incucyte software) to analyze the degree of cell growth inhibition, and the cell growth inhibition results after 72 hours were plotted using Graphpad prism software. 3A2.005 mRNA and 3A2.007 mRNA were prepared and used in the same manner as in Example 8.2.

[0314] As a result, as shown in Fig. 20, the 3A2.005 mRNA treatment group and the 3A2.007 mRNA treatment group each showed a cell growth inhibition effect in a concentration-dependent manner of mRNA treatment in all cancer cell lines. The 50% inhibitory concentration (GI) of the maximum cell proliferation of each cancer cell line 50 , nM) are listed in Table 12.

[0315] Cancer cell line GI 50 (nM)3A2.0053A2.007NSCLCA5491.580.37SW9000.340.21

[0316] Example 16. Confirmation of the specific SOX2 protein degradation ability and cell growth inhibition effect of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments in human non-small cell lung cancer cell lines.

[0317] The SOX2 proteolytic and cell growth inhibitory effects of 3A2 single-domain antibody, E3 ubiquitin ligase protein fragment (alone), or 3A2-E3 ubiquitin ligase fusion protein were compared in human non-small cell lung cancer cell lines (A549 cells, SW900 cells).

[0318] mRNA encoding 3A2 single domain antibody, E3 ubiquitin ligase protein (alone) (3A2.005 E3 only, 3A2.007 E3 only) or 3A2-E3 ubiquitin ligase fusion protein (3A2.005, 3A2.007), respectively, was transfected into human non-small cell lung cancer cell lines (A549 cells, SW900 cells) using Lipofectamine-MessengerMax. At this time, A549 cells were treated with 0.3 nM of mRNA, and SW900 cells were treated with 1 nM of mRNA, respectively. The mRNA of the 3A2 single domain antibody and the mRNA of the E3 ubiquitin ligase protein fragment were respectively represented by SEQ ID NO: 112 (3A2) and SEQ ID NO: 117 (SPOP). 167-374 ) and sequence number 119 (CHIP 128-303 ) was produced and used using the method of Example 8.2. In addition, 3A2.005 mRNA and 3A2.007 mRNA were also produced and used using the same method as Example 8.2.

[0319] The expression of SOX2 protein in transduced cells and the cancer cell growth inhibition effect were confirmed using the same methods as in Example 7.3.2 and Example 7.2, respectively. For the detection of single domain antibodies, E3 ubiquitin ligase fragments, and 3A2-E3 ubiquitin ligase fusion proteins, anti-FLAG antibody (Sigma, Cat no. F1804) was used as the primary antibody, and anti-mouse-HRP antibody (Proteinsimple, Cat no. 042-206) was used as the secondary antibody.

[0320] As a result, as shown in Figures 21a and 21b, in the single-domain antibody (binder only) mRNA treatment group and the E3 ubiquitin ligase protein fragment (only) (E3 only) mRNA treatment group, no degradation of SOX2 protein (Figure 21a) or inhibition of cancer cell growth (Figure 21b) was observed. On the other hand, in both the 3A2.005 mRNA treatment group and the 3A2.007 mRNA treatment group, degradation of SOX2 protein and inhibition of cancer cell growth were observed.

[0321] Example 17. Confirmation of SOX2 protein degradation ability of fusion protein containing 3A2 and E3 ubiquitin ligase fragments in human cancer cell line transplant tumor mouse model

[0322] The tumor suppressive efficacy of the 3A2-E3 ubiquitin ligase fusion protein was confirmed using a mouse tumor model transplanted with a non-small cell lung cancer cell line (A549 cells). The mouse tumor model was prepared using the same method as in Example 10.

[0323] The tumor size of each individual mouse model above was approximately 150-200 mm. 3 Upon reaching the target, mice were randomly assigned to groups of three per group. A single intravenous injection of lipid nanoparticles containing 3A2.012 mRNA was administered at a concentration of 2 mg / kg. Tumor tissue was excised from the mice 16 hours later. PBS was administered intravenously to the control group. 3A2.012 mRNA was prepared and used according to the method described in Example 8.2.

[0324] The extracted tumor tissue was treated with cell lysis buffer and finely pulverized using a TissueLyser. Then, the pulverized material was sonicated (10 seconds, 10 times) and centrifuged (15,000 rpm, 10 minutes) to obtain a tumor tissue extract. The amount of SOX2 protein in the tumor tissue was confirmed using the same method as in Example 6. At this time, the primary antibody used was an anti-SOX2 antibody (CST, Cat no. 3579) or an anti-GAPDH antibody (Abcam, Cat no. 8245).

[0325] As a result, as shown in Fig. 22, it was confirmed that SOX2 protein was reduced in the tumor tissues of all individuals (3 mice) in the 3A2.012 mRNA treatment group compared to the control group.

[0326] Example 18. Evaluation of SOX2 protein degradation ability according to treatment time of fusion protein containing 3A2 and E3 ubiquitin ligase fragments in human non-small cell lung cancer cell lines.

[0327] After expression of the 3A2-E3 ubiquitin ligase fusion protein in human non-small cell lung cancer cell lines, the extent of SOX2 protein degradation over time was determined.

[0328] Specifically, 3A2.005 mRNA and 3A2.012 mRNA were transduced at a concentration of 2 nM into human non-small cell lung cancer cell line (SW900 cells) using Lipofectamine-MessengerMax, respectively. The transduced cells were cultured for various time periods (4, 8, and 24 hours), and cell extracts were obtained to confirm the expression of SOX2 protein using the same method as in Example 7.3.2. The results were expressed as a graph using Graphpad Prism software. 3A2.005 mRNA and 3A2.012 mRNA were produced and used using the same method as in Example 8.2.

[0329] As a result, as shown in Fig. 23, SOX2 protein degradation was observed 4 hours after treatment with 3A2.005 mRNA or 3A2.012 mRNA, and it was confirmed that the degree of SOX2 protein degradation increased over time.

[0330] Example 19. Expression of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments and evaluation of the persistence of SOX2 protein degradation in human non-small cell lung cancer cell lines.

[0331] After treating human non-small cell lung cancer cell lines with mRNA encoding a 3A2-E3 ubiquitin ligase fusion protein, the duration of the fusion protein's ability to degrade SOX2 protein was confirmed.

[0332] Specifically, 3A2.005 mRNA, 3A2.007 mRNA, and 3A2.012 mRNA were transduced into human non-small cell lung cancer cell line (SW900 cells) at a concentration of 1 nM using Lipofectamine-MessengerMax, respectively. The transduced cells were cultured for each time period (0, 4, 8, 24, 48, 72, 96, 120, 144, and 168 hours) to obtain cell extracts. The intracellular SOX2 protein of each treatment group was confirmed using the same method as in Example 6, and represented as a graph using Graphpad prism software. 3A2.005 mRNA, 3A2.007 mRNA, and 3A2.012 mRNA were prepared and used using the same method as in Example 8.2.

[0333] As a result, as shown in Figures 24a and 24b, when 3A2.005, 3A2.007, and 3A2.012 were treated, the SOX2 degradation ability was highest 24 hours after treatment, and the degradation effect was confirmed to last until the 7th day. Inversely, the expression level of the fusion protein was highest at 24 or 48 hours and decreased thereafter.

[0334] Example 20. Induction of cancer cell growth inhibition by combined use of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments and doxorubicin in doxorubicin-resistant lung cancer cell lines.

[0335] The combined effect of 3A2-E3 ubiquitin ligase fusion protein was confirmed to enhance the therapeutic effect of standard treatment drugs by inducing SOX2 protein degradation in human non-small cell lung cancer cell line H69 cells and doxorubicin-resistant lung cancer cell line H69-DoxR cells.

[0336] Specifically, 3A2.005 mRNA, 3A2.007 mRNA, and 3A2.012 mRNA were transfected into H69 cell lines or H69-Dox at a concentration of 5 nM each using Lipofectamine-MessengerMax. R Transduced into cells. Simultaneously with transduction, H69-Dox R Cells were treated with doxorubicin serially diluted 1 / 4 from 50 uM, and H69 cells were treated with doxorubicin serially diluted 1 / 4 from 30 uM, and then cultured. To confirm the degree of cell growth inhibition, at the end of culture, Cell Counting Kit-8 reagent (Dojindo, CK04) was treated and reacted, and then the absorbance was measured using a microplate reader (BioTek, Synergy HTX). The results were expressed as a graph using Graphpad prism software. The clinical dose indicated on the graph was converted to the cell area standard and indicated in the corresponding concentration range. 3A2.005 mRNA, 3A2.007 mRNA, and 3A2.012 mRNA were produced and used in the same manner as in Example 8.2.

[0337] As a result, as shown in Figs. 25 and 26, H69-Dox RIn cells, it was confirmed that the growth of cancer cells was inhibited when doxorubicin and 3A2.005 mRNA, 3A2.007 mRNA, or 3A2.012 mRNA were combined in a concentration range where cancer cell growth inhibition did not occur (single treatment dose range: 8.3 uM to 10.3 uM; combination treatment dose range: 5.5 uM to 10.3 uM) by acquiring doxorubicin resistance.

[0338] In addition, in H69 cells without resistance, it was confirmed that cancer cell growth was further inhibited when doxorubicin and 3A2.005 mRNA, 3A2.007 mRNA, or 3A2.012 mRNA were combined, compared to when doxorubicin was treated alone at the lowest concentration of the clinical dose range, 5.5 uM or less.

[0339] Through the above results, it was confirmed that combined administration of the fusion protein of the present invention and doxorubicin can improve (increase) the efficacy of doxorubicin.

[0340] Example 21. Induction of cancer cell growth inhibition by combined use of a fusion protein containing 3A2 and E3 ubiquitin ligase fragments and tamoxifen in tamoxifen-resistant breast cancer cell lines.

[0341] Human breast cancer cell line MCF7 and tamoxifen-resistant breast cancer cell line MCF7-Tam R We confirmed the combined effect of 3A2-E3 ubiquitin ligase fusion protein in cells to enhance the therapeutic effect of standard treatment drugs by inducing SOX2 protein degradation.

[0342] Specifically, 3A2.005 mRNA, 3A2.007 mRNA, and 3A2.012 mRNA were transfected into MCF7 cells or MCF7-Tam cells at a concentration of 5 nM each using Lipofectamine-MessengerMax. RTransduced into cells. After culturing for 24 hours after transduction, tamoxifen was diluted from 27 uM to 3 uM in 3 uM intervals and treated to each cell, and cultured for an additional 48 hours. The degree of cell growth inhibition was measured in the same manner as in Example 20. The indicated clinical dose was converted to a cell area basis and indicated in the corresponding concentration range. 3A2.005 mRNA, 3A2.007 mRNA, and 3A2.012 mRNA were produced and used in the same manner as in Example 8.2.

[0343] As a result, as shown in Figs. 25 and 27, MCF7-Tam R In cells, it was confirmed that cell growth was inhibited when tamoxifen and 3A2.005 mRNA, 3A2.007 mRNA, or 3A2.012 mRNA were co-treated in a concentration range (2.01 uM to 4.08 uM) where cell growth inhibition due to tamoxifen resistance did not occur.

[0344] In addition, it was confirmed that cell growth was inhibited by more than 50% in the combination treatment group of tamoxifen and 3A2.005 mRNA, 3A2.007 mRNA, or 3A2.012 mRNA compared to the tamoxifen alone treatment group in the clinical dose concentration range (2.01 uM to 4.08 uM) in MCF7 cells without resistance.

[0345] Through the above results, it was confirmed that the combined administration of the fusion protein of the present invention and tamoxifen can improve (increase) the efficacy of tamoxifen.

Claims

1. An anti-SOX2 single domain antibody or antigen-binding fragment thereof comprising a heavy chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 63, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 87, and SEQ ID NO: 94; HCDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 84, and SEQ ID NO: 89; and HCDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 91, and SEQ ID NO:

96.

2. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 56; HCDR2 comprising the amino acid sequence of SEQ ID NO: 58; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

60.

3. In paragraph 2, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

5.

4. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 63; HCDR2 comprising the amino acid sequence of SEQ ID NO: 65; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

67.

5. In paragraph 4, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

6.

6. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 69; HCDR2 comprising the amino acid sequence of SEQ ID NO: 71; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

67.

7. In paragraph 6, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

7.

8. In paragraph 1, An anti-SOX2 antibody or fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 63; HCDR2 comprising the amino acid sequence of SEQ ID NO: 72; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

67. An anti-SOX2 single domain antibody or antigen-binding fragment thereof.

9. In paragraph 8, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

8.

10. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 73; HCDR2 comprising the amino acid sequence of SEQ ID NO: 74; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

67.

11. In paragraph 10, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

9.

12. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 63; HCDR2 comprising the amino acid sequence of SEQ ID NO: 75; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

67.

13. In paragraph 12, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

10.

14. In paragraph 12, An anti-SOX2 single domain antibody or a fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

11.

15. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 63; HCDR2 comprising the amino acid sequence of SEQ ID NO: 76; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

67.

16. In paragraph 15, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

12.

17. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 82; HCDR2 comprising the amino acid sequence of SEQ ID NO: 84; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

85.

18. In paragraph 17, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

13.

19. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 78; HCDR2 comprising the amino acid sequence of SEQ ID NO: 79; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

81.

20. In paragraph 19, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

14.

21. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 87; HCDR2 comprising the amino acid sequence of SEQ ID NO: 89; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

91.

22. In paragraph 21, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

15.

23. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 94; HCDR2 comprising the amino acid sequence of SEQ ID NO: 89; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

91.

24. In paragraph 23, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

16.

25. In paragraph 1, An anti-SOX2 single domain antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 94; HCDR2 comprising the amino acid sequence of SEQ ID NO: 89; and HCDR3 comprising the amino acid sequence of SEQ ID NO:

96.

26. In paragraph 25, An anti-SOX2 single domain antibody or a fragment thereof, wherein the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:

17.

27. A polynucleotide encoding an anti-SOX2 single domain antibody or an antigen-binding fragment thereof according to any one of claims 1 to 26.

28. An expression vector loaded with the polynucleotide of clause 27.

29. A transformed cell into which the expression vector of clause 28 has been introduced. 30.i) A step of culturing the transformed cell of clause 29; and ii) A method for producing an anti-SOX2 single domain antibody or an antigen-binding fragment thereof, comprising: a step of obtaining an anti-SOX2 single domain antibody or an antigen-binding fragment thereof from the culture solution.

31. A fusion protein comprising the anti-SOX2 single domain antibody of claim 1 or an antigen-binding fragment thereof; and a fragment of E3 ubiquitin ligase or a variant thereof.

32. In paragraph 31, A fusion protein, wherein the fragment of the above E3 ubiquitin ligase or a variant thereof is any one selected from the group consisting of β-TrCP (Beta-transducin repeats-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 ZNFR1 (Zinc and ring finger 1).

33. In paragraph 31, A fusion protein, wherein the fragment of the E3 ubiquitin ligase or a variant thereof comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO:

31.

34. In paragraph 31, A fusion protein, wherein the above fusion protein comprises a linker.

35. In paragraph 34, The above fusion protein is a fusion protein having the following structural formula (I) or (II): N'-TB-(L)n-SR-C' (I) N'-SR-(L)n-TB-C' (II) At this time, in the structural formulas (I) and (II), The above N' is the N-terminus of the fusion protein, The above C' is the C-terminus of the fusion protein, The above TB is an anti-SOX2 single domain antibody or an antigen-binding fragment thereof, The above SR is a fragment of E3 ubiquitin ligase or a variant thereof, wherein L is a peptide linker, The above n is 0 or 1.

36. A fusion protein according to any one of claims 31 to 35, wherein the fusion protein comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 164 to SEQ ID NO:

184.

37. A polynucleotide encoding a fusion protein of any one of claims 31 to 36.

38. An expression vector loaded with the polynucleotide of item 37.

39. A transformed cell into which the expression vector of clause 38 has been introduced.

40. A method for producing a fusion protein, comprising the step of culturing the transformed cell of clause 39.

41. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a fusion protein of any one of claims 31 to 36; a polynucleotide of claim 37; or a vector of claim 38.

42. In paragraph 41, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is any one selected from the group consisting of breast cancer, colon 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.

43. Use of a fusion protein according to any one of claims 31 to 36; a polynucleotide according to claim 37; or a vector according to claim 38 for the prevention or treatment of cancer.

44. A method for preventing or treating cancer, comprising administering to a subject a fusion protein of any one of claims 31 to 36; a polynucleotide of claim 37; or a vector of claim 38.

Images