fusion proteins

Abstract

To provide a technique useful for cancer treatment targeting at least a chemoresistant cancer cell.SOLUTION: A fusion protein includes an anthrax protective antigen sequence, a target protein sequence, and a protease recognition sequence. The anthrax protective antigen sequence includes a sequence representing domain 1 of the anthrax protective antigen sequence. The sequence representing domain 1 has the protease recognition sequence and the target protein sequence inserted thereto in order from the N-terminal side.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present disclosure relates to fusion proteins, in particular, fusion proteins useful for cancer therapy targeting chemotherapy-resistant cancer cells. [Background technology]

[0002] Initial chemotherapy for cancer is often effective, but in most cases, cancer cells that are resistant to chemotherapy develop, causing future recurrence. High-grade serous adenocarcinoma is the most common type of ovarian cancer, and although initial chemotherapy is highly effective, most cases recur. If recurrence occurs within a short period of time after initial chemotherapy, the same drug cannot be used. Therefore, drugs that target chemotherapy-resistant cancer cells are thought to be a therapy that can prevent future recurrence. However, such drugs have not yet been developed, in part because it is difficult to extract remaining cancer cells from actual cancer patients after chemotherapy, and the characteristics of cancer cells that exhibit chemotherapy resistance remain largely unknown.

[0003] It has been known that anthrax protective antigen (PA) forms a heptamer on the cell membrane of a target cell, and that the lethal factor binds to the lethal factor binding site of the heptamerized PA, resulting in the toxicity of anthrax. In addition, PA mutants that have been created to contain either a cleavage site recognized by urokinase-type plasminogen activator (uPA) or matrix metalloproteinase (MMP) in the lethal factor binding site of PA in order to enhance cell specificity cannot form a functional heptamer with either PA mutant alone, so that binding with the lethal factor is inhibited and the toxicity to cancer cells cannot be exerted. Therefore, it has been shown that the anticancer effect is low when only either PA mutant is administered. On the other hand, it has been shown that when these two types of PA mutants are administered together, a functional heptamer can be formed, and while the toxicity to mice is reduced due to the high cell specificity, the effect is specific to transplanted cancer cells (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nat. Biotechnol., 2005;23(6):725-730 [Non-Patent Document 2] Biochemistry, 2014;53:2166-2171 [Non-Patent Document 3] Journal for ImmunoTherapy of Cancer, 2015;3:16 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present disclosure is to provide at least a technique useful for cancer treatment targeting chemotherapy-resistant cancer cells. [Means for solving the problem]

[0006] The inventors have found through single-cell gene expression analysis of ovarian cancer clinical specimens before and after chemotherapy, and gene expression analysis of cells separated and recovered by flow cytometry, that mesenchymal cancer cells expressing a group of genes associated with the properties of mesenchymal cells, including the mesenchymal marker THY1, appear in non-hemocyte cells remaining after chemotherapy. From the gene expression of this cell population, it was found that the anthrax toxin receptor ANTXR, as well as the proteases MMP and uPA, were specifically expressed. Furthermore, since these cells hardly expressed proliferation markers and were in the stationary phase, it was found that the effects of conventional anticancer drugs could not be expected.

[0007] Here, the present inventors developed a molecule for treating quiescent cancer cells, PA-DT, targeting chemotherapy-resistant cancer cells remaining after chemotherapy. PA-DT is a molecule formed by fusing anthrax protective antigen (PA) and diphtheria toxin (DT). The present inventors found that PA-DT is activated near cancer cells expressing matrix metalloproteinase (MMP) and urokinase-type plasminogen activator (uPA) and binds to ANTXR, and that the PA-DT bound to ANTXR is then delivered into the cells and induces cell death in the cancer cells. No adverse events were observed in mice when PA-DT was administered intraperitoneally.

[0008] Furthermore, the inventors developed a molecule for detecting quiescent cancer cells, PA-Clover, to label chemotherapy-resistant cancer cells remaining after chemotherapy. PA-Clover is a molecule formed by fusing protective antigen (PA), an anthrax toxin, with mClover (Clover), a fluorescent dye. The inventors found that PA-Clover is activated in the vicinity of cancer cells expressing MMP or uPA, and can label cancer cells by binding to ANTXR, or by subsequently delivering PA-Clover bound to ANTXR into the cells.

[0009] In other words, the present disclosure is as follows. [1] A fusion protein comprising an anthrax protective antigen sequence, a target protein sequence, and a protease recognition sequence, the anthrax protective antigen sequence comprises a sequence representing domain 1 of the anthrax protective antigen sequence, the protease recognition sequence and the target protein sequence are inserted in this order from the N-terminus into the sequence representing domain 1; Fusion proteins. [2] The fusion protein described in [1], wherein the insertion is made at any position in the region corresponding to positions 153 to 173 of the amino acid sequence of the anthrax protective antigen represented by SEQ ID NO:1. [3] The anthrax protective antigen sequence is the amino acid sequence represented by SEQ ID NO:1, or A sequence in which the glycine residue at position 342 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tryptophan residue. The fusion protein according to [1] or [2]. [4] The fusion protein according to any one of [1] to [3], wherein the target protein sequence is a sequence into which a second protease recognition sequence has been inserted. [5] The target protein sequence includes, in order from the N-terminus, a diphtheria toxin C domain (DT-C) sequence, the second protease recognition sequence, and a diphtheria toxin T domain (DT-T) sequence. [4] A fusion protein according to the invention. [6] The DT-C sequence is the amino acid sequence represented by SEQ ID NO: 2, and / or The DT-T sequence is an amino acid sequence represented by SEQ ID NO: 3. [5] A fusion protein according to the invention. [7] The protease recognition sequence and the second protease recognition sequence are recognized by the same or different proteases, and the protease is selected from matrix metalloproteases (MMPs) and urokinase-type plasminogen activators (uPAs). [5] or [6]. A fusion protein according to [5] or [6]. [8] The target protein sequence includes a sequence of a fluorescent protein; The fusion protein according to any one of [1] to [3]. [9] The protease recognition sequence is a sequence encoding a matrix metalloprotease (MMP) or a uroprotease. The fusion protein according to [8], which is recognized by kinase-type plasminogen activator (uPA).

[10] The fusion protein according to [8] or [9], wherein the fluorescent protein is mClover.

[11] A composition for inducing cell death in a cancer cell that expresses an anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, the composition comprising a fusion protein according to any one of [1] to [7] or an expression vector comprising a polynucleotide encoding the fusion protein.

[12] A composition for treating cancer, including cancer cells expressing anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, comprising the fusion protein according to any one of [1] to [7] or an expression vector comprising a polynucleotide encoding the fusion protein.

[13] A composition for labeling cancer cells expressing anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, the composition comprising the fusion protein according to any one of [8] to

[10] .

[14] The cancer labeling kit comprising the composition described in

[13] .

[15] A polynucleotide encoding the fusion protein according to any one of [1] to

[10] . Effect of the Invention

[0010] The present disclosure can provide at least a technique useful for cancer treatment targeting chemotherapy-resistant cancer cells.

[0011] Anticancer drugs that have been on the market so far have mainly been drugs that act on proliferating cancer cells to induce cell death or molecular targeted drugs that utilize cancer mutations, but these anticancer drugs have been difficult to show effects on cancer cells in the quiescent phase. By using the gene expression profile of quiescent chemotherapy-resistant cancer cells, the PA-DT disclosed here makes it possible to specifically damage these cells. PA-DT is stable in the absence of proteases, but is cleaved and activated when exposed to proteases. Activated PA-DT is rapidly degraded. Therefore, PA-DT is stable until it comes close to chemotherapy-resistant cancer cells, but when it comes close, it is activated by being cleaved by proteases such as MMP and uPA, binds to ANTXR, and is taken up into the cells, releasing toxins into the cells and inducing cell death. Here, if the activated PA-DT is not taken up into the cancer cells, the stability of the activated PA-DT decreases and it is degraded before acting on the surrounding normal tissues, so there are few side effects. In addition, the toxin portion of the molecule of the present disclosure is not limited to diphtheria toxin, and can be replaced with any toxin, etc., as necessary.

[0012] Similarly, PA-Clover is stable in the absence of proteases, but is cleaved and activated when exposed to proteases. This allows it to be used as a test method for labeling chemotherapy-resistant cancer cells. It can also be used as a cancer cell detection technology that leads to future recurrence. For example, clinical specimens after chemotherapy are extremely difficult to evaluate because of the large number of dead cells present, but by labeling resistant cancer cells with PA-Clover, it becomes easy to evaluate the risk of future recurrence. In addition, the mClover portion of the molecule disclosed herein can be replaced with any fluorescent substance as necessary. [Brief description of the drawings]

[0013] [Figure 1] Using single cell expression analysis, we analyzed the composition of the cell population that forms ovarian cancer using two initial untreated specimens, one specimen after initial chemotherapy, and one recurrent specimen. In the figure, black dots indicate expression of the MKI67 gene, a proliferation marker. The shade of black indicates the degree of expression, with darker black indicating a higher level of expression. [Diagram 2]The results of single-cell expression analysis of two initial untreated specimens, one initial post-chemotherapy specimen, and one recurrent specimen are shown in one diagram. In the diagram, black dots indicate the expression of each marker gene (EPCAM, PTPRC, THY1, MYC, MKI67, ANTXR1, MMP2, PLAU). The shade of black indicates the degree of expression, with darker black indicating higher expression. [Diagram 3] Schematic diagram of the design of the therapeutic molecule "PA-DT" and the detection molecule "PA-Clover" that target quiescent cancer cells. [Figure 4] FIG. 1 is a schematic diagram of the mechanism of action of PA-DT. [Diagram 5] A is a schematic diagram of PA-DT. B is a diagram showing the performance evaluation of PA-DT, in which MMP2, MMP9, or uPA were reacted with PA-DT, PA-DT with G342W substitution in the PA sequence (shown as PA-DT G342W in the figure), and PA-DT without MMP recognition sequence or uPA recognition sequence (shown as PA-DT no cut in the figure), electrophoresed on SDS-PAGE gel, transferred to a transfer membrane, and detected with HRP-labeled M2 antibody (photograph). [Figure 6] 1 shows the evaluation of the cell-killing effect of PA-DT, in which PA-DT was added to mouse ovarian cancer cell line ID8 luc2 and human normal prostate cell PNT2, and the cell viability was compared. [Figure 7] The results of functional analysis of PA-DT using PA-DT mutants are shown below. PA-DT (indicated as "original PADT" in the figure), PA-DT with G342W substitution in the PA sequence (indicated as "G342W" in the figure), and PA-DT without MMP recognition sequence and uPA recognition sequence (indicated as "nocut" in the figure) were added to mouse ovarian cancer cell line ID8 luc2, and cell viability was compared. [Figure 8]This is a photograph showing the evaluation of the cytocidal effect of PA-DT against cancer cells (human ovarian cancer cells (Kuramochi), human cervical cancer cells (HeLa), human castration-resistant prostate cancer cells (CWR22Rv1), and mouse melanoma cells (B16F10)). PA-DT was added to each type of cell, and the number of viable cells after a certain number of days was compared with that of the control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The configurations and combinations thereof in each embodiment are merely examples, and addition, omission, substitution, and other modifications of the configurations are possible as appropriate without departing from the spirit of the present invention. The present disclosure is not limited to the embodiments, but is limited only by the scope of the claims. Each feature disclosed herein may be combined with any other feature disclosed herein.

[0015] One embodiment of the present disclosure is a fusion protein comprising an anthrax protective antigen sequence, a target protein sequence, and a protease recognition sequence, wherein the anthrax protective antigen sequence includes a sequence representing domain 1 of the anthrax protective antigen sequence, and the protease recognition sequence and the target protein sequence are inserted, in that order from the N-terminus, into the sequence representing domain 1.

[0016] The fusion protein of this embodiment has an anthrax protective antigen sequence and is therefore capable of binding to both anthrax protective antigen receptor (ANTXR) 1 and ANTXR2. The anthrax protective antigen sequence is not particularly limited as long as it does not impede the effect of this embodiment, and an example of the sequence is the amino acid sequence shown in SEQ ID NO: 1. The anthrax protective antigen sequence of SEQ ID NO: 1 is an amino acid sequence excluding the methionine of the start codon. Hereinafter, in this embodiment, the positions of amino acids in the anthrax protective antigen sequence of SEQ ID NO: 1 are defined as positions excluding the start codon. In addition, the anthrax protective antigen sequence may be any sequence as shown in SEQ ID NO:1, so long as it can bind to ANTXR. It may be an amino acid sequence that contains a sequence that is 90% or more, preferably 95% or more, and more preferably 98% or more identical to the full length of the amino acid sequence shown in SEQ ID NO: 1, or it may be an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1. "One or several" may be, for example, 1 to 118, preferably 1 to 54, and more preferably 1 to 27. The identity between amino acid sequences can be calculated by methods known to those skilled in the art, for example, using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional adjustments: Conditional compositional score matrix adjustment) of blastp.

[0017] The anthrax protective antigen sequence may have any amino acid substitution as long as the fusion protein can bind to ANTXR, for example, the glycine residue at position 342 of the amino acid sequence shown in SEQ ID NO: 1 or a position equivalent thereto may be substituted with tryptophan, isoleucine, valine, leucine, etc. By using a sequence in which the glycine residue at position 342 of the amino acid sequence or a position equivalent thereto is substituted with any amino acid, for example, tryptophan, isoleucine, valine, leucine, etc., it is possible to reduce the binding of the anthrax protective antigen to ANTXR2 and, on the other hand, to increase the specificity for ANTXR1 (Non-Patent Document 2). The amino acid mutation in this embodiment has the advantage that the selectivity for ANTXR1 is increased by substituting a tryptophan residue for the glycine residue at position 342 or a position equivalent thereto in the amino acid sequence represented by SEQ ID NO: 1. This reduces the binding of anthrax protective antigen to ANTXR2 and reduces cell death mediated by ANTXR2. That is, the specificity for ANTXR1 can be increased by substituting a tryptophan residue for the glycine residue at position 342 or a position equivalent thereto in the amino acid sequence represented by SEQ ID NO: 1, and therefore cells expressing ANTXR1 can be specifically killed. In other words, when the fusion protein of this embodiment is intended to specifically bind to ANTXR1, it is preferable that the glycine residue at position 342 of the amino acid sequence represented by SEQ ID NO: 1 or a position equivalent thereto is substituted with any amino acid; when the fusion protein is intended to bind to both ANTXR1 and ANTXR2, a fusion protein without such amino acid substitution is used, and the fusion protein can be used according to the purpose.

[0018] The target protein sequence is a sequence for producing a specific action or effect attributable to the target protein in a cell expressing ANTXR by binding the target protein to the cell or by introducing the target protein into the cell. The target protein sequence may be any protein sequence depending on the purpose. The target protein sequence is not particularly limited as long as it does not interfere with the effect of this embodiment, and may be, for example, a toxin protein sequence such as diphtheria toxin or botulinum neurotoxin, a complement sequence such as C5, a fluorescent protein sequence, or a sequence in which a tag sequence is repeated multiple times. By repeating the tag sequence multiple times, the target cell can be clearly labeled.

[0019] A protease recognition sequence is a sequence that is recognized by a protease present in a cell expressing ANTXR and / or a protease present in the environment of said cell. The protease recognition sequence is not particularly limited, so long as it is a sequence that is recognized by proteases that are abundantly present around cells expressing ANTXR. The protease that recognizes the sequence may be a protease produced by cells expressing ANTXR, or a protease produced by the surrounding cells, but is preferably a protease produced by cells expressing ANTXR. Such proteases are not particularly limited as long as they do not impair the effects of the present embodiment, and examples thereof include matrix metalloproteases (MMPs) and urokinase-type plasminogen activators (uPAs). Preferred examples of MMPs include MMP2 and MMP9.

[0020] The fusion protein of this embodiment contains a sequence representing domain 1 of the anthrax protective antigen sequence, and the protease recognition sequence and the target protein sequence are inserted into the sequence representing domain 1, in that order from the N-terminus. The insertion of these sequences into the sequence representing domain 1 of the anthrax protective antigen sequence is not particularly limited as long as it does not impair the effects of this embodiment, and may be, for example, at any position in the region corresponding to positions 153 to 173 of the amino acid sequence of the anthrax protective antigen represented by SEQ ID NO: 1. Preferably, the insertion is made on the C-terminal side of the amino acid corresponding to position 163 of the amino acid sequence of the anthrax protective antigen represented by SEQ ID NO: 1. The method for inserting these sequences into domain 1 of the anthrax protective antigen sequence is not particularly limited as long as it does not interfere with the effects of this embodiment, and any method known to those skilled in the art can be used.

[0021] In this embodiment, the target protein sequence may be a sequence into which a second protease recognition sequence has been inserted. For example, when the target protein is a protein that produces a specific effect, such as a toxic activity, when a part of the protein is cleaved, such as diphtheria toxin, botulinum neurotoxin, or complement, the target protein sequence preferably has a second protease recognition sequence inserted into the region where the target protein is cleaved to produce its effect. For the second protease recognition sequence, the above description regarding the protease recognition sequence can be used.

[0022] When the target protein is diphtheria toxin, the target protein sequence is not particularly limited as long as it does not interfere with the effects of this embodiment, but may include, in order from the N-terminus, a diphtheria toxin C domain (DT-C) sequence, the second protease recognition sequence, and a diphtheria toxin T domain (DT-T) sequence.

[0023] When the target protein is diphtheria toxin, the DT-C sequence may be the amino acid sequence represented by SEQ ID NO:2, and the DT-T sequence may be the amino acid sequence represented by SEQ ID NO:3, although there are no particular limitations thereon as long as they do not interfere with the effects of this embodiment. Furthermore, the DT-C sequence and the DT-T sequence may be amino acid sequences that contain sequences that are 90% or more, preferably 95% or more, and more preferably 98% or more identical to the full-length amino acid sequences shown in SEQ ID NOs: 2 and 3, respectively, so long as they are capable of being taken up into cells and releasing diphtheria toxin into the cells, or may be amino acid sequences in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequences shown in SEQ ID NOs: 2 and 3, respectively. "One or several" may mean, for example, 1 to 18, preferably 1 to 9, and more preferably 1 to 5.

[0024] The protease recognition sequence and the second protease recognition sequence may be sequences recognized by the same protease or may be sequences recognized by different proteases. From the viewpoint of selectivity, the protease recognition sequence and the second protease recognition sequence are preferably sequences recognized by different proteases. The protease recognition sequence and the second protease recognition sequence are preferably sequences recognized by a protease selected from matrix metalloprotease (MMP) and urokinase-type plasminogen activator (uPA), respectively.

[0025] In this embodiment, the target protein sequence includes a sequence of a fluorescent protein. Good too. The protease recognition sequence is preferably a sequence recognized by matrix metalloprotease (MMP) or urokinase-type plasminogen activator (uPA).

[0026] The fluorescent protein is not particularly limited as long as it does not interfere with the effects of this embodiment, and examples thereof include mClover, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), mCherry, blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc.

[0027] The fusion protein of this embodiment may have any secretory signal sequence, such as an Igκ leader sequence, added to the N-terminus, any tag sequence, such as a 3×FLAG tag, added to the C-terminus, as long as the effect of this embodiment is not impaired. Furthermore, any linker sequence, such as a (GGGGS (SEQ ID NO: 4))×3 linker, may be included on the C-terminus of the target protein.

[0028] <Composition for inducing cell death in cancer cells> Another embodiment of the present disclosure is a composition for inducing cell death in a cancer cell that expresses anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, comprising any of the fusion proteins described above or an expression vector comprising a polynucleotide encoding the same. The polynucleotides may be modified polynucleotides having a base sequence in which one or several bases have been substituted, deleted, inserted, and / or added, so long as the fusion protein encoded by each of them can be expressed. Furthermore, the polynucleotides may have a structure in which some of the sugars or phosphates contained in the nucleotides have been substituted, so long as the fusion protein encoded by each of them can be expressed. The expression vector is not particularly limited as long as it can incorporate a target polynucleotide and express the protein in a host cell, and any expression vector known to those skilled in the art can be used. For example, a plasmid vector or a virus vector can be used, and a commercially available vector can be used. The method for incorporating a polynucleotide of interest into an expression vector is not particularly limited as long as it does not interfere with the effects of this embodiment, and can be carried out by any method known to those skilled in the art.

[0029] The fusion protein contained in the composition of this embodiment or the fusion protein expressed by the expression vector binds to ANTXR expressed on cancer cells. The fusion protein bound to ANTXR is activated by cleaving the protease recognition sequence by a protease such as MMP or uPA. The activated fusion protein forms a heptamer on the cell membrane and is endocytosed into cancer cells. After that, in the cancer cells, the fusion protein binds to a lysosome and the pH in the endosome decreases, releasing the toxin in the fusion protein into the cytoplasm, inducing cell death. For example, when the toxin in the fusion protein is diphtheria toxin, the diphtheria toxin C region is released into the cytoplasm, which inhibits the function of peptide chain elongation factor 2 (EF-2), inhibiting protein synthesis, and inducing cell death in cancer cells.

[0030] In this embodiment, the cancer cells are cells that express an anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein. The cancer cells are not particularly limited as long as they express a protease that recognizes the protease recognition sequence in ANTXR and the fusion protein, but may be, for example, cancer cells that are resistant to chemotherapy, cancer cells that have undergone epithelial-mesenchymal transition, THY1-positive cancer cells, and / or quiescent cancer cells.

[0031] The tissue from which the cancer cells are derived is not particularly limited as long as it exhibits the effects of this embodiment. The cancer cells are preferably cancer cells that express ANTXR1, and examples of such cancer cells include ovarian cancer, cervical cancer, prostate cancer, melanoma, etc. Furthermore, when the cancer cells are ovarian cancer, they are preferably high-grade serous adenocarcinoma.

[0032] In this embodiment, the fusion protein can be used alone as a pharmaceutical composition, but it may be mixed with a carrier or additive. Here, examples of the carrier include pharmacologically acceptable solvents, diluents, excipients, binders, etc., and for example, water, physiological saline, buffer solutions, etc. are used to prepare liquid pharmaceutical compositions. Examples of the additives include stabilizers, pH adjusters, thickeners, antioxidants, isotonicity agents, buffers, solubilizing agents, suspending agents, preservatives, cryoprotectants, cryoprotectants, lyophilization protectants, bacteriostatic agents, etc.

[0033] The dosage form of the medicament of the present disclosure is exemplified by liquids, tablets, etc., and is preferably liquids. When the medicament is a liquid, the concentration of the fusion protein is not particularly limited, and is, for example, 0.1 mg / mL to 10 mg / mL.

[0034] In this embodiment, the dosage of the composition is not particularly limited as long as it can induce cell death in cancer cells. The dosage is adjusted appropriately depending on the type, symptoms, and severity of cancer, and the age, sex, and weight of the patient, and the dosage per administration is, for example, 0.001 mg to 100 mg per kg of body weight. It may be administered once or multiple times. In the case of multiple administration, for example, a method of administering the composition 2 to 10 times in total every 1 to 60 days can be mentioned. The method of administration of the composition is not particularly limited, and includes oral administration, intravenous administration, local administration, etc., but it is preferable to administer the composition locally to the diseased site by injection, etc. Local administration by injection may be performed using a needle syringe or a needleless syringe. The subject to which the composition is administered is a mammal such as a human or a mouse, and preferably a human.

[0035] <Composition for treating cancer> Another embodiment of the present disclosure is a composition for treating cancer, including cancer cells expressing anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, comprising any of the fusion proteins described above, or comprising an expression vector comprising a polynucleotide encoding the same. The composition of this embodiment is not particularly limited with respect to the polypeptide, expression vector, cancer cells, dosage form, content of the fusion protein in the composition, dosage of the composition, administration schedule, administration method, administration target, etc., as long as it can treat cancer, and the matters described in the section <Composition for inducing cell death in cancer cells> can be cited.

[0036] In this embodiment, the treatment may be achieved, for example, by inducing cell death in cancer cells, and includes not only the elimination of all cancer cells present in the cancer tissue to be treated, but also the damaging of cancer cells, the reduction of the number of cancer cells, the inhibition of cancer cell proliferation, and the prevention of cancer recurrence.

[0037] <Composition for labeling cancer cells> Another embodiment of the present disclosure is a composition for labeling cancer cells expressing anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, comprising any of the fusion proteins described above. The composition of this embodiment is not particularly limited in terms of the polypeptide, expression vector, cancer cells, dosage form, content of the fusion protein in the composition, dosage of the composition, administration schedule, administration method, administration target, etc., as long as it can label cancer cells, and the matters described in the section <Composition for inducing cell death in cancer cells> can be cited.

[0038] In this embodiment, the label is the above-mentioned "composition for inducing cell death in cancer cells". As in the case above, the composition may bind to ANTXR in a target cancer cell, be taken up into the cell, and emit fluorescence inside the cell, or the composition may bind to ANTXR in a target cancer cell, but may not be taken up into the cell, and emit fluorescence on the cell membrane.

[0039] The composition for labeling cancer cells of this embodiment can be used for diagnosing cancer, and can also be used to confirm and / or identify the site of cancer during surgery.

[0040] Another embodiment of the present disclosure is a kit for labeling cancer, comprising the above-mentioned "composition for labeling cancer cells." In addition to the composition for labeling cancer cells, the kit may optionally contain a negative control composition, an instrument for administering or applying the composition, a buffer or diluent, and the like.

[0041] Another embodiment of the present disclosure is a polynucleotide encoding any of the fusion proteins described above.

[0042] In this embodiment, the polynucleotide may be a modified polynucleotide having a base sequence in which one or several bases have been substituted, deleted, inserted, and / or added, so long as the fusion protein encoded by each polynucleotide can be expressed. In this embodiment, the polynucleotides may have structures in which some of the sugars or phosphates contained in the nucleotides are substituted, so long as the fusion proteins encoded by the polynucleotides can be expressed. EXAMPLES

[0043] The present disclosure will be described in more detail below with reference to examples, but it goes without saying that the scope of the present disclosure is not limited to the examples.

[0044] Example 1: Single cell analysis of ovarian cancer clinical specimens after chemotherapy Single cell expression analysis was performed using samples from four ovarian cancer patients, including two cases of primary ovarian cancer, one case of recurrent ovarian cancer, and one case of ovarian cancer after primary chemotherapy, using methods known to those skilled in the art. Specifically, cancer tissues were collected from ovarian cancer (serous adenocarcinoma) patients who had given their consent, and the cancer tissues were disrupted in collagenase solution using gentleMACS™ (Miltenyi Biotec). After preparing a single cell suspension, PI (Propidium Iodide) staining was performed, and only PI-negative cells, i.e., live cells, were collected using a flow cytometer. A library for single cell expression analysis was prepared using Chromium Controller from 10Xgenomics, and the sequence was decoded using Hiseq X. The obtained data was analyzed using Cellranger, and figures were created using Loupe. The results showed that MKI67 was highly expressed in recurrent cancer, indicating active cell proliferation, whereas MKI67 was hardly expressed in post-chemotherapy cancer, indicating that cell proliferation had almost ceased (Figure 1). Additionally, single cell expression analysis of samples from four ovarian cancer patients, two with primary ovarian cancer, one with recurrent ovarian cancer, and one with ovarian cancer after primary chemotherapy, are shown in a single figure (Figure 2). The figure shows that mesenchymal cell marker THY1-positive cells, circled in the figure, are present in the ovarian cancer tissue. These THY1-positive cells did not express the proliferation marker MKI67, but instead expressed the anthrax toxin receptor ANTXR1 and the proteases MMP2 and uPA (shown as PLAU in the figure). Furthermore, after chemotherapy, cancer cells positive for the epithelial cell marker EPCAM disappeared, but THY1-positive or THY1-positive and EPCAM-positive quiescent cancer cells remained. This suggested that these THY1-positive quiescent cancer cells may be the cause of future recurrence. In addition, it was shown that PTPRC is a gene that encodes CD45, which is expressed in blood cells, but is not expressed in THY1-positive cells.

[0045] <Example 2> Preparation of PA-DT protein The sequence of PA-DT was designed by designing the amino acid sequence (SEQ ID NO: 5), and then codon optimization for humans was performed to prepare a base sequence. The DNA sequence of the PA-DT prepared was artificially synthesized by a method known to those skilled in the art. The synthesized PA-DT sequence was incorporated into a mammalian expression plasmid by a method known to those skilled in the art. The PA-DT expression plasmid has a sequence in which the PA-DT sequence is inserted downstream of the CAG promoter, specifically, the sequence is CAG promoter-PA-DT-IRES-puro resistance gene-polyA. The PA-DT expression plasmid was introduced into 293FT cells (Thermo Fisher Scientific) using Lipofectamine (registered trademark) 2000 (Thermo Fisher Scientific), and the protein was expressed in the 293FT cells. Then, a 293FT cell extract was prepared, and the PA-DT protein was purified by M2 agarose gel using the Flag tag, and eluted from the gel by the Flag peptide. The protein concentration was measured by Qubit. PA-DT protein was also produced using Expi293 cells instead of 293FT cells. The Expi293 cells were purchased from Thermo Fisher Scientific, and the DPH1 and DPH2 genes were disrupted using the CRISPR / Cas9 system known to those skilled in the art to confer DT resistance, followed by selective culture with a high concentration of DT. The PA-DT we created is structured as shown in the upper diagram of Figure 3. Since a signal peptide (Igκ leader) is bound to PA-DT, it is secreted from cells into which the gene has been introduced. The PA-DT protein binds to THY1-high expressing cancer cells that specifically express ANTXR1, MMP, and uPA, and is taken up into the cells and activated, inducing cell death in the cancer cells. In PA-DT, two regions other than the receptor-binding domain of diphtheria toxin: the C region (DT-C) and the T region (DT-T) are inserted into domain 1 of the PA protein. This diphtheria toxin region in PA-DT is activated by cleavage between the C and T regions by the protease uPA, inducing cell death in the cancer cells. Furthermore, domain 1 on the amino terminal side of PA is also cleaved by the protease MMP, and the therapeutic molecule is taken up into the cell via ANTXR1. In this way, for PA-DT to function in cells, the presence of the three elements MMP, uPA, and ANTXR1 is the minimum requirement. Since there are few normal tissues in which these three proteins are simultaneously expressed, PA-DT is able to target chemotherapy-resistant cancer cells that cannot be damaged by conventional anticancer drugs without harming normal cells. As with PA-DT, PA-Clover was also obtained by artificially synthesizing a DNA sequence using a method known to those skilled in the art, incorporating it into a mammalian expression plasmid, and then introducing the PA-DT expression plasmid into 293FT cells to produce and generate it. As shown in the lower diagram of Figure 3, PA has PA that binds to ANTXR1, and Clover, a fluorescent protein, is contained in PA domain 1, making it possible to detect quiescent cancer cells.

[0046] Figure 4 shows the mechanism of action of PA-DT. After binding to ANTXR1 on the cell membrane, PA-DT is activated by cleaving its respective protease recognition sites with MMP and uPA. Activated PA-DT forms a heptamer and is endocytosed. It then binds to lysosomes within the cell, and the pH in the endosomes drops, releasing the diphtheria toxin C domain into the cytoplasm. Diphtheria toxin inhibits protein synthesis by suppressing the function of EF-2, causing cell death.

[0047] <Example 3 Performance evaluation of PA-DT protein> The performance of the PA-DT protein was evaluated by a cleavage assay of the PA-DT protein. PA-DT, PA-DT with a G342W substitution in the PA sequence (in the figure, PA-DT G342W) and PA-DT not having the MMP recognition sequence and uPA recognition sequence (indicated as PA-DT no cut in the figure) were expressed in 293FT cells, respectively, and the proteins were purified. The purified proteins were added with 20 ng / μL of MMP2 (Biolegend, 554306), MMP9 (Biolengend, 550506) or uPA (Biolegend, 755302), and cleaved by incubating for a predetermined time (1, 2, 4 or 6 hours) at 37°C in a buffer containing 100 mM Tris-HCl pH 7.5 and 150 mM NaCl. Thereafter, the proteins were electrophoresed on an SDS-PAGE gel, transferred to a transfer membrane, and detected with HRP-labeled M2 antibody by a method known to those skilled in the art. The results are shown in Figure 5. PA-DT was cleaved by MMP2, MMP9, and uPA as designed. It was shown that the cleaved and activated PA-DT was almost completely degraded after 4 hours. On the other hand, it was shown that if PA-DT was not cleaved by these proteases, it was not degraded at all and was stable for 6 hours at 37°C (indicated as "no enzyme" in the figure). This suggests that PA-DT functions only in the vicinity of mesenchymal cancer cells that express these proteases. Similar results were shown for PA-DT, which has a G342W substitution in the PA sequence (PA-DT G342W). Moreover, PA-DT, which does not have either the MMP or uPA recognition sequence (PA-DT no cut), was not degraded at all in the presence of MMP2, MMP9, or uPA, indicating that MMP2, MMP9, and uPA specifically cleave the recognition sequence as designed.

[0048] Example 4: Evaluation of the cytocidal effect of PA-DT PA-DT was administered to 80% confluent mouse ovarian cancer cell line ID8 luc2 (prepared by the method described in Non-Patent Document 3) and human normal prostate cell line PNT2 (The European Collection of Authenticated Cell Cultures: ECACC) as a negative control at concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 μg / mL (solvent containing 50 mM Tris pH 7.4, 150 mM NaCl, 10% Glycerol, and 150 μg / mL 1×flag peptide) and cultured for 24 hours. After that, each sample was collected and cell viability was measured by MTS assay (n=3, error bars represent standard deviation). The mouse ovarian cancer cell line ID8 luc2 is a cell that weakly expresses ANTXR1 and strongly expresses ANTXR2, and is a cell that expresses MMP and uPA. As a result, it was shown that PA-DT inhibited cell proliferation in a dose-dependent manner in mouse ovarian cancer cell line ID8 luc2 (Figure 6). On the other hand, no inhibitory effect on cell proliferation was observed in human normal prostate cell line PNT2. In addition, when the anticancer drug cisplatin (indicated as Carboplatin in the figure) was administered at various concentrations (0, 62.5, 125, 250, 500, 1000, 2000μg / mL) and cultured for 24 hours, cell proliferation was inhibited in a dose-dependent manner in both mouse ovarian cancer cell line ID8 luc2 and human normal prostate cell PNT2. In other words, it was suggested that PA-DT has a cytotoxic activity specific to cancer cells.

[0049] <Example 5: Analysis of PA-DT Function Using PA-DT Mutants> Figure 7 shows the results of functional analysis of PA-DT when PA-DT mutants were used. PA-DT (indicated as "original PADT" in the figure), PA-DT with a G342W substitution in the PA sequence (indicated as "G342W" in the figure), and PA-DT without the MMP recognition sequence and uPA recognition sequence (indicated as "nocut" in the figure) were added to 80% confluent mouse ovarian cancer cell line ID8 luc2 at concentrations of 0, 3.125, 6.25, 12.5, and 25 μg / m L (culture medium) and cultured for 24 hours. After that, each sample was collected and cell viability was measured by MTS assay (n=3, error bars represent standard deviation). Mouse ovarian cancer cell line ID8 luc2 weakly expresses ANTXR1 and strongly expresses ANTXR2, and expresses MMP and uPA. As a result, when PA-DT was used at a concentration higher than 6.25 μg / mL, the cell viability decreased in a dose-dependent manner. When PA-DT with a G342W substitution in the PA sequence was used, there was no difference in cell viability at a concentration of 6.25 μg / mL, but at a concentration higher than 12.5 μg / mL, the cell viability decreased in a dose-dependent manner. In other words, PA-DT with a G342W substitution has a weaker binding to ANTXR2. On the other hand, when PA-DT without the MMP recognition sequence or uPA recognition sequence was used, no decrease in cell viability was observed at any concentration.

[0050] Example 6: Cytotoxicity Assay of PA-DT Cancer cells (human ovarian cancer cells (Kuramochi), human cervical cancer cells (HeLa), human castration-resistant prostate cancer cells (CWR22Rv1), mouse melanoma cells (B16F10)) were seeded on a 24-well plate, and 20 μg of purified PA-DT was added to 500 μL of culture medium and cultured. After a certain period of time had elapsed for each cell (6 days, 3 days, 5 days, and 3 days, respectively), cell images were taken (Figure 8). The control was 50 mM Tris pH 7.4, 150 mM NaCl, 10% Glycerol, 150 μg / mL. The solvent containing 1× flag peptide was added. The results showed that PA-DT produced a significant cell proliferation inhibitory effect in all types of cancer cells compared to the control.

Claims

1. A fusion protein comprising an anthrax protective antigen sequence, a protein of interest sequence, and a protease recognition sequence, the anthrax protective antigen sequence comprises a sequence representing domain 1 of the anthrax protective antigen sequence, the protease recognition sequence and the target protein sequence are inserted in this order from the N-terminus into the sequence representing domain 1, The target protein sequence is a sequence into which a second protease recognition sequence has been inserted. Fusion proteins.

2. The fusion protein according to claim 1, wherein the protease recognition sequence and the target protein sequence are inserted at any position in a region corresponding to positions 153 to 173 of the amino acid sequence of the anthrax protective antigen represented by SEQ ID NO:

1.

3. The anthrax protective antigen sequence is The amino acid sequence represented by SEQ ID NO: 1, or A sequence in which the glycine residue at position 342 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a tryptophan residue. A fusion protein according to claim 1 or 2.

4. the target protein sequence includes, in this order from the N-terminus, a diphtheria toxin C domain (DT-C) sequence, the second protease recognition sequence, and a diphtheria toxin T domain (DT-T) sequence; A fusion protein according to any one of claims 1 to 3.

5. The DT-C sequence is the amino acid sequence represented by SEQ ID NO:2, and / or The DT-T sequence is an amino acid sequence represented by SEQ ID NO:

3. The fusion protein of claim 4.

6. The protease recognition sequence and the second protease recognition sequence are recognized by the same or different proteases, and the protease is selected from matrix metalloproteases (MMPs) and urokinase-type plasminogen activators (uPAs). A fusion protein according to any one of claims 1 to 5.

7. A fusion protein comprising an anthrax protective antigen sequence, a target protein sequence, and a protease recognition sequence, the anthrax protective antigen sequence comprises a sequence representing domain 1 of the anthrax protective antigen sequence, the protease recognition sequence and the target protein sequence are inserted in this order from the N-terminus into the sequence representing domain 1, The anthrax protective antigen sequence is an amino acid sequence represented by SEQ ID NO: 1 in which the glycine residue at position 342 is replaced with a tryptophan residue. Fusion proteins.

8. The fusion protein described in claim 7, wherein the insertion is made at any position in the region corresponding to positions 153 to 173 of the amino acid sequence of the anthrax protective antigen represented by sequence number 1.

9. The target protein sequence includes a sequence of a fluorescent protein; A fusion protein according to claim 7 or 8.

10. The fusion protein according to any one of claims 7 to 9, wherein the protease recognition sequence is recognized by matrix metalloprotease (MMP) or urokinase-type plasminogen activator (uPA).

11. The fusion protein of claim 9 or 10, wherein the fluorescent protein is mClover.

12. A composition for inducing cell death in a cancer cell expressing an anthrax toxin receptor (ANTXR) and a protease that recognizes a protease recognition sequence in the fusion protein, the composition comprising an expression vector comprising a polynucleotide encoding the fusion protein according to any one of claims 1 to 8.

13. A composition for treating cancer, including cancer cells expressing anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, comprising the fusion protein according to any one of claims 1 to 8 or an expression vector comprising a polynucleotide encoding the same.

14. A composition for labeling a cancer cell expressing an anthrax toxin receptor (ANTXR) and a protease that recognizes the protease recognition sequence in the fusion protein, the composition comprising the fusion protein according to any one of claims 9 to 11.

15. A kit for labeling cancer comprising the composition of claim 14.

16. A polynucleotide encoding the fusion protein according to any one of claims 1 to 11.

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