JP2014105158A - Probe for detecting dead cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide molecular imaging probes which accumulate specifically and with high sensitivity at tumor parts in vivo, and enabling quantitative analysis.SOLUTION: There is provided a molecular imaging probe for detecting apoptotic cells and/or necrotic cells that comprises a fusion protein in which a protein or a polypeptide capable of forming a dimer is bound to a C-terminus of a Tim4 protein, and a mucin domain and the domain of the C-terminal end of the Tim4 protein are substituted by a polypeptide comprising an amino acid sequence of following a) or b): a) a sequence included in the amino acid sequence of the mucin domain of a wild type Tim4 protein whose length is 30 to 120 amino acid residues, b) a sequence having 80% or higher identity to a).

Description

  The present invention relates to a probe that detects dead cells such as apoptotic cells and necrotic cells and enables molecular imaging of the cells.

  Detection of cell death in vivo and molecular imaging are important for diagnosis of diseases such as cancer and determination of therapeutic effects of drugs. For imaging diagnosis of tumor tissue, positron tomography (PET), single photon emission tomography (SPECT), etc. have been established, and various molecular imaging probes are used.

  Such molecular imaging probes include molecules that recognize and bind to phosphatidylserine (hereinafter also referred to as PS). This is based on the fact that cells exhibiting apoptosis or necrosis express PS on the cell membrane surface, and detect the cells in tumor tissue (Non-patent Document 1). Specifically, it has been proposed to use a radionuclide-labeled C2A domain of Annexin A5 or Synaptotagmin I which is a PS-binding molecule as a tracer for SPECT or PET.

By the way, Tim4 (T cell immunoglobulin protein and mucin domain 4) is known as one of proteins related to immune function and cell survival (Patent Document 1). Tim4 is a cell surface molecule having conserved IgV and mucin domains, and has been confirmed to be a PS receptor (Non-Patent Document 6).
Tim4 is dimerized with a single transmembrane protein and binds to PS. As the tertiary structure has been found to have a metal ion pocket (Non-Patent Documents 2 and 3), unlike the Annexin A5 requiring Ca ²⁺ for binding to PS, Ca ²⁺ independent binds to PS It has also been confirmed (Non-Patent Documents 4 and 5).
There has been no example of using Tim4 protein as a molecular imaging probe so far.

Japanese Patent No. 4572276

Apotosis, (2010) 15, 1072-1082 Immunity, (2007) 27 (6), 941-951 Immunological Reviews, (2010) 235, 172-189 Nature (2007) 450, 435-439 Immunity (2007) 27, 927-940 Nature, 2007, Nov. 15, p.435-9

Annexin A5 and Synaptotag known as PS binding probes
It has been pointed out that the C2A domain of min I accumulates non-specifically in the liver or kidney, and there is a problem that when applied as a nuclide-labeled probe, there is a concern about the influence of radioactivity on the living body. In addition, these molecules contain a large amount (2.5 mM or more) of Ca ²⁺ upon binding to PS.
Therefore, it is not suitable for performing quantitative image analysis in vivo.
In view of such circumstances, an object of the present invention is to provide a molecular imaging probe that accumulates specifically and highly sensitively at a tumor site exhibiting cell death in a living body, and further enables quantitative analysis.

As a result of intensive studies to solve the above problems, the present inventors have found that a fusion protein of a PS-binding molecule Tim4 and a protein or polypeptide that forms a dimer is highly sensitive to apoptotic cells and / or necrotic cells. It was found that it can be detected. Furthermore, the inventors have found that by setting the size of the mucin domain in the Tim4 protein to be smaller than that of the wild type, the specificity of accumulation in tumor tissue exhibiting apoptosis can be increased, and the present invention has been completed.
That is, the present invention is as follows.
[1] A protein for a probe for detecting apoptotic cells and / or necrotic cells,
A fusion protein in which a dimer-forming protein or polypeptide is bound to the C-terminus of the Tim4 protein;
A protein (hereinafter also referred to as “protein of the present invention”) in which the mucin domain of the Tim4 protein and the domain on the C-terminal side thereof are substituted with a polypeptide having the amino acid sequence of a) or b) below.
a) a sequence having a length of 30 to 120 amino acid residues contained in the amino acid sequence of the mucin domain of the wild type Tim4 protein b) a sequence having 80% or more identity with a) [2] The mucin domain of the Tim4 protein and its C The protein according to [1], wherein the terminal domain is substituted with a polypeptide having the amino acid sequence of a) or b) below.
a) Sequence from the N-terminal to the 30th to 120th amino acid residues of the amino acid sequence of the mucin domain of the wild-type Tim4 protein b) A sequence having 80% or more identity with a) [3] Forming the dimer The protein according to [1] or [2], wherein the protein or polypeptide to be processed is a human IgG Fc region protein.
[4] The protein according to any one of [1] to [3], wherein the wild-type Tim4 protein is a human Tim4 protein.
[5] The protein according to any one of [1] to [4], wherein the fusion protein has a molecular weight of 100 kDa to 250 kDa as measured by SDS-PAGE under non-reducing conditions.
[6] A labeled probe containing the protein according to any one of [1] to [5] (hereinafter also referred to as “probe of the present invention”).
[7] A diagnostic imaging agent for tumors comprising the probe according to [6].
[8] A diagnostic imaging kit comprising the tumor diagnostic imaging agent according to [7].
[9] A method for detecting apoptotic cells and / or necrotic cells using the probe according to [6].

  The present invention provides a molecular imaging probe that can detect dead cells with high sensitivity, specifically accumulates in a tumor site exhibiting cell death in a living body, and enables quantitative analysis. The probe of the present invention can accurately detect and image cell death in a living body, and can be used for disease diagnosis and drug sample effect determination.

PET image of a mouse 48 hours after administration of mTim4-Fc or mTim4-Δ230-Fc (photograph, maximum projection display). Immunostained image (photograph) of an excised tumor section. A to C: MMC-administered tumor isolated, D to E: Saline-administered tumor, A and B: Immunostained images with anti-activated caspase 3 antibody (red), B and E: TUNEL stained images (green) , C and F: Composite images of immunostained image with anti-activated caspase 3 antibody, TUNEL stained image, and nuclear stained image with Hoechist 33258 (blue). The dot blotting image (photograph) which shows the binding property of each hTim-Fc with respect to PS. The dot blotting image (photograph) which shows the binding property of hTim4-187-Fc with respect to various phospholipids. Immunostaining images of apoptosis-inducing cells with hTim4-187-Fc or Annexin A5 (photos). A: immunostained image with hTim4-187-Fc and PI, B: immunostained image with Annexin A5 and PI. Left column: hTim4-187-Fc or Annexin A5 stained image, middle column: PI stained image, right column: synthesized image of fluorescence image and transmitted light image, respectively. The upper and lower stages are images of different fields of view under the same conditions.

  The probe protein of the present invention is a fusion protein in which a protein or polypeptide that forms a dimer is bound to the C-terminus of the Tim4 protein.

Wild-type Tim4 protein is composed of a signal sequence, an IgV domain, a mucin domain, a transmembrane domain, and a cytoplasmic region in this order from the N-terminal side. The IgV domain is a PS binding site, and the mucin domain is a sugar chain binding site.
As for the amino acid sequence of the wild-type Tim4 protein, the human one is shown in SEQ ID NO: 2 and the mouse one is shown in SEQ ID NO: 4, both of which have 48% identity in the amino acid sequence.

The mucin domain of the Tim4 protein and its C-terminal domain in the protein of the present invention are replaced with a polypeptide that is reduced in size by deleting the C-terminal and / or N-terminal of the wild-type mucin domain. The region to be replaced is a region that includes at least the full-length mucin domain that is a part or all of the mucin domain, transmembrane domain, and cytoplasmic region of the Tim4 protein. From the viewpoint of ease of production of the fusion protein, preferably, all of the mucin domain, transmembrane domain, and cytoplasmic region are replaced.
Specifically, the polypeptide reduced in size by deleting the C-terminal and / or N-terminal of the wild-type mucin domain is specifically a) 30-120, preferably included in the amino acid sequence of the mucin domain of the wild-type Tim4 protein Consists of an amino acid sequence having a length of 45-80, more preferably 50-60 amino acid residues, or b) a) an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with a) It is a polypeptide. Or c) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the sequence a). In the present specification, “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but is within a range that does not significantly impair the effects of the present invention. -50, preferably 2-30, more preferably 2-10, particularly preferably 2-5.
In order to reduce the size of the wild-type mucin domain, it is preferable to delete the C-terminal side from the viewpoint of ease of preparation. That is, a ′) the amino acid sequence of the mucin domain of the wild-type Tim4 protein is substituted with a sequence from the N-terminal to the 30th to 120th, preferably 45 to 80th, more preferably 50 to 60th amino acid residues. Is preferred. Alternatively, an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with b ') a'), or c ') a') in the sequence of 1 'or several amino acids It may be substituted with a polypeptide comprising an amino acid sequence that is deleted, substituted, inserted, and / or added.

By labeling the Tim4 protein in the fusion protein into a modified Tim4 protein that has been shortened and shortened in the amino acid sequence length of the mucin domain, a labeled protein (probe) is labeled on an individual having a site / tissue exhibiting cell death such as a tumor. When it is administered as, it specifically accumulates in a site / tissue having a target dead cell and suppresses non-specific accumulation in other sites.
The amino acid sequence of the mucin domain of human Tim4 protein is shown in SEQ ID NO: 6, and the amino acid sequence of the mucin domain of mouse Tim4 protein is shown in SEQ ID NO: 8. When the probe of the present invention is applied to clinical image diagnosis or the like, it is particularly preferable to use a human Tim4 protein.

The IgV domain of the Tim4 protein in the protein of the present invention may be wild type or modified. Here, the modified type means an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the wild-type amino acid sequence and have PS binding ability. The IgV domain preferably has a wild type sequence from the viewpoint of maintaining binding to PS.
Although it is known that the inner region in the IgV domain is mainly involved in the binding of the Tim4 protein to PS (Non-patent Documents 2 and 3), the sites near both ends of the IgV domain have a β-sheet structure. It is thought that it forms and is involved in maintaining the tertiary structure of the Tim4 protein and indirectly contributes to the binding to PS. Therefore, in the modification in the modified IgV domain, in addition to the binding property to PS, from the viewpoint of maintaining the tertiary structure of the Tim4 protein, the PS binding site in the IgV domain and β sheet structure forming sites at both ends should be retained. Is preferred.

The protein or polypeptide that forms a dimer in the protein of the present invention is bound to the C-terminus of the modified Tim4 protein described above. The protein forming this dimer is introduced to promote dimerization when the Tim4 protein binds to PS.
Examples of the protein or polypeptide forming the dimer include IgG Fc region protein, protein or polypeptide having a leucine zipper structure, and the like. In addition, a case where an arbitrary protein or polypeptide is fused to the Tim4 protein and the protein or polypeptide of two molecules of the fusion protein is SS-bonded to form a fusion protein is also included in the protein of the present invention. Among these, human IgG-Fc protein is preferable from the viewpoint of suppressing non-specific accumulation of the probe other than the target site as an appropriate molecular weight, and from the viewpoint of ease of protein purification in probe preparation described later.

The molecular weight of the protein of the present invention is preferably 100 kDa to 250 kDa, more preferably 130 kDa to 200 kDa, and even more preferably 150 kDa to 180 kDa, as measured by SDS-PAGE under non-reducing conditions.
In general, probe molecules administered in vivo tend to accumulate nonspecifically in the liver when the molecular weight is large, and in the kidney when the molecular weight is small. Therefore, it is preferable that the fusion protein in the present invention has the above-mentioned size from the viewpoint of specific accumulation in a site / tissue having a target dead cell and suppression of non-specific accumulation in other sites.

The protein of the present invention can be prepared by any method including a well-known gene manipulation method, and is not particularly limited.
For example, a DNA encoding wild-type Tim4 is modified by deleting a region encoding the C-terminal or N-terminal side of the mucin domain in the DNA so that a modified mucin domain having a desired amino acid length remains. A DNA fragment encoding type Tim4 is obtained and ligated with a DNA fragment encoding a protein or polypeptide that forms a dimer by a known method to produce a DNA fragment encoding a fusion protein. DN encoding the resulting fusion protein
The A fragment is introduced into an appropriate expression vector, E. coli is transformed with the vector, the fusion protein is expressed and recovered by a known method, and purified appropriately by chromatography or the like.
In addition, mutations such as deletion, substitution, insertion, and / or addition can be introduced into a wild-type sequence by a known method.

The protein of the present invention becomes a probe by labeling and can be applied to detect apoptotic cells and / or necrotic cells. For easy detection of a tumor site / tissue having apoptotic cells and / or necrotic cells, the type of labeling is not particularly limited as long as it is a detectable mode. The labeling can be appropriately performed by a known method. For example, a fluorescent labeling method using FITC, an enzyme labeling method using peroxidase, an RI labeling method using a nuclide usually used for diagnostic imaging such as PET and SPECT, biotin Examples include labeling methods.
From the viewpoint of application to widely used diagnostic imaging methods such as PET and SPECT, the probe of the present invention is particularly preferably labeled with RI. Examples of the PET nuclide include, but are not limited to, ⁶⁴ Cu, ⁸⁹ Zr, ⁶⁸ Ga, ¹²⁴ I, ¹⁸ F and the like having a half-life of several days. Examples of SPECT nuclides include, but are not limited to, ¹²³ I, ¹¹¹ In, and ^99m Tc with a half-life of several days.

The protein of the present invention can specifically bind to PS among various phospholipids present in the cell membrane. In addition, the binding ability is Annexin which is a known PS binding protein.
Equivalent to A5. In general, the labeled body shows a lower binding ability to the target molecule than before the labeling, but the protein of the present invention is maintained in the form of the probe labeled with RI or the like without decreasing the binding ability to PS. The
Based on the above properties, the probe of the present invention binds to the PS of a cell expressing PS on the cell membrane surface, thereby detecting the cell. In general, in living cells, PS is present inside the cell membrane, but in dead cells, PS is expressed on the cell membrane surface, so that the probe of the present invention can specifically detect dead cells. Specifically, apoptotic cells and necrotic cells can be detected, and cells that are in a stage of dying due to induction of apoptosis or cells that have already undergone cell death can be detected.

In addition, the modified Tim4 protein is used as a fusion protein, so that the protein of the present invention does not cause non-specific accumulation in the kidney when administered to a living body, and to a site / tissue having apoptotic cells and / or necrotic cells. Accumulate specifically.
Furthermore, since the protein of the present invention binds to PS without depending on the Ca ²⁺ concentration, quantitative analysis is possible even when the probe of the present invention is administered to a living body.

Therefore, the protein of the present invention can detect dead cells with high sensitivity by adopting a probe mode, and specifically accumulates in a tumor site exhibiting cell death in a living body, and further performs quantitative analysis. It can be a molecular imaging probe. In particular, by appropriately labeling with RI or the like, it can be used as a tracer in diagnostic imaging such as PET or SPECT. Therefore, the probe of the present invention can be included in a diagnostic imaging agent for tumors, and the diagnostic imaging agent can be used as a diagnostic imaging kit.
Examples of usage when the probe of the present invention is used as a tracer in diagnostic imaging such as PET and SPECT include application to diagnosis of diseases such as cancer and determination of therapeutic effects of drugs. The type of cancer or tumor tissue to be targeted is not particularly limited.

When performing the method of detecting apoptotic cells and / or necrotic cells, or tumor tissue containing these using the probe of the present invention, it is performed according to a general technique.
In vitro, for example, the probe of the present invention may be added to a sample cell and allowed to react for an appropriate time, and then detection may be performed by a technique according to the label.
In a living body, the probe of the present invention is administered by means such as injection (local or systemic) or infusion into the vein of a subject individual, and after an appropriate time has elapsed, for example, 1 to 24 hours after administration, the radiation is emitted by SPECT or PET. Is used for diagnosis and judgment. Diagnosis, examination methods and procedures are basically the same as normal cancer diagnosis and examinations or normal diagnostic imaging methods and procedures.

The amount of application when the probe of the present invention is administered to an individual is not particularly limited, but is preferably 10 to 100 μg / individual for humans, and more preferably 15 to 74 μg / individual. Alternatively, in the case of an RI-labeled probe, administration is performed so as to be 50 to 250 MBq / individual, preferably 40 to 200 MBq / individual, more preferably 37 to 185 MBq / individual.
In addition, the dose to mice is preferably 500 to 700 μg / kg, and in the case of an RI-labeled probe, 10 to 25 MBq / individual is preferable.

<Preparation of wild-type or modified mTim4-Fc>
A fusion protein of wild-type or modified mouse Tim4 and human IgG Fc region protein was prepared by the following procedure.
Fusion protein of wild type mouse Tim4 and human IgG Fc region protein (mTi
m-Fc) is encoded by plasmid pTim4-Fc (provided by Professor Shigekazu Nagata, refer to Non-Patent Document 6 for the production method), E. coli (JM109, Takara Bio) is transformed, and the plasmid vector is Amplified. The plasmid vector was purified using FASTPLASMID MINI KIT-250 PREPS (VPprime), and PCR was performed using Expand High Fidelity PCR System (Roche) using the obtained pTim4-Fc as a template. Using the primers of SEQ ID NOs: 11 and 12, mTim4-ΔM230-Fc (amino acid sequence from the N-terminal to the 96th amino acid of the mucin domain by deleting the C-terminal side of the mucin domain of wild-type mTim4 in the mTim4 part) A DNA fragment encoding a modified type leaving a) was prepared. In addition, using the primers of SEQ ID NOS: 11 and 13, mTim4-ΔM184-Fc (in the mTim4 part, the C-terminal side of the mucin domain of wild-type mTim4 was deleted, and the amino acid from the N-terminal of the mucin domain to the 50th amino acid A DNA fragment encoding a modified type leaving the amino acid sequence was prepared. These PCR products and pTim4-Fc were digested with restriction enzymes SalI and EcoRV (both Takara Bio), and the target band was excised and purified by agarose gel electrophoresis. The purified PCR product and pTim4-Fc were ligated and transformed into E. coli. Ampicillin-added LB plate (0.1 mg / mL ampicillin, 1.5% agarose, 40 capsules / L, Circle grow, Q-BIO
gene) was inoculated with E. coli transformed and incubated at 37 ° C. for 16 hours. Colony PCR was performed using the formed colonies as templates and primers of SEQ ID NOs: 14 and 15 and EmeraldAmp MAX PCR Master Mix (Takara Bio), and after confirming the transformants, DNA sequence analysis was performed. Each fusion protein was expressed by the following method using the target plasmid vectors each containing DNA encoding mTim4-Fc, mTim4-ΔM230-Fc, and mTim4-ΔM184-Fc. Table 1 shows the DNA sequence, amino acid sequence, and number of amino acid residues of the mucin domain of each expressed protein.

E. coli transformed with the plasmid vector encoding the fusion protein was cultured with shaking in 0.1 mg / mL ampicillin and 200 mL LB at 37 ° C. for 16 hours, and the amplified vector was purified using EndoFree Plasmid Maxi Kit (Qiagen). The purified vector was transfected into 293T cells by the calcium phosphate method, and the medium was replaced with DMEM medium 24 hours later. After 48 hours from the medium exchange, the medium was collected, centrifuged at 1000 × g for 5 minutes, and the supernatant was collected. 25 μL of Protein A agarose beads (Thermo Scientific) was added to 50 mL of the collected medium at 4 ° C.
And rotated overnight. The next day, the beads were collected, washed 5 times with PBS (150 mM NaCl, 20 mM Phosphate buffer, pH 7.0), suspended in 100 μL of 100 mM glycine buffer, pH 3.0, allowed to stand at room temperature for 5 minutes, and then centrifuged. The supernatant was collected by The collected supernatant was replaced with PBS using Amicon Ultra (Millipore, 30 kDa cut), concentrated, and stored at −20 ° C. until use. As a result, 100 to 150 μg of each fusion protein could be obtained from 200 mL of the culture supernatant.

<PET imaging>
The fusion proteins (mTim4-Fc, mTim4-ΔM230-Fc, and mTim4-ΔM184-Fc) obtained as described above were each labeled with RI by the following procedure.
The stock solution of each fusion protein was replaced with PBS (D-PBS, Wako Pure Chemical Industries). 10 mM p-SCN-Bn-NOTA (NOTA, Macrocyclics) aqueous solution was added to 0.1N Na.
What was adjusted to pH 7.9-8.4 with OH was added so that it might become 1000 equivalent with respect to each fusion protein, and it left still at 4 degreeC overnight. Thereafter, the fusion protein bound to NOTA and unreacted NOTA were separated by a desalting column (PD-10, Thermo Sci.) Equilibrated with PBS. The NOTA-binding fusion protein fraction was concentrated with Amicon Ultra, replaced with 100 mM acetate buffer (pH 6.5), 6 MBq of [ ⁶⁴ Cu] CuCl ₂ aqueous solution was added per μg of NOTA-binding fusion protein, and incubated at 40 ° C. for 30 minutes. Then, a chelate reaction of ⁶⁴ Cu was performed. After the reaction, unreacted ⁶⁴ Cu was washed three times with 300 μL of 100 mM glycine buffer (pH 6.5) using Amicon Ultra, and 0.05% Tween 20−
The protein concentration was measured by replacing with PBS (PBS-T). As a result, RI labeled bodies having a specific activity of about 600 to 800 MBq / nmol were obtained with a purity of 90% or more (Table 2).

In the PET imaging experiment, a model animal was prepared as follows.
5-6 weeks old male Balb / c-Ajcl-nu / nu mice (CLEA Japan) were prepared and divided into 2 groups (MMC administration group and Vehicle group, 3 mice each). The MMC administration group was inoculated with A431 cells in the left thigh 2 to 3 weeks before the PET experiment and the Vehicle group 10 to 2 weeks before, and the tumor size was 50 days before the RI label administration. Individuals that became ~ 120cm ³ were PE
Used for T experiments. One day, three days, and five days before RI-labeled substance administration, MMC (mitomycin C) was adjusted to 5 mg / 6.25 mL / kg in the MMC administration group and administered via the tail vein, and 6. 25 mL / kg of physiological saline was administered via the tail vein.
Each ⁶⁴ Cu labeled body prepared as described above is administered via the tail vein so as to be 0.2 to 0.25 mg / kg (15 to 25 MBq / body), and dynamic imaging is performed for 48 hours after the administration. (MicroPET Focus 220, Siemens). The obtained image is shown in FIG.
Mice were dissected after completion of all imaging, and the radioactivity of each tissue was measured with a γ-counter (Table 3).
As a result, although there was no difference between the MMC-administered animals administered with [ ⁶⁴ Cu] Tim4-Fc and the saline-administered animals, [ ⁶⁴ Cu] mTim4-ΔM230-Fc and [ ⁶⁴ Cu] mTim4-ΔM184 were observed. -In Fc, accumulation in tumors was observed in MMC-administered animals.

<Determination of apoptosis in target tissue>
After the above PET experiment was completed, tumors were removed from the mice and frozen sections were prepared. The prepared frozen sections were fixed with chilled methanol, stored at −20 ° C. until use, and used for experiments during 3-5 days after dissection.
Frozen sections were blocked with Protein Block, Serum-Free (Dako), and DeadEnd Fluorometric TUNEL System (Pr
primary antibody (Cleaved) diluted 1000 times after TUNEL staining using
Caspase-3 (Asp175) Antibody, CST) was reacted at room temperature for 2 hours, and a secondary antibody (Cy3-labeled anti-rabbit IgG antibody) was further reacted at room temperature for 2 hours. Nuclear staining was also performed using Hoechist 33256 (Dojindo). After the reaction, the sample was observed with a confocal laser microscope.
The results are shown in FIG. In a frozen section of a tumor collected from an MMC-administered individual, activated caspase 3 antibody and TUNEL positive cells were observed throughout. On the other hand, all of the saline-administered individuals were negative. From this, it was confirmed that apoptosis occurred in the tumor of the individual administered with MMC.

<Production of wild-type or modified hTim4-Fc>
A fusion protein of human Tim4 (wild type, modified type in which the length of the mucin domain was varied) and a human IgG Fc region protein was prepared by the following procedure.
A vector encoding the hTim4 sequence (pCMV6-XL5 Homo sapiens
T-cell immunoglobulin and mucin domain containing 4, transscript variant 1 as transfec
PCR was carried out using a primer (SEQ ID NO: 22) containing an EcoRV restriction enzyme site at the 5 ′ end and a primer containing a BglII restriction site (SEQ ID NO: 23) at the 3 ′ end using the template (ion-ready DNA, OriGene Technologies) as a template. It was. The obtained PCR product and pFUSE-hIgG1e3-Fc2 (InvivoGen) encoding human IgG-Fc region were digested with EcoRV and BglII, respectively, and transformed into E. coli (JM109, Takara Bio) to obtain a colony. It was. A plasmid was extracted from the obtained colonies and subjected to DNA sequence analysis, and a large number of colonies into which the hTim4 sequence had been inserted were cultured to obtain a vector. The obtained vector was amplified using the primers shown in SEQ ID NOs: 20 and 21, and inserted into a protein expression vector (pcDNA3.3-TOPO, Life technologies). The insertion vector is E. coli (T
OP10, Life technologies), and the insertion direction was confirmed by DNA sequence analysis. Using the obtained vector (phTim4-Fc) as a template, vectors having mucin domains of various lengths were prepared using the primers shown in Table 4. For deletion of the mucin-like domain, PrimeSTA Mutagenesis Basal Kit (Takara Bio) was used. Table 5 shows the DNA sequence and amino acid sequence of each produced expressed protein and the number of amino acid residues of the mucin domain.
Freestyle M was added to 30 mL of Freestyle 293F cells (Life technologies) prepared by adjusting 37.5 μg of the obtained vector to 1 × 10 ⁶ cells / mL.
The cells were transfected using AX Reagent (Life technologies), cultured under shaking at 37 ° C. and 8% CO ₂ for 4 days, and 10 mL of medium was added on the third day of culture. Protein A agarose beads (40 mL of collected culture supernatant)
Pierce) 250 μL was added and the expressed protein was recovered. The collected expressed protein was purified with a gel filtration column (Superdex 200 10/300 GL, GE healthcare), replaced with PBS pH 6.8, and stored at 4 ° C. until use. The purified protein was subjected to SDS-PAGE, and the molecular weight in a non-reduced state was calculated (Table 6).

<Evaluation of binding ability of wild type or modified hTim4-Fc to PS>
Dot blotting was performed according to the following procedure to examine the binding ability of each fusion protein (hTim4-Fc) prepared as described above to PS.
Blocking was performed by spotting 0.15 μg of PS on a 10 mm square PVDF membrane (GE Healthcare) and incubating in a 24-well plate with 500 μL of blocking buffer (5% skim milk-PBS-T) at room temperature for 1 hour. It was. Each fusion protein was added to the blocking buffer to a final concentration of 0.5 μg / mL and incubated overnight at 4 ° C. The control was only blocking buffer. Thereafter, the PVDF membrane was washed 5 times with 2 mL of PBS-T and incubated at room temperature for 2 hours in an HRP-labeled anti-human IgG-Fc antibody (Bethyl) solution diluted 10,000 times with 500 μL of blocking buffer. Then, it was washed 5 times with 2 mL PBS-T and ECL p
chemiluminescence with lime (GE healthcare), LAS3000 (Fujif
ilm).
As shown in FIG. 3, hTim4-240-Fc, hTim4-187-Fc, hTim4-184-Fc, hTim4-181-Fc, hTim4-178-Fc, hTim4-175-Fc and hTim4-162-Fc Binding to PS was confirmed.

<Evaluation of binding specificity of modified hTim4-Fc to PS>
Dot blotting was performed according to the following procedure, and the binding properties of the fusion protein of the present invention to various phospholipids were examined.
100 pmol / μL 3-sn-Ph on PVDF membrane (GE Healthcare)
osphatidylethanolamine (PE), L-α-Phosphatylylcholine (PC), L-α-Phosphatylylinositol (PI), and 3-sn-phosphatidyl-L-serine (Sigma A
ldrich) was spotted 1 μL each, and blocking was performed by incubating with 2 mL blocking buffer (5% skim milk-PBS) at room temperature for 1 hour. To each well, hTim4-187-Fc was added to the blocking buffer to a final concentration of 0.5 μg / mL and incubated at room temperature for 2 hours. Thereafter, the PVDF membrane was washed 5 times with 5 mL PBS-T, and then diluted 10000 times with 2 mL blocking buffer.
The reaction was carried out in a P-labeled anti-human IgG-Fc antibody (Bethyl) solution at room temperature for 2 hours. After the reaction, it was washed 5 times with 5 mL of PBS-T, chemiluminescent with ECL prime (GE healthcare), and detected with LAS3000 (Fujifilm).
As shown in the results shown in FIG. 4, it was confirmed that hTim4-187-Fc binds only to PS.

<Detection of apoptosis-inducing cells>
Immunostaining was performed according to the following procedure to examine the binding of hTim4-187-Fc or Annexin A5 to cells in which apoptosis was induced.
Apoptosis was induced by adding Etoposid at a final concentration of 100 μM to 1 × 10 ⁶ cells / mL Jurkat cells and culturing at 37 ° C. with 5% CO ₂ for 6 hours. The cells were collected, washed with cold PBS, and suspended in 2% FBS-PBS at 1 × 10 ⁷ cells / mL. hTi
The immunostaining with m4-187-Fc was performed at a final concentration of 0.5 μg / mL in the cell solution.
hTim4-187-Fc, 2.5 ng / mL CF488-labeled anti-human IgG-Fc antibody (Biotium), 20 μL / mL Propidium Iodide (PI) solution (BioVision) was added and incubated at room temperature for 5 minutes. Thereafter, it was washed with cold PBS and observed with a fluorescence microscope. Staining with Annexin A5 was performed on the cell solution using Annexin V-FITC Apoptosis Detection Kit (BioVision), and similarly observed with a fluorescence microscope.
The results are shown in FIG. Like the existing probe Annexin A5, hTim4-
It was confirmed that 187-Fc showed binding to apoptosis-inducing cells and did not bind to living cells. PI positive indicates dead cells, Annexin A5
Similarly, it was also confirmed that hTim4-187-Fc can detect apoptosis-induced cells and dead cells.

  According to the present invention, there is provided a molecular imaging probe that can detect dead cells with high sensitivity, specifically accumulates in a tumor site exhibiting cell death in a living body, and enables quantitative analysis. The Therefore, since cell death in a living body can be accurately detected and imaged and used for disease diagnosis and drug sample effect determination, it is very useful industrially.

Claims (9)

A protein for a probe for detection of apoptotic cells and / or necrotic cells,
A fusion protein in which a dimer-forming protein or polypeptide is bound to the C-terminus of the Tim4 protein;
A protein in which the mucin domain of the Tim4 protein and the domain on the C-terminal side thereof are substituted with a polypeptide comprising the amino acid sequence of a) or b) below.
a) a sequence of 30 to 120 amino acid residues in the amino acid sequence of the mucin domain of the wild-type Tim4 protein b) a sequence having 80% or more identity with a) The protein according to claim 1, wherein the mucin domain of the Tim4 protein and the domain on the C-terminal side thereof are substituted with a polypeptide having the amino acid sequence of a) or b) below.
a) Sequence from the N-terminal to the 30th to 120th amino acid residues of the amino acid sequence of the mucin domain of the wild-type Tim4 protein b) A sequence having 80% or more identity with a) The protein according to claim 1 or 2, wherein the protein or polypeptide forming the dimer is a human IgG Fc region protein.   The protein according to any one of claims 1 to 3, wherein the wild-type Tim4 protein is a human Tim4 protein.   The protein according to any one of claims 1 to 4, wherein the molecular weight of the fusion protein measured by SDS-PAGE under non-reducing conditions is 100 kDa to 250 kDa.   A probe comprising the protein according to any one of claims 1 to 5, which is labeled.   A diagnostic imaging agent for a tumor comprising the probe according to claim 6.   A diagnostic imaging kit comprising the tumor diagnostic imaging agent according to claim 7.   A method for detecting apoptotic cells and / or necrotic cells using the probe according to claim 6.