JP2011079758A - Complex of protein and fine particle, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a complex of a protein and a fine particle, and a method for producing the complex by solving the problems wherein, when producing the complex of the protein having cysteine and the fine particle, the losses of the protein and the fine particles are caused to reduce the yield of the obtained complex. <P>SOLUTION: The problem is solved by using a fused protein to which a linker having two or more cysteines is introduced. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a complex of protein and fine particles and a method for producing the same.

  A molecule that generates a physical signal to a lesion marker-binding molecule such as an antibody that binds to a lesion marker in order to detect an antigen (lesion marker) present at the lesion site (hereinafter sometimes abbreviated as a signal-transmitting molecule) Research and development of in vivo diagnostic contrast agents labeled with. Examples of these signal transmitting molecules include radionuclides, molecules that transmit MRI (Magnetic Resonance Imaging) signals, molecules that transmit ultrasonic signals, and molecules that transmit fluorescent signals. It is considered that a lesion site and a lesion marker can be detected by administering such a complex into the body and detecting a signal from a signal transmission molecule of the complex from outside the body.

  Attempts have been made to use fine particles as signal-transmitting molecules. As a method for binding a protein such as an antibody as a lesion marker binding molecule to a fine particle, for example, a method using a bond between a cysteine thiol group in a protein molecule and a maleimide group introduced on the surface of the fine particle is used. In this method, since cysteine can be introduced into an arbitrary site of the protein using genetic engineering, the protein can be bound to the fine particles without impairing the function of the protein. Arutselvan Natarajan et al. Can produce a complex of an antibody fragment and iron oxide microparticles by reacting an antibody fragment having one cysteine introduced at the carboxy terminus with iron oxide microparticles having maleimide groups on the surface. (Non-Patent Document 1).

  When the above method is used, the protein may dimerize because the cysteine of the protein forms a disulfide bond with the cysteine of another protein. For this reason, it is necessary to add a reducing agent such as dithiothreitol or tri (2-carboxyethyl) phosphine hydrochloride to bond the disulfide bond forming the dimer once before bonding with the fine particles. However, in order to efficiently bind proteins to the surface of the fine particles, it may be necessary to prepare an excessively high concentration protein solution with respect to the fine particles. Therefore, there is a possibility that the protein dimer is reformed by increasing the protein concentration. In addition, non-specific interaction between proteins and microparticles may form a complex of protein and microparticles with low stability that does not involve cysteine, which may cause loss of protein and microparticles. Thereby, the input protein and the fine particles cannot achieve their purpose and may cause loss. From the above, it is considered that the efficiency of protein binding to the microparticles decreases, and the yield of the resulting protein-microparticle complex also decreases.

Arutselvan Natarajan et al., 2008 Cancer Biotherapy & Radiopharmaceuticals, Vol. 23, pp. 82-91.

  As described above, when producing a complex of cysteine-containing protein and fine particles, protein loss and accompanying fine particle loss occur, and the yield of the resulting complex is reduced. Therefore, there is a demand for a composite that can eliminate this drawback and a method for producing the same.

  As a result of intensive studies, the present inventors have found that the above problem can be solved by introducing a linker having two or more cysteines between the protein and the fine particles. The protein / microparticle complex of the present invention is a complex composed of a protein and microparticle, and a linker that links the protein and the microparticle, wherein the linker has two or more cysteines. To do.

  The method for producing a complex of a protein and microparticles of the present invention includes a step of producing a protein into which a linker having two or more cysteines is introduced, a step of cleaving the disulfide bond bound by the cysteine with a reducing agent, And a step of mixing the protein and the fine particles.

  The protein-microparticle complex of the present invention using a protein having a linker having two or more cysteines is improved in yield compared to a conventional complex using a protein having a linker having one cysteine. Can be made.

It is a schematic diagram which shows one form of the composite_body | complex of protein of this invention and microparticles | fine-particles. SDS-polyacrylamide gel electrolysis under the condition that a single cysteine-introduced protein (scFv-Cys1) and two cysteine-introduced protein (scFv-Cys2) are present in the presence or absence of a reducing agent, respectively. It is a result of electrophoresis (SDS-PAGE). The complex of scFv-Cys1 and iron oxide microparticles and the complex of scFv-Cys2 and iron oxide microparticles are the results of gel filtration chromatography at 490 nm absorption.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In addition, embodiment disclosed separately is an example in which the composite_body | complex of the protein of this invention and microparticles | fine-particles and its manufacturing method are actually used, It is not limited to this.

(First embodiment)
The protein / microparticle complex of the present invention is a complex composed of a protein, a microparticle, and a linker having two or more cysteines that link the protein and the microparticle.
(Complex definition)
The “complex of protein and microparticle” refers to a compound in which a protein and microparticle are bound with a strong bond corresponding to a covalent bond or a covalent bond via a linker.

(Definition of protein)
The protein of the present invention is a protein that can bind to the linker with a strong bond corresponding to a covalent bond or a covalent bond, and includes various lesion marker binding molecules such as antibodies, peptides, and ligands for receptors.

The antibody includes not only an antibody having a naturally produced immunoglobulin structure but also an antibody fragment reduced in molecular weight while maintaining the target molecule binding property. Antibody fragments include Fab fragment, Fab ′ fragment, F (ab ′) ₂ , heavy chain variable (VH) domain alone, light chain variable (VL) domain alone, complex in which VH and VL are bound non-covalently (Fv ), A single chain antibody in which VH and VL are linked by a peptide linker, a camelized VH domain, a peptide containing an antibody complementarity determining region (CDR), and the like. Among these, in particular, single-chain antibodies can be prepared inexpensively and easily corresponding to various antigens, and furthermore, since they have a lower molecular weight than normal antibodies, they are easily excreted rapidly outside the body. It is easy to reach the lesion site. Therefore, the single chain antibody is preferably used for detection or treatment of a lesion site.

  Moreover, the peptide which mimics the binding site of an antibody, the peptide which has the lesion marker binding property acquired from the peptide library which arrange | positioned the peptide which consists of several amino acids at random, etc. can be utilized for the said peptide. Furthermore, as the ligand for the receptor, various ligands such as an EGF ligand for epidermal growth factor receptor (EGFR) and a VEGF ligand for vascular endothelial growth factor receptor (VEGFR) can be used.

  The lesion markers include epidermal growth factor receptor (EGFR) family and its ligand (EGF) family, vascular endothelial growth factor (VEGF) family and its receptor (VEGFR) family, prostate specific antigen (PSA), carcinoembryonicity It can be selected from various lesion markers such as antigen (CEA), matrix metalloprotease (MMP) family, integrin family, selectin family, endoglin or mucin (MUC) family. For example, human epidermal growth factor receptor 2 (HER2) of the EGFR family has many known monoclonal antibodies, antibody fragments, peptides and the like that bind to HER2, and can be used as a lesion marker.

(Linker definition)
The linker of the present invention refers to a covalently bonded compound used for binding the protein and the microparticles. The compound is not particularly limited as long as it is a compound capable of binding the protein and the fine particles. For example, if the linker has a thiol group bonded to a functional group such as a maleimide group or is composed of an amino acid, it preferably contains cysteine. In the present invention, a linker composed of a plurality of amino acids having two or more cysteines linked by peptide bonds is particularly preferred. The “fusion protein” in the present invention refers to a fusion protein in which the protein and the linker are bound with a strong bond corresponding to a covalent bond or a covalent bond.

(Linker introduction position)
The linker can be introduced from any site of the protein. For example, the carboxy terminus of a single-chain antibody (scFv) is far from the antigen binding site and therefore rarely inhibits the antigen-binding ability of the antibody. Therefore, in the case of a single chain antibody, it is preferable to introduce the linker at the carboxy terminus.

(Number of cysteines)
A feature of the present invention is to form a disulfide bond in the molecule of the linker or in the molecule of the fusion protein in order to prevent formation of a dimer by the fusion proteins before binding to the microparticles. is there. The dimer here mainly refers to a dimer between the fusion proteins of the present invention, but also includes a dimer formed by the linkers or a dimer of the linker and the fusion protein.
Therefore, in order to suppress the formation of an intermolecular dimer, in order to form a disulfide bond in the molecule of the linker introduced into the fusion protein, the linker has two or more cysteines. is there. Preferably, it is an even number of 2 or more. More preferably, it is two. Moreover, in order to make all the thiol groups in the linker or the fusion protein molecule into disulfide bonds, the number of thiol groups needs to be an even number. For example, in the case of the fusion protein containing the linker molecule composed of amino acids, an even number of cysteines are required. Therefore, the linker has two or more cysteines. Preferably, it is an even number of 2 or more. More preferably, it is two.
In order to make all the thiol groups in the linker or the fusion protein molecule into disulfide bonds, the number of thiol groups needs to be an even number. For example, in the case of the fusion protein containing the linker molecule composed of amino acids, an even number of cysteines are required. Therefore, the linker has two or more cysteines. Preferably, it is an even number of 2 or more.

  Since the fusion protein and the microparticle are bonded with a strong bond corresponding to a covalent bond or a covalent bond, in this embodiment, cysteine that can be covalently bonded to a functional group on the upper surface of the microparticle is introduced into the linker. . Regarding the number thereof, it is preferable to have two or more cysteines rather than one in order to increase the formation efficiency and stability of the fusion protein and the microparticles. Ultimately, the yield of the complex of the fusion protein of the present invention and the microparticles can be improved.

Furthermore, since the fusion protein having two or more cysteines can improve the efficiency of the binding reaction with the microparticles, a complex of the protein of the present invention and the microparticles is formed with a lower concentration of the fusion protein. be able to. In addition, by controlling the concentration of the fusion protein (including a linker or the like) at a low level, the dimer can be easily reformed, and the non-specific interaction between the fusion protein and the microparticle causes the fusion protein and the microparticle to Phenomena such as loss can be suppressed.
The loss of fusion protein and microparticles here means that if the stability of the dimer is low compared to the case where the fusion protein is a monomer, the fusion protein tends to aggregate with the dimerization of the fusion protein. There is a loss of the fusion protein due to becoming, and a loss due to precipitation of the fine particles accompanying the precipitation of the fusion protein dimer. Therefore, it is considered that suppressing the dimerization of the fusion protein can suppress the loss of the fusion protein and the microparticles.

  In addition, since there is a limit to the cysteine-bonded functional groups on the surface of each of the fine particles, even if more cysteines than the number of the functional groups are introduced into the fusion protein, a fusion protein that cannot bind to the fine particles may appear. There is. Therefore, the number of cysteines introduced into the linker of the present invention is preferably 2.

  In general, it is known that when a protein is produced using a microorganism or the like, the yield of the produced protein is reduced by introducing cysteine. The cause is thought to be the formation of incorrect disulfide bonds between different protein molecules in the cells of the microorganism. When two cysteines are introduced in a limited manner, cysteines in the same fusion protein molecule are disulfide bonded to each other, so that erroneous disulfide bonds between different protein molecules are less likely to occur. The introduction is effective from the viewpoint of ensuring the productivity of the fusion protein of the present invention.

(Cysteine distance and amino acid type between them)
For this reason, it is preferable to arrange two or more cysteines at a distance capable of forming a disulfide bond. In particular, the number of amino acid residues between cysteines is preferably about 0 to 50. When the number of amino acid residues between cysteines exceeds 50, the distance between cysteines becomes large, so that it is difficult for cysteines in the molecule to form disulfide bonds before binding to the fine particles. As a result, there is a possibility that the fusion proteins and the like are dimerized, so that the reaction efficiency with respect to the fine particles may be reduced.

  In addition, when the distance between cysteines is longer than the particle size of the fine particles, there is a possibility that cysteine cannot easily access the functional groups on the fine particle surfaces. Thereby, since the reaction efficiency to the fine particles may be lowered, the distance between cysteines is preferably shorter than the particle size of the fine particles. Therefore, for example, when the particle diameter of the fine particles is 20 nm, the number of amino acid residues between cysteines is most preferably about 0 to 50.

  FIG. 1 is a schematic diagram of one form of a complex of a protein and fine particles of the present invention. X and Z in the figure represent any 0 or more amino acids. When X and Z are 0 amino acids, cysteines are directly linked by a peptide bond, and an OH group is located in the Z portion. For example, as a constitution of a lesion marker binding molecule (probe) that can improve the yield of a complex without inhibiting the antigen binding ability of a single chain antibody, two cysteines are added to the carboxy terminus of the single chain antibody. It is particularly preferable to introduce it.

  Various amino acids other than cysteine can be introduced as the type of amino acid between two or more cysteines. For example, a combination of glycine, serine, and the like is preferable because it has high flexibility, and cysteines are likely to be close to each other and a disulfide bond is easily formed. Specifically, for example, a combination of glycine and serine such as CysGlyGlyGlyGlySerCys is preferable. In FIG. 1, X can be GGGGS, and Z can be a tag sequence for protein purification.

  In addition, it is conceivable that the linker may inhibit the function of the protein by increasing the length of the linker. For example, the molecular size of a single chain antibody is about 5 nm, and if the length of the linker is longer than that, the linker may inhibit the antigen-binding function of the single chain antibody. Therefore, the length of the linker is preferably a length that can maintain the antigen-binding ability of the protein. In particular, the number of amino acid residues (the number of cysteine, the sum of the X amino acids and the Z amino acids) is preferably in the range of 0 to 50, more preferably in the range of 0 to 20.

(Effect other than improving yield)
By using the fusion protein into which the linker having two or more cysteines is introduced, the possibility that the fusion protein and the microparticles are released can be reduced. For example, the binding to the microparticles via two or more cysteines can suppress the release by cleavage of the binding portion between the fusion protein and the microparticles compared to the binding to the microparticles via a single cysteine. It is considered possible.

  Further, if at least one cysteine out of two or more cysteines can bind to the microparticle, at least one remaining cysteine is a protein cysteine (two or more in a similar state on the surface of the microparticle). At least one cysteine among the cysteines may form a disulfide bond with at least one cysteine remaining in the protein bound to the microparticles. This may also reduce the likelihood of protein and microparticles being released.

  By reducing the possibility that the fusion protein and the microparticles are released in this way, when the prepared complex is administered into the living body, the effective amount of the complex is reduced due to the release of the fusion protein and the microparticles, It is considered that an increase in noise (background) from other than the lesion site can be avoided. In addition, since the storage stability of the produced composite is improved, it can be stored for a long period of time.

(Types of fine particles)
As the fine particles of the present invention, various fine particles such as fine particles of metal oxides such as iron oxide fine particles, gold fine particles, gold nanorods, platinum and silver, and metal oxides can be used. Among these, iron oxide fine particles are most preferable.

(Particle surface)
There is no particular limitation on the surface of the fine particle as long as it has a molecule or chemical group capable of binding to the thiol group of cysteine. In the present invention, it is preferable to have a functional group on the surface of the fine particles. In addition, the functional group may be purchased, and the molecule or chemical group on the surface of the fine particles may be converted into the functional group. Various functional groups such as a maleimide group and an iodoacetamide group can be used as the functional group capable of binding to the cysteine thiol group. For example, iron oxide fine particles Nanomag (registered trademark, product of Corefront) having amino groups on the surface and N- (4-maleimidobutyryloxy) succinimide (N- (4- By using Maleimidobutyryloxy) succinimide, a product of Dojindo Laboratories, iron oxide fine particles having maleimide groups can be prepared.

(Second embodiment)
The method for producing a complex of a protein and fine particles of the present invention includes a step of producing a fusion protein into which a linker having two or more cysteines is introduced, and a step of mixing the fusion protein and the fine particles.

  Here, the step of producing a fusion protein into which a linker having two or more cysteines is introduced can use a conventional method for producing a fusion protein, and is not particularly limited. In the present invention, it is preferable to produce a fusion protein according to the protein production mechanism in the cell. For example, the amino acid sequence of a linker having two or more cysteines is linked to the C-terminal of the amino acid sequence of the protein, a gene sequence encoding the linked amino acid sequence is generated, and prepared using a microorganism or the like according to the protein generation mechanism Can be mentioned. In addition, when a fusion protein is produced by such a method, in addition to the amino acid sequence and the like, components such as necessary enzymes may be in accordance with conventional techniques, and are not particularly limited here.

In the step of mixing the fusion protein and the microparticles, first, a reducing agent such as dithiothreitol or tri (2-carboxyethyl) phosphine hydrochloride (TCEP) is added to the fusion protein to cleave the disulfide bond. . The amount of the reducing agent to be added here is preferably a molar amount of 1: several tens with respect to the amount of the fusion protein, particularly preferably a molar amount of 1:10 to 1:20.
Next, the fusion protein with the disulfide bond cleaved and the microparticles are mixed and bound. Thereafter, the fusion protein and the protein other than the fusion protein that are not bound to the microparticles are removed by ultrafiltration or the like to obtain a complex of the protein of the present invention and the microparticles.
Here, in order to ensure the yield and storage stability of the obtained complex of the protein of the present invention and microparticles, the mixing molar ratio of the fusion protein and the microparticles is preferably 4: 1.

  As the linker, protein, fine particles and the like used in the method for producing a complex of protein and fine particles of the present invention, those defined above can be used.

  In order to further clarify the characteristics of the present invention, the present invention will be described with reference to examples. The types of proteins used in the present invention, the types and positions of linkers having two or more cysteines, and the types of fine particles These combinations can also use other combinations, and the present invention is not limited by this embodiment.

Example 1
(Production of single chain antibody)
A gene encoding a single chain antibody (scFv-Cys1) having a linker having one cysteine and a gene encoding a single chain antibody (scFv-Cys2) having a linker having two cysteines were prepared. The scFv-Cys1 is a tag sequence (hereinafter referred to as “6xHis tag”) in which six histidines for protein purification are connected to the C-terminus of a single chain antibody based on the variable region of immunoglobulin G that binds to HER2. And 2) glycine and 1 cysteine. The scFv-Cys2 is in the order of 3 alanines, CysGlyGlyGlyGlySerCys, leucine, glutamic acid, and 6xHis tag at the C-terminus.

  Plasmid pET-22b (+) (Novagen product) in which the gene encoding scFv-Cys1 and the gene encoding scFv-Cys2 are inserted downstream of the T7 promoter is introduced into Escherichia coli BL21 (DE3). Thus, a strain for expression was obtained. The obtained strain was pre-cultured in 4 ml of LB-Amp medium for about 8 hours, and then the whole amount was added to 250 ml of 2xYT medium, followed by shaking culture at 28 degrees and 120 rpm overnight (about 10 hours or more).

Subsequently, isopropyl-β-thiogalactopyranoside with a final concentration of 1 mM was added and cultured overnight at 28 ° C. The cultured Escherichia coli was centrifuged at 8000 × g for 30 minutes at 4 ° C., and the supernatant culture was collected. 60% by weight of ammonium sulfate was added to the obtained culture broth, and proteins were precipitated by salting out. The salted-out solution was allowed to stand at 4 ° C. overnight, and then centrifuged at 8000 × g for 30 minutes at 4 ° C. to collect a precipitate. The resulting precipitate was dialyzed against 1 L of dialysis buffer (20 mM Tris.HCl / 500 mM NaCl, pH = 8). The protein solution after dialysis was added to a column packed with His / Bind (registered trademark) Resin (Novagen) and purified by metal chelate affinity chromatography via Ni ions. Both purified scFv showed a single band by SDS-PAGE to which the reducing agent dithiothreitol was added, and both molecular weights were confirmed to be about 28 kDa (FIG. 2). In addition, when no reducing agent was added, a dimer band of about 56 kDa could be confirmed with scFv-Cys1, whereas a dimer band could not be confirmed with scFv-Cys2 (FIG. 2). From the above, it was confirmed that by introducing two cysteines, formation of a dimer can be substantially suppressed (dimer is hardly formed).

(Example 2)
(Preparation of a composite of scFv and iron oxide fine particles)
A scFv-Cys1 prepared above, scFv-Cys2 _KH 2 of NaCl / 1.47 mM of KCl / 137 mM phosphate buffer (2.68 mM containing 5mM EDTA and _PO 4/1 mM of _Na 2 _HPO 4 / 5mM Of EDTA, pH 7.4). Thereafter, reduction treatment was carried out with 10-fold molar amount of tri (2-carboxyethyl) phosphine hydrochloride (TCEP) at 25 ° C. for about 2 hours. This reduced scFv was treated with 25 mg iron oxide fine particles Nanomag-D-SPIO having a particle size of 20 nm containing PEG300 surface-modified with a quarter-fold molar amount of maleimide for about 2 hours (product of Corefront) And reacted. After the reaction, scFv that did not bind to the iron oxide fine particles was removed by ultrafiltration using 100 kDa pore size Amicon Ultra-4 (product of Nihon Millipore) to obtain a complex of scFv and iron oxide fine particles. .

(Evaluation of yield by gel filtration)
Gel filtration chromatography of the complex of scFv-Cys1 and iron oxide fine particles obtained above and the complex of scFv-Cys2 and iron oxide fine particles using Superdex200GL10 / 300 column (product of GE Healthcare Co., Ltd.), respectively. Each sample was compared with the peak area of absorbance at 490 nm. As a result, as shown in FIG. 3, it was confirmed that the peak area was increased in the complex of scFv-Cys2 and iron oxide fine particles as compared with the complex of scFv-Cys1 and iron oxide fine particles. Therefore, the complex of scFv-Cys2 and iron oxide fine particles improved the yield of the obtained complex about twice as compared with the complex of scFv-Cys1 and iron oxide fine particles.

The complex of the present invention can be used as an in-vivo diagnostic agent for detecting a lesion site and a lesion marker. For example, a complex of an antibody and iron oxide fine particles can be used as an in-vivo diagnostic agent for MRI. In addition, it is also possible to deliver a drug to a lesion site by introducing a drug such as an anticancer drug into the fine particles.

Claims (5)

A complex comprising a protein, fine particles, and a linker for linking the protein and the fine particles, wherein the linker has two or more cysteines.
The complex according to claim 1, wherein the protein is a single chain antibody.
The composite according to claim 1, wherein the fine particles are iron oxide fine particles.
The complex according to any one of claims 1 to 3, wherein the linker has the amino acid sequence CysGlyGlyGlyGlySlyCys.
Producing a fusion protein introduced with a linker having two or more cysteines;
Cleaving the cysteine-bonded disulfide bond with a reducing agent;
The method for producing a complex according to claim 1, further comprising a step of mixing the fusion protein and microparticles.