JP5665021B2 - Fusion MHC molecule-linked magnetic fine particles, antigen peptide screening method, recombinant vector, and transformant of magnetic bacteria - Google Patents

Description

  The present invention relates to a fusion MHC molecule-linked magnetic fine particle, a method for screening an antigen peptide, a recombinant vector, and a transformant of magnetic bacteria.

Cancer has been the leading cause of death in Japan since 1981. The number of patients is expected to increase in the future, and the development of more effective treatment methods and diagnostic methods is required.
Currently, immunotherapy is being developed as a cancer treatment method. A typical example of immunotherapy is peptide vaccine therapy.

Peptide vaccine therapy is a therapeutic method using a peptide (cancer antigen peptide) derived from a protein that is specifically or overexpressed in tumor cells as a vaccine. The cancer antigen peptide is used to induce immune cells specific to tumor cells and attack the tumor cells.
It is the MHC / antigen peptide complex that plays an important role as the starting point of the immune mechanism. T cells function by recognizing MHC / antigen peptide complexes on the surface of antigen-presenting cells or on the surface of tumor cells via the cell surface T-cell receptor (TCR). Therefore, for the development of peptide vaccine therapy, it is important to identify cancer antigen peptides simply and with high efficiency.

  MHC (major histocompatibility complex) is roughly classified into two types, class I and class II, and the cells to be expressed and the mechanism of antigen presentation are different. As for the antigenic peptides, 180 types of peptides presented on MHC class I and 75 types of peptides presented on MHC class II have been identified. Currently, an algorithm for predicting the sequence of an antigenic peptide presented on MHC from an antigenic protein has been developed and applied to the identification of the antigenic peptide (see Non-Patent Documents 1 and 2).

Currently, clinical trials of peptide vaccine therapy are being conducted on MHC class I binding antigen peptides identified using algorithms. However, the clinical effectiveness rate is only a few percent, and it is said that it is insufficient as a treatment method. Furthermore, there is a need for a vaccine design that can stimulate not only cytotoxic T lymphocytes (CTLs) but also helper T cells, and the use of MHC class II binding antigen peptides for vaccines is considered. .
Therefore, it is required to newly identify many MHC class I binding antigen peptides and MHC class II binding antigen peptides.

In addition, one of the steps often employed in current antigenic peptide identification methods is peptide screening using recombinant MHC protein (see Non-Patent Document 3). However, MHC has a problem that recombinant expression is difficult because it takes a huge structure composed of an α chain and a β chain. Furthermore, since it has a plurality of disulfide bonds, it is difficult to correctly construct the structure between the units or aggregation easily occurs, so that it is difficult to produce a soluble recombinant protein having the original function.
As a technique for solving the above problem, a method for introducing an α chain and a β chain into separate plasmids to express them has been reported in E. coli (see Non-Patent Document 4). However, the procedure is complicated and requires a lot of time.

  By the way, it is known that magnetic bacteria produce magnetic bacteria particles having a particle size of about 50 to 100 nm and hold them in the cells. Magnetic bacterial particles are covered with lipid bilayer membranes, and technologies for displaying various functional proteins on magnetic bacterial particles using membrane proteins present in these membranes as anchor molecules have been established (non- Patent Documents 5 and 6, and Patent Documents 1 to 3).

JP 2006-75103 A JP 2006-314314 A JP 2008-273926 A

J Pharmacol Sci 2007; 105: 299-316 Cancer Immunol Immunother 2004; 53: 196-203 J Mol Biol 2004; 340: 81-95 Immunome Res 2009; 5: 2 Appl Environ Microbiol 2004; 70: 2880-5 Anal Chim Acta 2008; 626: 71-7

  An object of the present invention is to provide a fusion MHC molecule-linked magnetic fine particle capable of screening an antigenic peptide with high efficiency and high sensitivity, and a screening method for antigenic peptide with high efficiency and high sensitivity. Furthermore, it is to provide a recombinant vector that can be used for the production of the fused MHC molecule-linked magnetic fine particles, and a transformant of magnetic bacteria containing the same.

  The first aspect of the present invention is a magnetic microparticle derived from a magnetic bacterium, a magnetic microparticle derived from a magnetic bacterium present on the magnetic microparticle, or a fragment thereof, and linked to the magnetic microparticle membrane protein or a fragment thereof. A fusion MHC molecule-linked magnetic comprising an α chain of a MHC molecule or a fragment thereof, a β chain or a fragment thereof, and a fusion MHC molecule having an antigen peptide-binding ability including a first linker peptide disposed therebetween Fine particles.

  The fused MHC molecule-linked magnetic fine particles of the present invention are preferably those in which the magnetic fine particle membrane protein or a fragment thereof and the fused MHC molecule are linked via a second linker peptide.

According to a second aspect of the present invention, the fused MHC molecule-linked magnetic fine particles are brought into contact with the sample containing the antigen peptide, and the fused MHC molecule-linked magnetic fine particles after the contact are changed into the fused MHC molecule-linked magnetic fine particles by the contact. Extracting the bound peptide as an antigenic peptide,
A method for screening an antigenic peptide comprising

The method for screening an antigenic peptide of the present invention may further include concentrating the fused MHC molecule-linked magnetic fine particles after contact using magnetism.
In the antigen peptide screening method of the present invention, it is preferable that the fused MHC molecule-linked magnetic fine particles have been treated to form a disulfide bond in the fused MHC molecule before contacting with the sample. Moreover, it is preferable that the fused MHC molecule-linked magnetic fine particles have been washed before being brought into contact with the sample.
The screening method for antigenic peptides of the present invention is suitable for screening for cancer antigenic peptides.

  The third aspect of the present invention is a DNA encoding a magnetic microparticle membrane protein derived from a magnetic bacterium or a fragment thereof, an α chain of a MHC molecule or a fragment thereof, a β chain or a fragment thereof, and disposed therebetween. A recombinant vector comprising a DNA encoding a fusion MHC molecule capable of binding to an antigen peptide, comprising a first linker peptide.

  The recombinant vector of the present invention preferably further comprises a DNA encoding a second linker peptide that links the magnetic fine particle membrane protein or a fragment thereof and the fusion MHC molecule.

  The fourth aspect of the present invention is a transformant of a magnetic bacterium containing the above recombinant vector.

  According to the present invention, it is possible to provide a fusion MHC molecule-linked magnetic fine particle capable of screening an antigen peptide with high efficiency and high sensitivity, and a method for screening an antigen peptide with high efficiency and high sensitivity. Furthermore, it is possible to provide a recombinant vector that can be used for the production of the fused MHC molecule-linked magnetic fine particles and a transformant of magnetic bacteria containing the same.

It is a conceptual diagram of the expression plasmid of Example 1 of the present invention. It is a conceptual diagram of the plasmid for expression of Example 2 of this invention. It is a photograph of the western blot of Example 4 of this invention. It is a photograph of the western blot of Example 4 of this invention. It is an analysis result of Nano-LC / MS of Example 9 of this invention. It is a Nano-LC / MS analysis result of Example 14 of the present invention.

<Fused MHC molecule-coupled magnetic fine particles>
The fused MHC molecule-linked magnetic fine particles of the present invention are linked to magnetic bacteria-derived magnetic fine particles, magnetic bacteria-derived magnetic fine particle membrane protein or fragments thereof present on the magnetic fine particles, and magnetic fine particle membrane proteins or fragments thereof. In addition, a fusion MHC molecule having an antigen peptide binding ability comprising an α chain of a MHC molecule or a fragment thereof, a β chain or a fragment thereof, and a first linker peptide arranged therebetween. .
By adopting such a configuration, the antigen peptide is highly efficient and highly sensitive by utilizing the characteristics of the fusion MHC molecule capable of reversibly binding and dissociating with the antigen peptide and the characteristics of magnetically inducible magnetic fine particles. It is possible to provide fused MHC molecule-linked magnetic fine particles that can be screened by
The fused MHC molecule-linked magnetic fine particles of the present invention are preferably those in which the magnetic fine particle membrane protein or a fragment thereof and the fused MHC molecule are linked via a second linker peptide.

  In the present invention, “antigen peptide binding ability” means that the antigen peptide binding site of the fusion MHC molecule retains the same three-dimensional structure as the antigen peptide binding site of a natural MHC molecule having an α chain and a β chain. Refers to the ability to bind antigenic peptides.

  In this specification, “binding” between an MHC molecule, a fused MHC molecule, or a fused MHC molecule-linked magnetic microparticle and an antigen peptide means that the antigen peptide is bound to an MHC molecule, a fused MHC molecule, or a fused MHC molecule-linked magnetic microparticle. It means that it is maintained at the same level as in natural MHC molecules.

(Fusion MHC molecule)
The natural MHC molecule is a heterodimer of α chain and β chain, but the fusion MHC molecule in the present invention is the first between the α chain of the MHC molecule or a fragment thereof and the β chain of the MHC molecule or a fragment thereof. This is a fusion protein in which the linker peptide is arranged.
As used herein, “fusion protein” refers to a protein not found in nature, in which two or more proteins or fragments thereof are linked. A fusion protein can be produced by ligating genes encoding respective proteins or fragments thereof in-frame and expressing them recombinantly.

  The fusion MHC molecule in the present invention may be constructed based on any natural MHC as long as it has the ability to bind to an antigenic peptide. The fusion MHC molecule in the present invention may lack a part of α chain and / or a part of β chain as long as it has the ability to bind to an antigen peptide. From the viewpoint of expression efficiency when expressed in, it can be appropriately designed. Specifically, an MHC in which an extracellular domain containing a peptide binding region and a region necessary for maintaining the structure of the peptide binding region is preserved and other domains (eg, transmembrane domain, intracellular domain) are deleted is used. obtain. Such a fusion MHC molecule can be produced by a gene recombination technique according to a procedure usually performed by those skilled in the art.

The type of natural MHC that forms the group constituting the fusion MHC molecule in the present invention is selected according to the type of antigen peptide to be screened. For example, MHC class I is selected for screening antigen peptides derived from endogenous antigens, and MHC class II is selected for screening antigen peptides derived from foreign antigens. In addition, MHC class I is selected to screen for antigenic peptides that can be recognized by CD8 ⁺ T cells, and MHC class II is selected to screen for antigenic peptides that can be recognized by CD4 ⁺ T cells. .

The natural MHC as a group constituting the fusion MHC molecule in the present invention is not limited to human origin, and various vertebrate origins can be used. From the point of clinical application to humans, human MHC can be selected to screen for antigenic peptides that can activate the human immune mechanism.
Human MHC is called human leukocyte antigen (HLA). MHC class I has three types, HLA-A, HLA-B, and HLA-C, and MHC class II has HLA-DR and HLA. -There are three types: DP and HLA-DQ. HLA is rich in polymorphism among individuals and races, and the genotypes common to Japanese are MHC class I, HLA-A24, HLA-A2, etc., MHC class II, HLA-DR4, HLA-DR1 Etc. are known, and these may be suitably selected in the present invention.

The fusion MHC molecule in the present invention is a fusion protein in which a first linker peptide is arranged between an α chain or a fragment thereof and a β chain or a fragment thereof.
The first linker peptide only needs to be arranged between the α chain or a fragment thereof and the β chain or a fragment thereof, and the α chain or a fragment thereof, the β chain or a fragment thereof on the N-terminal side of the first linker peptide. Any of the fragments may be arranged. That is, the C-terminus of the α chain or a fragment thereof and the N-terminus of the β chain or a fragment thereof may be linked via a first linker peptide, and the C-terminus of the β chain or a fragment thereof and the α chain or a fragment thereof May be linked to the N-terminus of each via a first linker peptide. In this case, the α chain or a fragment thereof and the β chain or a fragment thereof can be appropriately designed as described above from the viewpoints of binding to an antigen peptide and expression efficiency in magnetic bacteria.

  The length of the first linker peptide is not particularly limited, but is preferably composed of 5 to 50 amino acid residues from the viewpoint of the antigen peptide binding activity of the fusion MHC molecule. When the number of amino acid residues is 5 or more, it is presumed that the antigen peptide binding site formed from the α chain or a fragment thereof and the β chain or a fragment thereof can be arranged in an appropriate three-dimensional structure, and the antigen of the fusion MHC molecule Peptide binding activity is further improved. When the number of amino acid residues is 50 or less, it is presumed that the structure formation of the entire fusion MHC molecule is unlikely to be hindered because the higher order structure in the linker is difficult to form, and the antigen peptide binding activity of the fusion MHC molecule is further improved. To do. The number of amino acid residues is more preferably about 10 to 30, more preferably about 15 to 25, from the viewpoint of the antigen peptide binding activity of the fusion MHC molecule.

  The amino acid sequence of the first linker peptide may be any sequence. The first linker peptide is preferably a linker peptide in which an amino acid sequence composed of a plurality of amino acid residues is defined as one unit, and the amino acid sequence of the one unit is repeatedly arranged. The peptide sequence in one unit is preferably composed of a sequence of 5 amino acids, but is not limited thereto.

The amino acid constituting the first linker peptide is not particularly limited, but glycine (G) and serine (S) are preferable from the viewpoint of the antigen peptide binding activity of the fusion MHC molecule. If the amino acids constituting the peptide are glycine (G) and serine (S), the size of the amino acid itself is small, and it is difficult to form a higher-order structure in the linker. It is speculated not to be.
Specifically, a linker peptide composed of a peptide having 4 units of glycine and then 1 unit of (G ₄ S) in which serine is arranged side by side is preferable. Here, 5 amino acids in one unit are used. The arrangement of is not limited. That is, in one unit, the sequence of four glycines and one serine may be arbitrarily arranged.

In addition, as the first linker peptide, a linker peptide (hereinafter referred to as “(G ₄ S) _n polypeptide” or “a polypeptide comprising one unit composed of the amino acid sequences of the above four glycines and one serine”) is used. GS linker ”) is preferred. Here, the number of repetitions (n) of the one unit is not particularly limited and may be any number. In the present invention, the number of repetitions may be about 1 to 10 times, and 2 to 6 times. The degree is preferable, and about 3 to 5 times is more preferable.

In the present invention, the first linker peptide is preferably the above (G ₄ S) _n polypeptide, and in particular, 1 unit of (G ₄ S) repeats 3 to 5 times, (G ₄ S) ₃ poly Peptides, (G ₄ S) ₄ polypeptides, (G ₄ S) ₅ polypeptides are preferred. In the present invention, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is preferred.

As long as the fusion MHC molecule in the present invention has the ability to bind to an antigenic peptide, it preserves the extracellular domain including the MHC peptide binding region and the region necessary for maintaining the structure of the peptide binding region, and other domains (for example, In which the α chain or a fragment thereof and the β chain or a fragment thereof are linked via a (G ₄ S) ₃ polypeptide are preferable.

(Magnetic microparticles derived from magnetic bacteria)
In the present invention, the “magnetic bacterium” is a bacterium having an ability to accumulate magnetic fine particles (magnetic bacterial particles, also called BacMPs: Bacterial Magnetic Particles) in the body. For example, Magnetospirillum magneticum AMB-1 (FERM BP-5458, ATCC 70000264), MS-1 (IFO15272, ATCC31632, DSM3856), MSR-1 (IFO15272, DSM6361), MGT-1 (FERM P-16617), etc. Examples include, but are not limited to, species of magnetic bacteria such as Desulfovibrio sp. RS-1 (FERM BP-13283).
These magnetic bacteria have a structure in which about 10 to 20 magnetic fine particles having a particle diameter of about 50 to 100 nm are connected in a chain, and are covered with an organic film having a thickness of about 2 to 4 nm. In addition, the forms of BacMPs synthesized by magnetic bacteria include octahedral, hexagonal, and bullet shapes, and these forms have been observed to be species-specific.
BacMPs synthesized by AMB-1 is a magnetic fine particle made of magnetite (Fe ₃ O ₄ ) and is a ferromagnetic material having a single magnetic domain structure. Efficient magnetic concentration. BacMPs inherently have a lipid membrane (lipid bilayer membrane). Since it has this lipid bilayer membrane, BacMPs is excellent in dispersibility in an aqueous solution while being a ferromagnetic substance.
The lipid membrane can be removed from BacMPs by, for example, treatment with a surfactant. Further, the lipid constituting the lipid membrane can be exchanged with a molecule (polymer or the like) other than lipid. In the present invention, the BacMPs may be one obtained by removing the lipid membrane, or one obtained by exchanging the lipid constituting the lipid membrane with a molecule other than lipid. Examples of the polymer exchangeable with lipid include a block copolymer and a polymer having a phospholipid polar group. Examples of the block copolymer include a block copolymer composed of PMOXA-PDMS-PMOXA, and a phospholipid polar group. Examples of the polymer having an MPC polymer include MPC polymer.

  The term “magnetic fine particles” in the present invention means fine particles having magnetic responsiveness (sensitivity to a magnetic field), and shows sensitivity to a magnetic field such as being magnetized by a magnetic field or adsorbed to a magnet when an external magnetic field is present. Refers to fine particles.

  The shape of the magnetic fine particles is not particularly limited, but a polyhedral shape such as a spherical shape, an ellipsoidal shape, a granular shape, a rectangular parallelepiped shape, and a cubic shape can be preferably exemplified. The size of the magnetic fine particles is not particularly limited, and may be the size of magnetic fine particles obtained from magnetic bacteria.

(Magnetic particle membrane protein or fragment thereof)
In the present invention, the “magnetic fine particle membrane protein” refers to a protein that can inherently exist in the lipid membrane (also referred to as magnetic fine particle membrane). The magnetic fine particle membrane protein or a fragment thereof can be present on the magnetic fine particles even when the lipid membrane is removed or the lipid constituting the lipid membrane is replaced with a molecule other than lipid. In the present invention, as long as the magnetic fine particle membrane protein or a fragment thereof is present on the magnetic fine particles, the lipid film may be omitted. From the viewpoint that the magnetic fine particle film protein or a fragment thereof can be stably present on the magnetic fine particle, and the fused MHC molecule can be stably present on the magnetic fine particle, the magnetic fine particle is preferably provided with the lipid film.
In the present invention, the magnetic microparticle membrane protein derived from magnetic bacteria or a fragment thereof is present on the magnetic microparticle, and the fused MHC molecule is linked to all or a part of the magnetic microparticle membrane protein. All or part of the magnetic fine particle membrane protein can anchor the fused MHC molecule to the magnetic fine particle, and the fused MHC molecule can be efficiently localized on the surface of the magnetic fine particle.

  Examples of magnetic fine particle membrane proteins include Mms5, Mms6, Mms7, Mms13, Mms16, and the like, which are derived from magnetic spiroram species, and the DNA base sequences encoding these are access numbers AB096081 and AB096082. It is registered in databases such as DDBJ and GenBank.

As the magnetic fine particle film protein in the present invention, Mms13, Mms6, Mms16, Mms24 and the like which can be highly expressed in the magnetic fine particle film are preferable. Among these, Mms13 described in JP 2006-75103 A is more preferable.
The amino acid sequence of Mms13 is known (J Biol Chem 2003; 278: 8745-50). Mms13 is a membrane twice-penetrating protein consisting of 124 amino acids, and it is considered that the N-terminus and C-terminus are on the lipid membrane surface of magnetic bacteria. A fusion protein comprising the entire region of Mms13 can be present on magnetic microparticles. Further, there may be no region that is not a transmembrane domain on the C-terminal side of Mms13, and a fusion protein containing Mms13 lacking this region can also be present on the magnetic microparticle.

(Second linker peptide)
The fused MHC molecule-linked magnetic fine particles of the present invention are preferably those in which the fused MHC molecules and the magnetic fine particle membrane protein or fragments thereof are linked via a second linker peptide. More specifically, it is preferable that the C-terminal of the magnetic fine particle membrane protein or fragment thereof and the N-terminal of the fusion MHC molecule are linked via a second linker peptide. The fusion MHC molecule is linked to the magnetic fine particle membrane protein or a fragment thereof via the second linker peptide, so that nonspecific adsorption of various molecules and cells to the surface of the magnetic fine particle can be reduced. Peptide screening efficiency can be improved.

  The second linker peptide is preferably composed of at least 50 amino acid residues. By comprising at least 50 amino acid residues, nonspecific adsorption of various molecules and cells to the surface of the magnetic fine particles can be reduced, and the screening efficiency of the antigen peptide can be improved.

The amino acid sequence of the second linker peptide may be any sequence, and is preferably composed mainly of uncharged polar amino acids.
The second linker peptide is preferably a polypeptide in which an amino acid sequence composed of a plurality of amino acid residues is taken as one unit, and the amino acid sequence of the one unit is repeated. The peptide sequence in one unit is preferably composed of a sequence of 5 amino acids, but is not limited thereto.

  The amino acid constituting the second linker peptide is preferably an uncharged polar amino acid, and examples of the uncharged polar amino acid include asparagine (N), glutamine (Q), threonine (T), tyrosine (Y) and serine (S). Although arbitrarily selected, two or more are preferably selected from the group consisting of asparagine, glutamine and serine. The uncharged polar amino acid is also a hydrophilic amino acid, and the second linker peptide can be made hydrophilic by constituting the second linker peptide mainly with the uncharged polar amino acid.

More preferably, the second linker peptide is specifically arranged by arranging four first type amino acids (eg, asparagine) and then one second type amino acid (eg, serine). A polypeptide composed of a peptide having (N ₄ S) as one unit is preferable, but here, the sequence of five amino acids in one unit is not limited. That is, in one unit, the sequence of four first-type amino acids (asparagine) and one second-type amino acid (serine) may be arbitrarily arranged.
In the above description, the case where asparagine and serine are selected is described as an example, but the sequence of one unit can be an amino acid sequence composed of a combination arbitrarily selected from asparagine, glutamine, threonine, tyrosine and serine.

As the second linker peptide, a polypeptide in which one unit composed of the amino acid sequences of the above four asparagines and one serine is repeated (hereinafter referred to as “(N ₄ S) _n polypeptide” or “ NS Linker ") is preferred. In particular, asparagine is preferable from the viewpoint of imparting hydrophilicity and steric hindrance to the polypeptide, and serine can impart structural flexibility to the polypeptide. Here, the repeating number (n) of the one unit is not particularly limited and can take any value, but in the present invention, it is preferable to repeat at least several times, preferably about 10 times or more, particularly about 20 times. .

In the present invention, the above (N ₄ S) _n polypeptide is preferable, and (N ₄ S) ₂₀ polypeptide in which one unit of (N ₄ S) is repeated 20 times is particularly preferable. In the present invention, the polypeptide having the amino acid sequence represented by SEQ ID NO: 4 is preferred.

The second linker peptide may be provided with a sequence serving as a digestion site by a protease in the amino acid sequence. By designing the second linker peptide to have a site that is recognized and cleaved by the protease, the second linker peptide is cleaved using the protease, and the fusion protein containing the fusion MHC molecule is converted into the magnetic particle membrane protein. Alternatively, it can be isolated from the fragment. Examples of the protease include thrombin, trypsin, chymotrypsin, enterokinase, cathepsin and the like.
In this case, the position at which the sequence serving as the digestion site by the protease is provided in the second linker peptide is not particularly limited. For example, when trying to isolate a fusion protein with a relatively long NS linker on the fusion MHC molecule, the second linker peptide is a protease after repeating (N ₄ S) units 0 to several times. And (N ₄ S) unit is preferably repeated about 10 times or more, preferably about 15 to 20 times.
In addition, when trying to isolate a fusion protein in which the fusion MHC molecule does not have an NS linker or a relatively short NS linker, the second linker peptide has (N ₄ S) units about 10 times. As described above, an embodiment in which the protease recognition sequence is repeated after preferably repeating about 15 to 20 times and the (N ₄ S) unit is further repeated 0 to several times is preferable.

The fused MHC molecule-linked magnetic fine particles of the present invention preferably have Mms13 on magnetic bacteria-derived magnetic fine particles, and Mms13 and fused MHC molecules are linked via (N ₄ S) ₂₀ polypeptide. More preferably, the C-terminal of the MHC molecule and the N-terminal of the fusion MHC molecule are linked via an (N ₄ S) ₂₀ polypeptide.

(Method for producing fused MHC molecule-linked magnetic fine particles)
The method for producing the fused MHC molecule-linked magnetic fine particles of the present invention is not particularly limited, but can be produced using magnetic bacteria. Specifically, it may be a production method including at least the following steps. That is, an expression step in which a fusion protein in which a magnetic Membrane protein derived from magnetic bacteria or a fragment thereof and a fusion MHC molecule capable of binding to an antigen peptide are linked is expressed in the magnetic bacterium, and the magnetic microparticle is isolated from the magnetic bacterium. And an isolation step.

The expression step is preferably a step of expressing a fusion protein in which a magnetic microparticle membrane protein derived from magnetic bacteria or a fragment thereof and a fusion MHC molecule are linked via a second linker peptide.
More specifically, DNA for synthesizing these fusion proteins in magnetic bacteria is prepared, and the DNA is introduced into a vector such as a plasmid by a known gene technique to obtain a recombinant vector. The obtained recombinant vector is introduced into a magnetic bacterium comprising an expression system capable of expressing the fusion protein by known introduction techniques such as microinjection and electroporation, and a transformant of the magnetic bacterium is obtained. Get. Then, by culturing the magnetic bacterium by a known culture technique, the fusion protein can be expressed in the magnetic bacterium.

The recombinant vector includes at least (i) a DNA encoding a magnetic microparticle membrane protein derived from a magnetic bacterium or a fragment thereof, (ii) an α chain or a fragment thereof of an MHC molecule, a β chain or a fragment thereof, and between these A DNA encoding a fusion MHC molecule having an antigen peptide binding ability, comprising a first linker peptide arranged in Preferably, (i) and (ii) are arranged in the order of (i) and (ii) from the 5 ′ side.
The recombinant vector further comprises (iii) DNA encoding a second linker peptide that links the magnetic particle membrane protein or fragment thereof and the fusion MHC molecule from the 5 ′ side (i) (iii) The aspect arrange | positioned in order of (ii) is preferable.
Here, the details and preferred embodiments of the fused MHC molecule, the magnetic microparticle membrane protein derived from magnetic bacteria or a fragment thereof, and the second linker peptide are as described above.
In order to facilitate the expression in the magnetic bacterium, the DNA base sequence is preferably created based on the codon usage frequently used in the magnetic bacterium. For example, refer to the codon usage database of Magnetospirllum magneticum AMB-1 as a magnetic bacterium.

The DNA encoding the fusion MHC molecule of (ii) is not particularly limited as long as the protein encoded by the DNA has an antigen peptide binding ability. For example, the following DNAs are preferable.
Examples of DNA encoding a fusion MHC molecule derived from MHC class I include the following (a), (b) or (c) as preferred specific examples of DNA.
(A) DNA comprising a base sequence of SEQ ID NO: 7, a base sequence of SEQ ID NO: 2, and a base sequence of SEQ ID NO: 8 arranged in this order;
(B) DNA encoding a protein consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence encoded by the DNA of (a) is deleted, substituted or added, and having an antigen peptide binding ability;
(C) DNA that hybridizes with a DNA complementary to the DNA of (a) or (b) under stringent conditions and that encodes a protein having an antigenic peptide binding ability.
As the DNA encoding the fusion MHC molecule derived from MHC class II, the following (d), (e) or (f) is mentioned as a specific example of preferable DNA.
(D) DNA comprising the base sequence of SEQ ID NO: 9, the base sequence of SEQ ID NO: 3, and the base sequence of SEQ ID NO: 10 arranged in this order;
(E) a DNA encoding a protein having an antigen peptide binding ability, comprising an amino acid sequence in which one or several amino acids of the amino acid sequence encoded by the DNA of (d) are deleted, substituted or added;
(F) A DNA that hybridizes under stringent conditions with a DNA complementary to the DNA of (d) or (e), and that encodes a protein having an antigen peptide binding ability.

In this specification, the number of amino acids to be deleted when “one or several amino acids are deleted, substituted or added” means that the protein comprising the amino acid sequence has antigen peptide binding ability. It may be within a range, for example, about 1 to 9, and is preferably smaller from the viewpoint of antigen peptide binding ability, preferably about 1 to 6, and more preferably about 1 to 4.
In the present specification, “under stringent conditions” means that DNAs having high homology, for example, DNAs having a homology of 60% or more, preferably 80% or more hybridize, and DNA having a lower homology than that. A condition where they do not hybridize with each other can be mentioned. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 65 ° C, preferably 65 ° C.
In the present specification, when “homology” is indicated as a specific numerical value, for example, BLAST (Basic Local Alignment Search Tool) (NCBI or Altschul, SF et al. Biol., 215: 403-410 (1990)).

As the vector, for example, pRK415 described in JP-A-8-228782, pMS-T1 described in JP-A-11-285387, or a derivative thereof can be suitably used.
The vector preferably carries a promoter region sequence (promoter sequence) of DNA encoding a magnetic fine particle membrane protein. This promoter sequence is described in, for example, JP-A-2006-75103 and JP-A-2006-314314. Among these, Pmms16 promoter, Pmsp1 promoter, and Pmsp3 promoter having high transcription activity can be preferably used.
Further, the vector may contain an antibiotic resistance gene (for example, an ampicillin resistance gene) as a selection marker.

  The isolation step is a step of collecting fused MHC molecule-linked magnetic fine particles from a culture of magnetic bacteria according to a known technique. For example, the fused MHC molecule-linked magnetic microparticles can be isolated by crushing or lysing the cultured cells and then centrifuging them or using magnetism.

<Screening method of antigen peptide>
The method for screening an antigen peptide of the present invention comprises contacting the fused MHC molecule-linked magnetic microparticle of the present invention with a sample containing an antigen peptide, and linking the fused MHC molecule by the contact from the fused MHC molecule-linked magnetic microparticle after the contact. Extracting the peptide bound to the magnetic microparticles as an antigen peptide.
By adopting such a configuration, antigen peptides can be screened with high efficiency and high sensitivity.

  The method for bringing the fused MHC molecule-linked magnetic fine particles into contact with the sample containing the antigen peptide is not particularly limited. For example, incubation is performed in an appropriate buffer at room temperature (20 to 26 ° C.) for about 30 minutes to 10 hours. Is done by. At this time, the fused MHC molecule-linked magnetic fine particles may be added to the sample, or the sample may be put into a solution containing the fused MHC molecule-linked magnetic fine particles.

The method for extracting the peptide bound to the fused MHC molecule-linked magnetic fine particles by the contact as the antigen peptide from the fused MHC molecule-linked magnetic fine particles after the contact is not particularly limited. For example, by adjusting the environment in which the fused MHC molecule-linked magnetic fine particles are placed, specifically, the salt concentration and pH of the solution containing the fused MHC molecule-linked magnetic fine particles, It can be dissociated and extracted.
About the extracted antigenic peptide, it is possible to identify molecular weight, an amino acid sequence, a three-dimensional structure, etc. by a well-known analysis technique.

The method for screening an antigenic peptide of the present invention may further comprise concentrating the fused MHC molecule-linked magnetic fine particles after contact using magnetism. By utilizing the magnetic responsiveness of the magnetic fine particles constituting the fused MHC molecule-linked magnetic fine particles, it is possible to concentrate the fused MHC molecule-linked magnetic fine particles by magnetism and improve the screening efficiency of the antigen peptide. it can.
The method for concentrating the fused MHC molecule-linked magnetic fine particles using magnetism is not particularly limited and may be any method. Specific methods include the methods described in the examples.

  In the antigen peptide screening method of the present invention, it is preferable that the fused MHC molecule-linked magnetic fine particles have been subjected to a treatment for forming a disulfide bond in the fused MHC molecule before contacting with the sample. The ability to bind the antigenic peptide is improved by bringing the fused MHC molecule-linked magnetic fine particles, which have been treated to form disulfide bonds in the fused MHC molecule (hereinafter also referred to as “disulfide bond-forming treatment”), with the sample. In addition, screening efficiency can be further increased. This is presumably because the disulfide bond formation process forms a disulfide bond based on the amino acid sequence in the fused MHC molecule and stabilizes the fused MHC molecule in a more appropriate three-dimensional structure. It is not bound by the theory of

The disulfide bond forming treatment is not particularly limited, and examples thereof include oxidation treatment. By the oxidation treatment, the SH group in the fusion MHC molecule can be oxidized to form a disulfide bond.
The method for the oxidation treatment is not particularly limited, and any known method capable of forming a disulfide bond in the fusion MHC molecule may be used. For example, a method of incubating the fused MHC molecule-linked magnetic microparticles in a buffer containing appropriate concentrations of reduced glutathione and oxidized glutathione is preferable. In the method, the concentration of reduced glutathione is preferably 1 to 5 mM, and the concentration of oxidized glutathione is preferably 0.01 to 0.5 mM. More preferably, the concentration of reduced glutathione is 5 mM and the concentration of oxidized glutathione is 0.5 mM. The incubation temperature and time are not particularly limited, but may be about 30 minutes to 10 hours at 4 ° C. or room temperature (20 to 26 ° C.).
The method of incubating in the above buffer containing reduced glutathione and oxidized glutathione is described in, for example, Methods in Molecular Biology 2009; 524: 383-405, and specific methods are described in the Examples. The method is mentioned.

In the antigen peptide screening method of the present invention, the fused MHC molecule-linked magnetic fine particles are preferably washed before contacting with the sample. By using washed fused MHC molecule-coupled magnetic microparticles, non-specifically extracted molecules other than antigen peptides are reduced when the peptide bound to the fused MHC molecule-coupled magnetic microparticles by the contact is extracted as an antigen peptide. And the screening efficiency of the antigen peptide can be further increased.
The washing method is not particularly limited as long as the fused MHC molecules are not lost from the fused MHC molecule-linked magnetic fine particles. For example, there is a method of washing by leaving at room temperature or gently stirring in an extraction buffer that can be used when extracting peptides bound to fused MHC molecule-linked magnetic particles by the contact as antigenic peptides. Can be mentioned.
As the above buffer solution, for example, an acidic buffer solution, more specifically, about 0.05 to 0.2 mol / L, preferably about pH 3 containing about 0.1 to 0.15 mol / L citric acid. Examples of the phosphate buffer can be exemplified.

  Either the disulfide bond forming treatment or the washing may be performed first, but the washing is preferably performed first. A fusion MHC molecule having a more appropriate three-dimensional structure by the disulfide bond formation process by not including another process between the disulfide bond formation process and contacting the fused MHC molecule-linked magnetic fine particles with the sample. Is presumed to be able to contact the antigenic peptide while retaining its structure, and the screening efficiency of the antigenic peptide can be further increased.

  The sample in the antigen peptide screening method of the present invention is not particularly limited, and may be a natural product or an artificial synthetic product. For example, samples derived from various animals or plants, samples derived from cultured cells, fungi, bacteria or viruses The sample etc. are mentioned. These samples may be used as they are, or may be used after purification, or may be suspended in an appropriate solvent or may be appropriately diluted as necessary.

  The antigen peptide screening method of the present invention can be suitably used for screening for cancer antigen peptides. When screening for a cancer antigen peptide, examples of the sample include a sample derived from human whole blood such as serum, a sample derived from a cancer patient such as a lysate of cancer cells, and a culture supernatant of cancer cells.

  Examples of cancer include lung cancer, tracheal and bronchial cancer, oral epithelial cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, liver and intrahepatic bile duct cancer, kidney cancer, pancreatic cancer, prostate cancer, breast cancer, uterus Cancer, ovarian cancer, brain tumor, malignant melanoma (melanoma), epithelial cells such as skin cancer, malignant cancer and tumor, myoma, osteosarcoma, Ewing's sarcoma, etc., cells that constitute the supporting tissue muscle and bone Examples include, but are not limited to, cancers and tumors derived from hematopoietic cells such as cancers and tumors that have become malignant, leukemia, malignant lymphoma, myeloma, Burkitt lymphoma, and the like.

  According to the present invention, a kit for screening an antigenic peptide using fused MHC molecule-linked magnetic microparticles can be provided. The antigen peptide screening kit includes at least the fusion MHC molecule-linked magnetic fine particles of the present invention, and may include a means for concentrating the fused MHC molecule-linked magnetic fine particles bound to the antigen peptide using magnetism. The fused MHC molecule-linked magnetic microparticles may be provided in the form of a fused MHC molecule-linked magnetic microparticle, and in the form of an expression system comprising a recombinant vector or a transformant of magnetic bacteria used for the production of the fused MHC molecule-linked magnetic microparticle. May be provided.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples unless it exceeds the gist thereof. Unless otherwise specified, “%” is based on mass.
All the reagents were commercially available special grades for research or equivalents, and the reagents were appropriately prepared using distilled water or pure water obtained by treating distilled water with MilliQ Lab (Nihon Millipore).
Image analysis was performed using image analysis software Aqua cosmos (Hamamatsu Photonics).
N-terminal biotin-labeled peptides were synthesized by Bex Co., Ltd., and other peptides were synthesized by Gl Biochem (Shanghai) Ltd. Previous studies on MHC have shown that biotinylation of the N-terminus of a peptide does not prevent MHC-peptide binding (J Cell Biol 2004; 119: 531-42). In this example, A peptide in which the following biotinylated C6 linker was linked to the N-terminus of the peptide was used.

Example 1
<Construction of plasmid for fusion MHC class I molecule expression and transformation of magnetic bacteria>
A fusion protein obtained by fusing Mms13, NS linker, and HLA-A24 (actually, a fusion protein in which the β chain and α chain of HLA-A24 are linked via a GS linker) in this order (hereinafter referred to as “fusion protein”). The plasmid pUM13LA24 (see FIG. 1) for expression of “Mms13-NS linker-HLA-A24 fusion protein” was constructed as follows. The HLA-A24 gene is the most common gene among the MHC class I genes found in Japanese.

Plasmid (pUC18 (100)) containing gene (303 bp) (SEQ ID NO: 6) encoding NS linker ((N ₄ S) ₁₈ + LVPRGS + (N ₄ S)) (SEQ ID NO: 5) (TAKARA BIO Co., Ltd. The NS linker gene was excised from the order) using the restriction enzyme SspI and introduced into the SspI site of the plasmid pUMP16M13 containing the Mms13 gene to construct the plasmid pUM13L (100).
pUMGP16M13 is a plasmid in which a PCR product containing the Pmms16 promoter and the mms13 gene downstream thereof is ligated to pUMG digested with SspI (see Appl Environ Microbiol 2006; 72: 465-71, Method construction of expression vectors). PUMP16M13 contains an ampicillin resistance gene.

Next, the gene (45 bp) in which the β chain gene (SEQ ID NO: 7) and α chain gene (SEQ ID NO: 8) of HLA-A24 encode the GS linker ((G ₄ S) ₃ ) (SEQ ID NO: 1) ( A fused gene (SEQ ID NO: 12) encoding FLAG-tag (DYKDDDDK) (SEQ ID NO: 11) linked to the 3 ′ end of the gene (SEQ ID NO: 12) at the end (further stop codon tga (Ordered gene synthesis from MBL) was introduced downstream of the NS linker gene of pUM13L (100) to construct plasmid pUM13LA24.
Top10 (Invitrogen) was used as E. coli used for gene recombination.

The amino acid (LVPRGS) in the NS linker ((N ₄ S) ₁₈ + LVPRGS + (N ₄ S)) is the thrombin digestion site. In this example, a recognition site for thrombin was provided in the NS linker in preparation for the recovery of HLA-A24. The NS linker was a polypeptide (4 units of 4 asparagine and 1 serine repeated 20 times). (N ₄ S) ₂₀ polypeptide).

  The pUM13LA24 obtained above was introduced into the magnetic bacterium AMB-1 by electroporation to obtain a transformant.

(Example 2)
<Construction of plasmid for expression of fusion MHC class II molecule and transformation of magnetic bacteria>
A fusion protein obtained by fusing Mms13, NS linker, and HLA-DR4 (actually, a fusion protein in which an α chain and a β chain of HLA-DR4 are linked via a GS linker) in this order (hereinafter referred to as a fusion protein) The plasmid pUM13LDR4 (see FIG. 2) for expression of “Mms13-NS linker-HLA-DR4 fusion protein” was constructed as follows. The HLA-DR4 gene is the most common gene among MHC class II genes found in Japanese.

First, plasmid pUM13L (100) was constructed in the same manner as in Example 1.
Next, the α chain gene (SEQ ID NO: 9) of HLA-DR4 and the β chain gene (SEQ ID NO: 10) are linked via a gene (SEQ ID NO: 3) encoding a GS linker (SEQ ID NO: 1). A fusion gene in which a gene (SEQ ID NO: 13) encoding FLAG-tag (SEQ ID NO: 11) is linked to the 3 ′ end (additional stop codon tga is added at the end) (Order gene synthesis from MBL) ) Was introduced downstream of the NS linker gene of pUM13L (100) to construct plasmid pUM13LDR4.
Top10 (Invitrogen) was used as E. coli used for gene recombination.

In this example, as in Example 1, a recognition site for thrombin was provided in the NS linker with the intention of recovering HLA-DR4. The NS linker contained 20 units of 4 asparagines and 1 serine. The polypeptide can be repeated (N ₄ S) ₂₀ polypeptide).

  The pUM13LDR4 obtained above was introduced into the magnetic bacterium AMB-1 by electroporation to obtain a transformant.

Example 3
<Fabrication of fused MHC molecule-linked magnetic particles using magnetic bacteria>
The preculture of each transformant of magnetic bacterium AMB-1 obtained in Examples 1 and 2 was inoculated into 10 L of MSGM (magnetic spirillum growth medium) (J Bacteriol 1979; 140: 720-9) ( (Preculture / MSGM = 1/1000 (v / v)) and room temperature (24 to 26 ° C.) for about 5 to 7 days. At the time of inoculation, a microaerobic state was formed by bubbling MSGM with argon gas (5 to 10 minutes for pUM13LA24, 30 to 40 minutes for pUM13LDR4). In addition, ampicillin was added to MSGM (5.0 μg / ml).

Separation of fused MHC molecule-linked magnetic fine particles from magnetic bacteria was performed according to the method of Tanaka et al. (Anal Chem 2000; 72: 3518-22). That is, the culture obtained by stationary culture was centrifuged (9000 G, 4 ° C., 10 minutes), and the cells of magnetic bacteria were collected. The cells were suspended in HEPES buffer (10 mM, pH 7.4) and crushed three times at a pressure of 1500 kg / cm ² using a French press (Otake Seisakusho, 5501M).
This cell disruption liquid is transferred to a glass container, and an Nd-Fe-B (neodymium-iron-boron) magnet is installed outside the glass container to accumulate the fused MHC molecule-linked magnetic fine particles. The supernatant was removed (hereinafter, the separation of the accumulation using a magnet and the supernatant was referred to as “magnetic separation”). The magnetic separation after adding HEPES buffer and suspending was repeated 10 times, and the fused MHC molecule-linked magnetic fine particles were washed. The washed fused MHC molecule-linked magnetic fine particles were suspended in PBS buffer and stored at 4 ° C.
As described above, Mms13-NS linker-HLA-A24 fusion protein linked magnetic particles (hereinafter simply referred to as “HLA-A24 linked magnetic particles”) and Mms13-NS linker-HLA-DR4 fusion protein were linked. Magnetic fine particles (hereinafter simply referred to as “HLA-DR4 linked magnetic fine particles”) were obtained.

  Hereinafter, when referring to the mass of the fused MHC molecule-linked magnetic fine particles, the mass is a value obtained by dispersing the fused MHC molecule-linked magnetic fine particles in an appropriate amount of PBS, measuring the absorbance (660 nm), and converting it to a dry mass using a calibration curve. .

Example 4
<Evaluation of MHC expression on magnetic particles>
30 μl and 25 μl of 1% SDS solution were added to 1.0 mg of HLA-A24-coupled magnetic microparticles obtained in Example 3 and 8.0 mg of HLA-DR4-coupled magnetic microparticles, respectively. Each was boiled with stirring by a Voltex mixer and ultrasonic waves (100 ° C., 20 to 30 minutes). Next, the supernatant was recovered by magnetic separation to obtain each sample. Add 3 x SDS sample buffer to each sample (15 μl for HLA-A24 sample, 12.5 μl for HLA-DR4 sample), boil for 5 minutes, and then perform SDS-PAGE (acrylamide concentration 12.5%) It was. Subsequently, the gel after electrophoresis was transferred to a PVDF membrane by a semi-dry method. Anti-FLAG monoclonal antibody derived from alkaline phosphatase (ALP) -labeled mouse against FLAG sequence (Sigma Aldrich Japan) (1 μg / ml, PBS-T) (15 ml for HLA-A24 sample, HLA-DR4) 10 ml) was reacted (room temperature, 1 hour). After washing with PBS-T for 10 minutes three times, NBT / BCIP-Blue Liquid Substrate (SIGMA, USA) was added as a substrate for color development.

The results for HLA-A24 linked magnetic fine particles are shown in FIG. 3, and the results for HLA-DR4 linked magnetic fine particles are shown in FIG. 3 and 4, M is a marker lane, 1 is a sample lane of a fused MHC molecule-linked magnetic particle, and 2 is a sample lane of a magnetic particle obtained from a wild-type magnetic bacterium AMB-1.
The Mms13-NS linker-HLA-A24 fusion protein was 75 kDa, and a band was confirmed at this size. The Mms13-NS linker-HLA-DR4 fusion protein was 74 kDa, and a band was confirmed at this size. This result confirmed the expression of HLA-A24 and the expression of HLA-DR4 on the magnetic fine particles.

(Example 5)
<Evaluation of MHC class I expression on magnetic particles by ELISA>
100 μl (10 μg / ml) of an ALP-labeled mouse-derived anti-FLAG monoclonal antibody was added to 100 μg of the HLA-A24-coupled magnetic fine particles obtained in Example 3, and reacted at room temperature for 30 minutes. After the reaction, the plate was washed with PBS (200 μl × 3 times), and 50 μl of the magnetic fine particle suspension was transferred to a 96-well microtiter plate. 50 μl of Lumifos 530 (Lumigen PPD, 4-Methyoxy-4 (3-phosphatephenyl) spiro [1,2-dioxeteane-3,2'adamantine] disodium salt, 3.3 × 10 ⁻⁴ M) (Wako Pure Chemicals) The mixture was further reacted at room temperature for 2 minutes, and the luminescence intensity (kcounts / sec) was measured using a luminescence plate reader (Lucy2 luminescence reader, Aloka). The results are shown in Table 1.

  As shown in Table 1, the emission intensity per 100 μg of wild-type magnetic fine particles was 157 kcounts / sec, whereas the emission intensity per 100 μg of HLA-A24 linked magnetic fine particles was 993 kcounts / sec. Since FLAG-tag is present at the C-terminal site of HLA-A24, the C-terminal of the HLA-A24 fusion protein is localized on the surface of the magnetic microparticle on the HLA-A24-linked magnetic microparticle obtained in Example 3. It was confirmed that

(Example 6)
<Functional evaluation of fused MHC class I molecularly linked magnetic fine particles>
[Evaluation of binding ability of HLA-A24-linked magnetic fine particles using biotin-labeled cancer antigen peptide]
1 ml of biotin-labeled cancer antigen peptide prepared at 50 μg / ml with PBS was added to 100 μg of HLA-A24-linked magnetic fine particles obtained in Example 3, and reacted at 4 ° C. for 3 hours. As the cancer antigen peptide, MAGE1 peptide (NYKHCCFEI) (SEQ ID NO: 14) and CDC27 peptide (FSWAMDLDPKGA) (SEQ ID NO: 15) were used.
After the reaction, it was washed with PBS (200 μl × 3 times), 1 ml of ALP-labeled streptavidin (Roche, diluted 1000 times with PBS) was added, and the mixture was reacted at room temperature for 30 minutes. Next, the sample was washed with PBS (200 μl × 3 times) and suspended in 50 μl of PBS as a sample. The plate was transferred to a 96-well microtiter plate, 50 μl of Lumiphos 530 was added and reacted at room temperature for 2 minutes, and the luminescence intensity (kcounts / sec) was measured using the luminescence plate reader. The results are shown in Table 2.

  The MAGE1 peptide used here is a cancer antigen peptide that has been reported to bind to HLA-A24 (Int J Cancer 1999; 80: 169-72). The CDC27 peptide is a cancer antigen peptide that has been reported to bind to MHC class II (Science 1999; 284: 1351-4).

  As shown in Table 2, when HLA-A24-linked magnetic fine particles were reacted with CDC27 peptide, almost no luminescence was observed as much as wild-type magnetic fine particles. When HLA-A24 linked magnetic fine particles were reacted with MAGE1 peptide, strong luminescence intensity was observed. From this, it was confirmed that the HLA-A24-coupled magnetic fine particles have an MHC class I type cancer antigen peptide binding ability.

(Example 7)
<Functional evaluation of fused MHC class I molecularly linked magnetic fine particles>
[Evaluation of change in binding ability of HLA-A24-coupled magnetic fine particles by glutathione treatment]
In Example 6, the PBS used for preparation of the biotin-labeled peptide was replaced with PBS containing 5 mM reduced glutathione and 0.5 mM oxidized glutathione, and MAGE1 peptide (SEQ ID NO: 14), CDC27 peptide (sequence) were used as cancer antigen peptides. No. 15) and CMVpp65 peptide (QYDPVAALF) (SEQ ID NO: 16) were used, and luminescence intensity (kcounts / sec) was measured in the same manner as in Example 6. The results are shown in Table 3.
The CMVpp65 peptide is a cancer antigen peptide that has been reported to bind to HLA-A24 (Immunol Lett 2004; 95: 199-205).

As shown in Table 3, since the HLA-A24-linked magnetic fine particles showed strong luminescence intensity when reacted with the MAGE1 peptide and CMVpp65 peptide, it was confirmed that the HLA-A24 linked magnetic fine particles have the ability to bind cancer antigen peptides of MHC class I type. .
In addition, HLA-A24-coupled magnetic microparticles increase in luminescence intensity when treated with PBS containing glutathione, confirming that the amount of binding between HLA-A24-coupled magnetic microparticles and cancer antigen peptide increases under oxidizing conditions. It was done. This suggested that the disulfide bond between cysteines present in HLA-A24 was strengthened under oxidizing conditions, and the ability to bind to a cancer antigen peptide was improved.

(Example 8)
<Functional evaluation of fused MHC class I molecularly linked magnetic fine particles>
[Structural evaluation of HLA-A24 during peptide binding by ELISA]
To 100 μg of the HLA-A24-coupled magnetic fine particles obtained in Example 3, 1 ml of a cancer antigen peptide prepared to 50 μg / ml with PBS or PBS containing 5 mM reduced glutathione and 0.5 mM oxidized glutathione was added at 4 ° C. Reacted for hours. As the cancer antigen peptide, MAGE1 peptide (SEQ ID NO: 14), CDC27 peptide (SEQ ID NO: 15), and CLCA2 peptide (LLGNCLPTV) (SEQ ID NO: 17) were used.
After the reaction, the plate was washed with PBS (200 μl × 3 times), 50 μl (10 μg / ml, PBS) of mouse-derived anti-HLA-ABC monoclonal antibody (W6 / 32) was added as a primary antibody, and the mixture was reacted at room temperature for 30 minutes. After the reaction, the plate was washed with PBS (200 μl × 3 times), 50 μl (10 μg / ml, PBS) of ALP-labeled goat-derived anti-mouse IgG antibody (Invitrogen) was added as a secondary antibody, and the mixture was reacted at room temperature for 30 minutes. Thereafter, the sample was washed with PBS (200 μl × 3 times) and suspended in 50 μl of PBS to prepare a sample. The plate was transferred to a 96-well microtiter plate, 50 μl of Lumiphos 530 was added, and the mixture was reacted at room temperature for 2 minutes. Then, the luminescence intensity (kcounts / sec) was measured using the microplate reader. The results are shown in Table 4.

The CLCA2 peptide has been identified as a cancer antigen peptide that binds to HLA-A2 belonging to the same MHC class I as HLA-A24 (J Immunol 2002; 169: 540-7).
It has been reported that the mouse-derived anti-HLA-ABC monoclonal antibody W6 / 32 forms a complex with an antigen peptide and specifically binds only to HLA in which the three-dimensional structure is maintained (Cell 1978; 14: 9- 20).

  As shown in Table 4, when HLA-A24-linked magnetic fine particles were reacted with an antigen peptide prepared with PBS, the emission intensity was as weak as when no peptide was added. On the other hand, when reacted with MHC class I-binding antigen peptides (MAGE1 peptide, CLCA2 peptide) prepared with PBS containing glutathione, high luminescence intensity was observed. From this, the HLA-A24 expressed on the magnetic fine particles has an enhanced disulfide bond between cysteines present in HLA-A24 under oxidizing conditions, and the mouse-derived anti-HLA-ABC monoclonal antibody (W6 / 32) It was suggested that a recognizable structure was formed. It was suggested that HLA-A24 on HLA-A24-linked magnetic microparticles has the same structure as natural HLA-A24 expressed on human cells and binds to MHC class I-binding antigenic peptides.

Example 9
<Functional evaluation of fused MHC class I molecularly linked magnetic fine particles>
[Screening of HLA-A24 binding antigen peptide]
50 mg of citrate buffer (0.06 mol / L Na ₂ HPO ₄ , 0.13 mol / L citrate, pH 3.0) was added to 1 mg of HLA-A24-linked magnetic fine particles obtained in Example 3, and incubated at room temperature for 30 minutes. By doing so, the HLA-A24 linked magnetic fine particles were washed. Thereafter, the supernatant was removed by magnetic separation, and HLA-A24-linked magnetic fine particles were recovered.
Peptide prepared by mixing MAGE1 peptide (SEQ ID NO: 14) and CDC27 peptide (SEQ ID NO: 15) prepared at 50 μg / ml with PBS in a ratio of 1: 1 or 1:10 to the washed HLA-A24-coupled magnetic fine particles 1 ml of each solution was added and reacted at 4 ° C. for 3 hours. After the reaction, the supernatant was removed by magnetic separation, and HLA-A24 linked magnetic fine particles were recovered. Next, 50 μl of the citrate buffer was added to the HLA-A24-coupled magnetic microparticles and incubated at room temperature for 30 minutes. The supernatant was collected by magnetic separation, the supernatant was diluted 10-fold with the following solution A, and the peptides contained in the supernatant were analyzed by Nano-LC / MS. The results are shown in FIG.

In Nano-LC / MS analysis, solution A (2% acetone, 0.1% formic acid) was used as the stationary phase, and solution B (80% acetone, 0.1% formic acid) was used as the mobile phase. As the Nano-LC / MS analyzer, a direct nanoflow LC system (DiNa; KYA Technologies, Tokyo, Japan) and an ESI IT Mass spectrometer (LCQ-DECA XP; Thermo Fisher Scientific, San Jose, CA, USA) were used. The column used was a C18 reverse phase column (C18 Trap Column; 1 mm in length; id, 0.5 mm; C18 Separation Column; 50 mm in length; id, 0.1 mm). Separation by LC was performed under the following conditions. Prior to each analysis, the column was equilibrated with solution A for at least 10 minutes. And after inject | pouring a sample, Nano-LC / MS was switched to Injection mode and the analysis was started after flowing A solution for 10 minutes. The peptide solution separated by the column was directly introduced into a mass spectrometer and analyzed.
-Temperature: Room temperature-Flow rate: 300 nL / min
Gradient: A solution; 100%; 2.1 minutes, B solution; 0-8% Linear Gradient; 3 minutes, B solution; 8-45% Linear Gradient; 30 minutes, B solution; 45-100% Linear Gradient; 5 minutes, solution B; 100%; 10 minutes, solution A; 100%; 20 minutes / measurement time: 70 minutes

The MAGE1 peptide has a molecular weight of 1489.79. In addition, since the MAGE1 peptide contains one basic amino acid, lysine and histidine, two protons are easily added, and a peak of a divalent ion is predicted as a peak observed by the above analysis.
As shown in FIG. 5, when the peptide mixing ratio was 1: 1, the strongest peak was detected at m / z 1489.59 corresponding to the MAGE1 peptide. Further, when the peptide mixing ratio was 1:10, the strongest peak was detected at m / z 745.62 corresponding to the mass-to-charge ratio when two protons were added to the MAGE1 peptide.
From this, it was shown that the antigen peptide can be concentrated and extracted by using HLA-A24-linked magnetic fine particles, and the antigen peptide can be identified by Nano-LC / MS or the like.

(Example 10)
<Functional evaluation of fused MHC class II molecularly linked magnetic particles>
[Evaluation of sample concentration and assay time using biotin-labeled peptide]
The biotin-labeled peptide to be used was adjusted to 5 mM with a DMSO solution and then adjusted to each concentration with PBS.
100 μl of biotin-labeled cancer antigen peptide prepared at 0 to 60 μM (0, 12.5, 25, 50, and 60 μM) with PBS was added to 100 μg of the HLA-DR4-linked magnetic fine particles obtained in Example 3, and the room temperature was 90 minutes. It was made to react with. The CLIP peptide (PKPPKPVSKMMATPLLMQA) (SEQ ID NO: 18) was used as the positive control peptide, and the HER2 peptide (TYLPTNASL) (SEQ ID NO: 19) was used as the negative control peptide.
Thereafter, the plate was washed with PBS (100 μl × 3 times), 100 μl of ALP-labeled streptavidin (1000-fold diluted with PBS) was added, and the mixture was reacted at room temperature for 30 minutes. Subsequently, the sample was washed with Tris-HCl (pH 7.4) (150 μl × 2 times) and suspended in 50 μl of Tris-HCl to prepare a sample. The plate was transferred to a 96-well microtiter plate, 50 μl of lumiphos 530 was added, and the mixture was reacted at room temperature for 5 minutes. Then, the luminescence intensity (kcounts / sec) was measured using the luminescence plate reader. The results are shown in Table 5.

  In addition, 100 μl of biotin-labeled CLIP peptide (SEQ ID NO: 18) prepared to 50 μM with PBS was added to 100 μg of HLA-DR4 linked magnetic microparticles obtained in Example 3, and 0 to 6 hours (0, 1, 3, (5 and 6 hours). After each reaction time, ALP-labeled streptavidin was added and reacted in the same manner as described above, and the luminescence intensity (kcounts / sec) was measured. The analysis results of the emission intensity are shown in Table 6.

  CLIP used here has been identified as a peptide that binds between the α unit and β unit, which are antigen-binding sites, in the process of generating MHC class II in the cell (Nature 1992; 358: 764- 8). The HER2 peptide is a cancer antigen peptide that binds to HLA-A belonging to MHC class I (Blood 2002; 99: 3717-24).

As shown in Table 5, when the HLA-DR4 linked magnetic fine particles were reacted with the CLIP peptide, the luminescence intensity was about 4 times maximum as compared with the case of reacting with the HER2 peptide. From this, it was confirmed that the HLA-DR4-linked magnetic fine particles bind to the antigenic peptides presented on MHC class II.
As shown in Table 6, as a result of examining the peptide reaction time, the maximum amount of peptide binding was observed at the reaction time of 5 hours.

(Example 11)
<Functional evaluation of fused MHC class II molecularly linked magnetic particles>
[Evaluation of binding ability of HLA-DR4-linked magnetic fine particles using biotin-labeled cancer antigen peptide]
100 μl of biotin-labeled cancer antigen peptide prepared to 50 μM with PBS was added to 100 μg of HLA-DR4-linked magnetic fine particles obtained in Example 3, and reacted at room temperature for 90 minutes. As the cancer antigen peptide, HER2 peptide (SEQ ID NO: 19), NY-ESO-1 119-143 peptide (PGVLLKEFTSGSGNILTIRTAADHR) (SEQ ID NO: 20), MAGE-A3 146-160 peptide (FFPVIFSASSSSLQL) (SEQ ID NO: 21) were used. .
Thereafter, the plate was washed with PBS (100 μl × 3 times), 100 μl of ALP-labeled streptavidin (1000-fold diluted with PBS) was added, and the mixture was reacted at room temperature for 30 minutes. Next, the sample was washed with PBS (100 μl × 3 times), exchanged with Tris-HCl for buffer, and suspended in 50 μl of Tris-HCl as a sample. The plate was transferred to a 96-well microtiter plate, 50 μl of lumiphos 530 was added, and the mixture was reacted at room temperature for 5 minutes. Then, the luminescence intensity (kcounts / sec) was measured using the luminescence plate reader. The results are shown in Table 7.

  Note that MAGE-A3 146-160 peptide (Cancer Res 2001; 61: 4773-8) and NY-ESO-1 119-143 peptide (Cancer Res 2000; 60: 4946-52) bind to HLA-DR4. Is a reported cancer antigen peptide.

  As shown in Table 7, when the HLA-DR4 linked magnetic fine particles were reacted with the HER2 peptide, almost no luminescence was observed as in the case where no peptide was added. Strong luminescence intensity was observed when reacted with MAGE-A3 146-160 peptide and when reacted with NY-ESO-1 119-143 peptide. From this, it was confirmed that the HLA-DR4-linked magnetic fine particles have an MHC class II type cancer antigen peptide binding ability.

(Example 12)
<Functional evaluation of fused MHC class II molecularly linked magnetic particles>
[Structural evaluation of HLA-DR4 during peptide binding by ELISA]
100 μl of cancer antigen peptide prepared to 50 μM with PBS was added to 100 μg of HLA-DR4-linked magnetic fine particles obtained in Example 3, and reacted at room temperature for 90 minutes. As cancer antigen peptides, HER2 peptide (SEQ ID NO: 19), NY-ESO-1 119-143 peptide (SEQ ID NO: 20), and MAGE-A3 146-160 peptide (SEQ ID NO: 21) were used.
Thereafter, 100 μl of mouse-derived anti-HLA-DR monoclonal antibody L243 (Biodesign International) suspended in PBS as a primary antibody was added, and reacted at room temperature for 30 minutes. After the reaction, the plate was washed with PBS (100 μl × 3 times), 100 μl of ALP-labeled goat-derived anti-mouse IgG antibody (Invitrogen) was added as a secondary antibody, and the mixture was reacted at room temperature for 30 minutes. Thereafter, the sample was washed with PBS (100 μl × 3 times) and suspended in 50 μl of PBS to prepare a sample. The plate was transferred to a 96-well microtiter plate, 50 μl of Lumiphos 530 was added, and the mixture was reacted at room temperature for 5 minutes. Then, the luminescence intensity (kcounts / sec) was measured using the microplate reader. The results are shown in Table 8.

  MHC class II is known to retain a complex structure (αβ heterodimer) by binding an antigenic peptide to the peptide binding region between the α and β chains. The anti-HLA-DR monoclonal antibody L243 has been reported to specifically bind to the HLA-DR / peptide complex in the antigen presenting state (J Biol Chem 1995; 270: 971-7).

  As shown in Table 8, when the HLA-DR4 linked magnetic fine particles were reacted with the HER2 peptide, it was found that the HLA-DR4 linked magnetic fine particles were not conjugated to L243 because it was as weak emission intensity as when no peptide was added. . On the other hand, when reacted with the MAGE-A3 146-160 peptide and when reacted with the NY-ESO-1 119-143 peptide, strong luminescence intensity was observed, indicating that it was bound to L243. I understood. From this, it was suggested that HLA-DR4 expressed on the magnetic fine particles has the same structure as natural HLA-DR and presents an antigenic peptide.

(Example 13)
<Functional evaluation of fused MHC class II molecularly linked magnetic particles>
[Evaluation of antigen-specific binding ability of HLA-DR4-linked magnetic fine particles using biotin-labeled cancer antigen peptide]
100 μl of biotin-labeled cancer antigen peptide prepared to 50 μM with PBS was added to 200 μg of HLA-DR4-linked magnetic fine particles obtained in Example 3, and reacted at room temperature for 90 minutes. Cancer antigen peptides include MAGE-A3 146-160 peptide (SEQ ID NO: 21), MAGE-A3 146-158 peptide (FFPVIFSKASSSL) (SEQ ID NO: 22), and MAGE-A3 146-156 peptide (FFPVIFSKASS) (SEQ ID NO: 23). ) Was used.
Thereafter, the plate was washed with PBS (100 μl × 3 times), 100 μl of ALP-labeled streptavidin (1000-fold diluted with PBS) was added, and the mixture was reacted at room temperature for 30 minutes. Thereafter, the sample was washed with PBS (100 μl × 3 times), exchanged with a buffer for Tris-HCl, and then suspended in 50 μl of Tris-HCl as a sample. The plate was transferred to a 96-well microtiter plate, 50 μl of Lumiphos 530 was added, and the mixture was reacted at room temperature for 5 minutes. Then, the luminescence intensity (kcounts / sec) was measured using the microplate reader. The results are shown in Table 9.

  The MAGE-A3 146-156 peptide and the MAGE-A3 146-158 peptide used here are peptides containing amino acids considered as MHC binding sites in the MAGE-A3 146-160 peptide sequence.

  As shown in Table 9, when HLA-DR4 linked magnetic microparticles were reacted with MAGE-A3 146-160 peptide, the strongest emission intensity was observed, followed by MAGE-A3 146-158 peptide, MAGE-A3 146- Strong luminescence intensity was observed in the order of 156 peptides. This correlates with the experimental result of T cell activation (Cancer Res 2001; 61: 4773-8) examined in identifying the antigen peptide of MAGE-A3. From this, it was shown that the binding properties of antigen peptides to MHC class II can be evaluated using HLA-DR4 linked magnetic microparticles.

(Example 14)
<Functional evaluation of fused MHC class II molecularly linked magnetic particles>
[Screening of HLA-DR4 binding antigen peptide]
100 μl of MAGE-A3 146-160 peptide (SEQ ID NO: 21) prepared to 50 μM with PBS was added to 1 mg of HLA-DR4-linked magnetic fine particles obtained in Example 3, and reacted at room temperature for 90 minutes. After the reaction, the supernatant was removed by magnetic separation, and HLA-DR4-linked magnetic fine particles were recovered. Next, 50 μl of citrate buffer (0.06 mol / L Na ₂ HPO ₄ , 0.13 mol / L citric acid, pH 3.0) was added to the HLA-DR4 linked magnetic microparticles and incubated at room temperature for 30 minutes. The supernatant was collected by magnetic separation, and the peptides contained in the supernatant were analyzed by Nano-LC / MS in the same manner as in Example 9. The results are shown in FIG.

  As shown in FIG. 6, the strongest peak was detected at m / z 836.23 corresponding to the mass-to-charge ratio when two protons were added to the MAGE-A3 146-160 peptide (molecular weight 1670.98). From this, it was shown that antigen peptides can be concentrated and extracted by using HLA-DR4-linked magnetic fine particles, and that antigen peptides can be identified by Nano-LC / MS or the like.

As shown in the results of the Examples, the use of fused MHC class I molecule-linked magnetic microparticles enables screening of antigenic peptides that bind to MHC class I with high efficiency and high sensitivity. If fused MHC class II molecule-linked magnetic fine particles are used, screening of antigenic peptides that bind to MHC class II can be performed with high efficiency and high sensitivity.
Therefore, by using the fused MHC molecule-linked magnetic fine particles of the present invention, a highly efficient and highly sensitive antigen peptide screening method can be provided.

Claims (10)

Magnetic microparticles derived from magnetic bacteria,
A magnetic bacterium-derived magnetic particle film protein or fragment thereof present on the magnetic particle;
A fused MHC molecule linked to the magnetic particulate film protein or fragment thereof and made into a single chain by a first linker peptide ;
A second linker peptide that links the magnetic particle membrane protein or fragment thereof and the fusion MHC molecule;
Equipped with a,
The fusion MHC molecule is
An α chain of a MHC class I molecule or a fragment thereof , comprising a fragment comprising an α ₁ domain, an α ₂ domain, and an α ₃ domain, a β chain of an MHC class I molecule, and a first linker disposed therebetween A fusion MHC molecule having the ability to bind an antigenic peptide consisting of a peptide;
An α chain of an MHC class II molecule or a fragment thereof, which includes an α ₁ domain and an α ₂ domain, and a β chain of an MHC class II molecule or a fragment thereof, which includes a β ₁ domain and a β ₂ domain A fusion MHC molecule having an antigen peptide binding ability comprising: a first linker peptide disposed between them;
Either
The second linker peptide is the polypeptide represented by SEQ ID NO: 4 or the polypeptide represented by SEQ ID NO: 5.
Fusion MHC molecule-linked magnetic fine particles. The fused MHC molecule-linked magnetic fine particle according to claim 1, wherein the first linker peptide is a polypeptide having an amino acid sequence in which (glycine-glycine-glycine-glycine-serine) is repeated 2 to 6 times. Contacting the fused MHC molecule-linked magnetic microparticle according to claim 1 or 2 and a sample containing an antigen peptide;
Extracting the peptide bound to the fused MHC molecule-linked magnetic fine particles by the contact as an antigen peptide from the fused MHC molecule-linked magnetic fine particles after the contact;
A method for screening an antigenic peptide comprising:   The method for screening an antigenic peptide according to claim 3, further comprising concentrating the fused MHC molecule-linked magnetic fine particles after contact using magnetism.   The method for screening an antigenic peptide according to claim 3 or 4, wherein the fused MHC molecule-linked magnetic fine particles have been subjected to a treatment for forming a disulfide bond in the fused MHC molecule before contacting with the sample.   The method for screening an antigenic peptide according to any one of claims 3 to 5, wherein the fused MHC molecule-linked magnetic fine particles are washed before contacting with the sample.   The method for screening an antigenic peptide according to any one of claims 3 to 6, wherein the antigenic peptide is a cancer antigenic peptide. DNA (i) encoding a magnetic microparticle membrane protein derived from magnetic bacteria or a fragment thereof;
DNA (ii) encoding a fusion MHC molecule single-stranded by a first linker peptide ;
DNA (iii) encoding a second linker peptide that links the magnetic particle membrane protein or fragment thereof and the fusion MHC molecule;
Only including,
The fusion MHC molecule is
An α chain of a MHC class I molecule or a fragment thereof , comprising a fragment comprising an α ₁ domain, an α ₂ domain, and an α ₃ domain, a β chain of an MHC class I molecule, and a first linker disposed therebetween A fusion MHC molecule having the ability to bind an antigenic peptide consisting of a peptide;
An α chain of an MHC class II molecule or a fragment thereof, which includes an α ₁ domain and an α ₂ domain, and a β chain of an MHC class II molecule or a fragment thereof, which includes a β ₁ domain and a β ₂ domain A fusion MHC molecule having an antigen peptide binding ability comprising: a first linker peptide disposed between them;
Either
The second linker peptide is the polypeptide represented by SEQ ID NO: 4 or the polypeptide represented by SEQ ID NO: 5.
Recombinant vector. The DNA (i), the DNA (ii), and the DNA (iii) are sequentially arranged in the order of DNA (i), DNA (iii), and DNA (ii) from the 5 ′ side. Item 9. The recombinant vector according to Item 8.   A transformant of a magnetic bacterium comprising the recombinant vector according to claim 8 or 9.