JP4524414B2 - Method for analyzing sugar-binding phage-displayed antibody - Google Patents

Description

  The present invention relates to a method for analyzing the sugar binding specificity of a phage-displayed antibody.

  Complex carbohydrate molecules or cell surface sugar chains are involved in various life phenomena. In particular, from the knowledge that the structure or distribution of sugar chains changes with diseases such as cancer, it is expected that investigation of sugar chains will lead to the elucidation of the cause of disease and the mechanism of progression. Therefore, it is important to detect specific sugar chains distributed in molecules or tissues simply and with high accuracy.

  Conventionally, in order to detect specific sugar chains in tissues or molecules, lectins and antibodies that recognize target sugar chains have been used. However, the specificity of lectins for sugars is low, and it is not suitable for detailed analysis of sugar chain structures, such as identifying the binding mode of sugar residues and oligosaccharide structures. Moreover, although the antibody produced by the immunological method has high specificity, the antibody with respect to the sugar chain which has antigenicity cannot be obtained, and the kind is also limited.

  On the other hand, since sugar-binding phage-displaying antibodies using the phage display method are produced without using immunological techniques, many types of antibodies can be obtained regardless of immunogenicity. It is expected to be able to identify sugar chains with high specificity and complex structure. However, in order to apply a phage-displaying antibody having sugar binding ability, it is necessary to clarify the sugar binding specificity.

  Conventionally, in order to analyze the specificity of a lectin or antibody that binds to a sugar, a technique for examining the binding to an artificial or natural glycoconjugate immobilized on the surface of a solid phase has been used. In particular, an artificial glycolipid in which a sugar chain and a lipid are chemically bound is used as a sugar chain probe for analyzing sugar binding specificity. However, there have been no reports on useful sugar-binding specificity analysis methods for phage-displayed antibodies so far. That is, an object of the present invention is to provide a method for qualitatively or quantitatively analyzing the sugar-binding specificity of a phage-displayed antibody.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors fixed an artificial glycolipid on a silica gel plate for thin-layer chromatography, and acted on this with a phage-displayed antibody. It has been found that the sugar-binding specificity of the phage-displayed antibody can be analyzed by detection and quantification, and the present invention has been completed.

  That is, according to the present invention, there is provided a method for analyzing the binding specificity of an antibody to a sugar chain, which comprises bringing a phage-displayed antibody into contact with a sugar chain immobilized on a carrier made of an inorganic substance. .

The carrier made of an inorganic substance is preferably a silica gel plate.
The sugar chain immobilized on a carrier made of an inorganic substance is preferably a glycolipid, more preferably an artificial glycolipid.

  The type of sugar component constituting the sugar chain is preferably D-mannose (D-Man), L-fucose (L-Fuc), DN-acetylglucosamine (D-GlcNAc), D-glucose (D- Glc), D-galactose (D-Gal), or DN-acetylgalactosamine (D-GalNAc).

  The phage-displayed antibody is preferably a human-type single chain phage library. More preferably, the phage-displayed antibody is obtained by amplifying a fragment containing CDR1 and CDR2 regions of a VH or VL region of an immunoglobulin gene and a fragment containing CDR3 region by PCR using a human cDNA library as a template, As a template, the VH or VL region is amplified by the PCR method, and these amplified DNA fragments are each incorporated into a non-expression type phagemid vector to prepare a VH library and a VL library. These E. coli VH libraries And VL library are infected with helper phage, converted to phage library, and these phage type VH and VL libraries are co-infected with Escherichia coli expressing Cre recombinase and assembled between VH and VL vectors in E. coli. Perform a replacement reaction, It is obtained by generating phage expressing full-length single chain antibodies to chromatography di infected. More preferably, the phage-displayed antibody is recovered from a human-type single-chain phage library as described above using the binding ability to an artificial glycolipid having a predetermined sugar chain structure as an index.

  According to another aspect of the present invention, there is provided a kit for performing the above-described analysis method of the present invention, comprising a carrier made of an inorganic substance having a sugar chain immobilized thereon. Preferably, the carrier made of an inorganic substance on which sugar chains are immobilized is a silica gel plate.

  By using an artificial glycolipid whose synthesis and purification technology has already been established as a sugar chain probe to be immobilized on the solid phase surface, sugar chains having various structures can be immobilized on the solid surface. In addition, this immobilization work does not require special equipment, and can be performed only with equipment that is routinely used in the laboratory. Furthermore, by spotting and immobilizing artificial glycolipids having various sugar chain structures on a single plate, the affinity of the phage-displayed antibody for various sugar chains can be analyzed simultaneously.

Hereinafter, embodiments of the present invention will be described more specifically.
(1) Artificial glycolipid As the glycolipid used in the present invention, for example, an artificial glycolipid described in Japanese Patent No. 2828391 can be used. Although the kind of saccharide | sugar component which comprises an oligosaccharide is not specifically limited, For example, D-mannose (D-Man), L-fucose (L-Fuc), DN-acetylglucosamine (D-GlcNAc), D-glucose Examples include monosaccharides such as (D-Glc), D-galactose (D-Gal), and DN-acetylgalactosamine (D-GalNAc).

  Furthermore, ganglioside, which is a glycosphingolipid having a sialic acid residue, can also be used. Gangliosides are known to exist on the cell membrane surface and regulate cell proliferation, differentiation, or signal transduction. Gangliosides interact with other cells and tissues in cancer cell invasion and metastasis. It is known to be involved in the action process.

  In the oligosaccharide, each constituent saccharide is bonded by α1 → 2 bond, α1 → 3 bond, α1 → 4 bond, α1 → 6 bond, β1 → 4 bond, or the like, or a combination thereof. For example, mannose may form a straight chain by the above bond, or may have a branched structure by a combination of α1 → 3 bond and α1 → 6 bond. The number of monosaccharides in the oligosaccharide is preferably 2 to 11.

  Specific examples of oligosaccharides include mannobiose (M2), mannotriose (M3), mannotetraose (M4), mannopentaose (M5), mannohexaose (M6), and mannoheptaose (M7). ), Various mixed oligosaccharides such as M5 (Chemical Formula 1) and RN (Chemical Formula 2) shown below.

  Furthermore, as oligosaccharides containing glucose, those having the structure shown in Chemical Formula 3 can be mentioned, and as oligosaccharides containing N-acetylglucosamine, those shown in Chemical Formula 4 can be mentioned, and as oligosaccharides containing fucose The thing shown to Chemical formula 5 can be mentioned.

These oligosaccharides all have one reducing terminal aldehyde group. Therefore, this aldehyde group can be used as a means for immobilizing oligosaccharides. That is, a Schiff base is formed by a reaction between the aldehyde group and a lipid having an amino group, and then the Schiff base is reduced according to a conventional method, preferably a chemical reduction, for example, NaBH ₃ CN is used to convert an oligosaccharide and a lipid. Can be combined (Tetsuo Mizuochi, Munehiro Nakata, Glycobiology Experimental Protocol, pages 42-47, Shujunsha, 1996).

  The lipid having an amino group is preferably a phospholipid having an amino group, and for example, dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE) and the like can be used. In the present invention, the conjugate of oligosaccharide and lipid obtained as described above may be referred to as artificial glycolipid.

  The glycolipid used in the present invention may be the above-mentioned artificial glycolipid, and further, natural products such as glycolipids obtained from bovine brain, cancer cells, or other animals and plants, and these glycolipids are modified. For example, a product to which a glycosyltransferase is allowed to act to add a sugar chain or a chemically synthesized product can be used. Specific examples include gangliosides (GT1a, GD1a, GM1, GM2, GM3, GM4, GQ1b, GT1b, GD1b, GD2, GD3, GP1c, GQ1c, GT1c, GT2 and GT3).

(2) Immobilization of artificial glycolipid In the present invention, a carrier made of an inorganic substance is used as a solid phase for immobilizing the artificial glycolipid. As a carrier made of an inorganic substance, for example, a silica gel plate (for example, a silica gel plate for thin layer chromatography) can be used. As a result of immobilizing artificial glycolipids on solid phases made of various materials and examining the binding of phage-displayed antibodies, the present inventors have surprisingly found that specific binding is observed only on silica gel plates. That is, in the present invention, by selecting a carrier made of an inorganic substance as a solid phase for immobilizing the artificial glycolipid, it becomes possible to analyze the specific binding between the sugar chain and the phage-displayed antibody. It was.

  The solvent for dissolving the artificial glycolipid is not particularly limited, but the solubility varies depending on the structure of the sugar chain portion of the artificial glycolipid. For example, the artificial sugar such as water or a mixture of chloroform / methanol / water Select a suitable solvent for lipid dissolution. 0.5 to 1 μl of the artificial glycolipid solution having various sugar chain structures prepared in this manner is spotted on a carrier (such as a silica gel plate) made of an inorganic substance at an appropriate interval (for example, 0.5 cm interval), Air dry. A carrier (silica gel plate or the like) made of an inorganic substance having a sugar chain immobilized in this manner can be stored in a desiccator until use.

(3) Phage-displayed antibodies Phage-displayed antibodies can be prepared by known methods ((Marks JD et al., J. Mol. Biol. 222, 581-597, 1991; Nissim A et al., EMBO J 13: 692-698, 1994; Satoshi Takayanagi, Riyo Okui, Nobuyoshi Shimizu, Super Repertoire Artificial Antibody Library, Application No. Japanese Patent Application No. 2001-358602 (International Publication No. WO 03/04419)).

  The phage-displayed antibody library is an application of a system that displays (displays) an antibody on the surface of a phage by fusing the antibody to a coat protein of a filamentous phage. Specifically, a phage display library can be obtained by amplifying antibody genes by PCR to prepare a library containing many types of antibody genes and displaying them on phages. Although the specific example of the preparation method of a phage display type | mold single chain antibody is shown below, it is not limited to this.

  Using a human peripheral blood and spleen cDNA library as a template, a fragment containing the CDR1 and CDR2 regions of the VH or VL region of an immunoglobulin gene and a fragment containing the CDR3 region are each amplified by PCR. These are mixed and used as a template to amplify the VH or VL region by PCR. Each of these amplified DNA fragments is incorporated into a non-expression type phagemid vector to prepare a VH library and a VL library. These E. coli VH libraries and VL libraries are infected with helper phages and converted into phage libraries. These phage type VH and VL libraries are co-infected with Escherichia coli expressing Cre recombinase, and a recombination reaction is carried out between the VH / VL vectors in E. coli. This E. coli can be infected with M13KO7 helper phage to generate a phage that expresses a full-length single chain antibody. In order to remove non-recombinants contaminating these phages, this phage is infected with E. coli so that multiple infections do not occur, and further helper phages are superinfected, and glucose containing ampicillin, kana machine, chloramphenicol Incubate at 25 ° C. in a medium containing no Thereby, a phage expressing a recombinant single chain antibody in the supernatant can be obtained. The phage in the supernatant can be precipitated with polyethylene glycol and resuspended to obtain an artificial antibody library having a repertoire of 10 11 or more.

  Furthermore, the phage-displaying antibody having the target sugar-binding property is selectively recovered from the human-type single-stranded phage library prepared by the above method using the binding ability to the artificial glycolipid having the sugar chain structure as an index. be able to. This operation may be referred to as panning.

(4) Binding Specificity Analysis Method for Sugar-Binding Phage Displayed Antibody A silica gel plate on which a sugar chain is immobilized is immersed in a plexi gum solution for 30 seconds and immediately air-dried. Next, in order to eliminate non-specific adsorption of the phage-displayed antibody, the plate is blocked by immersing in a buffer containing 3% gelatin for 1 hour at room temperature, for example. This plate is overlaid with a suspension of phage-displayed antibody prepared to an appropriate concentration with a buffer solution, and gently stirred. After sufficient reaction (eg, 2 hours at 37 ° C.), the plate is washed with buffer.

  In order to detect and quantify the phage-displayed antibody bound to the sugar chain, an aqueous solution of anti-phage antibody labeled with a substance suitable for detection is overlaid on the plate and reacted, washed with a buffer, and then the labeled substance is detected. Detection and quantification with the most suitable method. At this time, the labeling substance of the anti-phage antibody is not particularly limited, but for example, labeling with horseradish peroxidase is desirable in terms of simplicity. The detector used for detection is not particularly limited, and a reflection absorption measuring device, a fluorescence detector, a light emission detector, or the like can be used. In particular, it is desirable to detect light emission with a CCD camera-mounted image analyzer that can detect and quantify with high sensitivity.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1 Preparation of Artificial Glycolipid First, an oligosaccharide solution was prepared by adding 600 μl of distilled water to 2.5 mg of mannotriose (M3) or mannopentaose (M5) and dissolving it. Next, DPPE was dissolved in a chloroform / methanol (1: 1, volume ratio) mixture to a concentration of 5 mg / ml to prepare a DPPE solution. Further, a NaBH ₃ CN solution was prepared by dissolving NaBH ₃ CN in methanol at a concentration of 10 mg / ml. To 600 μl of each oligosaccharide solution, 9.4 ml of the DPPE solution and 1 ml of the NaBH ₃ CN solution were added and mixed. This mixed solution was kept at 60 ° C. for 16 hours to produce an artificial glycolipid.

  The artificial glycolipid synthesized as described above was purified by high performance liquid chromatography (HPLC) using silica gel column and ODS column. A silica gel column was used at room temperature using Shim-pack PREP-SIL. After equilibrating the column with chloroform / methanol / 50 mM acetic acid (65: 30: 5, volume ratio), a sample containing an artificial glycolipid dissolved in the same solvent was injected, and the ratio of chloroform, methanol and 50 mM acetic acid in 60 minutes. A linear gradient elution was performed with a ratio of 50:55:18. The flow rate was 2 ml / min, and the eluate was fractionated by 4 ml. The ODS column was equilibrated with 50 mM acetic acid, a sample containing artificial glycolipid dissolved in the same solvent was added to the column, the column was further washed with the same solvent, and then 50% acetonitrile, methanol, and chloroform / methanol / 50 mM acetic acid. (105: 100: 28, volume ratio) was sequentially flowed to elute the artificial glycolipid.

(Example 2) Selection of solid phase material (part 1)
In determining the solid phase material, the following five types of materials were examined. That is, various artificial glycolipid solutions were spotted on silica gel plates, nitrocellulose membranes, nylon membranes, positively charged nylon membranes and polyvinylidene fluoride (PVDF) membranes. First, the binding of artificial glycolipids in each material was confirmed by a staining method with lectin. FIG. 1 shows an example. It was confirmed that M3-DPPE and M5-DPPE were immobilized on both membranes by the action of concanavalin A, a lectin that recognizes mannose.

(Reference Example 1) Selection of solid phase material (Part 2)
As described above, an organic solvent, a mixed solution of an organic solvent and water, and water are selectively used as a solvent for preparing the artificial glycolipid solution, depending on the structure of the sugar chain portion of the artificial glycolipid. As shown in FIG. 2, the PVDF membrane was considered to be unsuitable for immobilization of artificial glycolipids because the spot size varies depending on the solvent.

(Example 3) Preparation of phage-displayed single-chain antibody A fragment containing CDR1 and CDR2 regions of a VH or VL region of an immunoglobulin gene and a fragment containing a CDR3 region, respectively, using human peripheral blood and spleen cDNA libraries as templates Amplified by PCR. VH or VL region was amplified by PCR method using them as a template.

  Each of these amplified DNA fragments was incorporated into a non-expression type phagemid vector to prepare a VH library and a VL library. Each was prepared so as to contain 10 6 and 10 5 or more colonies. These E. coli VH library and VL library were infected with M13KO7 helper phage and converted into a phage library. These phage type VH and VL libraries were co-infected with Escherichia coli expressing Cre recombinase, and a recombination reaction was carried out between VH and VL vectors in E. coli. This E. coli was infected with M13KO7 helper phage to generate a phage expressing a full-length single chain antibody. In order to remove non-recombinants contaminated with these phages, this phage was infected with E. coli XL1-Blue strain so as not to cause multiple infections, and further superinfected with M13KO7 helper phage, and then ampicillin / kanamachine chloram The cells were cultured at 25 ° C. in a medium containing phenicol and no glucose. As a result, a phage expressing the recombinant single chain antibody in the supernatant was obtained. The phage in the supernatant was precipitated with polyethylene glycol and resuspended to obtain an artificial antibody library having a repertoire of 10 11 or more. It was stored frozen at −80 ° C. until use.

Example 4 Panning Operation 250 μl of human single-stranded phage library (1.8 × 10 ¹⁵ pfu / ml) was added with 1.3 ml of TBS, 1.3 ml of 3% BSA / TBS and 30 μl of 10% Tween20 TBS containing was mixed and kept at 37 ° C. for 1 hour.

  To the antigen-coated plate, 50 μl of the above-treated phage library is added per well, and the mixture is incubated at 37 ° C. for 1 hour. The well was washed three times with TBS containing 0.2% Tween 20 (hereinafter referred to as TBS-T) and twice with TBS, and then the remaining liquid was carefully removed. 50 μl of 100 mM triethylamine (hereinafter referred to as TEA) was added to each well and allowed to stand at room temperature for 10 minutes to release bound phages.

  On the other hand, a neutralization solution (hereinafter referred to as NR) prepared by mixing 1M Tris-HCl buffer (pH 7.4) and 3% BSA / TBS at a ratio of 2: 1 was added to a phage recovery plate at 100 μl per well. added. In this well, TEA containing the phage released by the above operation was recovered. Again, TEA was added to the wells of the antigen-coated plate and allowed to stand at room temperature for 20 minutes, and then recovered in NR.

  To the collected phage solution, 100 μl of E. coli TG-1 suspension was added per well and incubated at 37 ° C. for 1 hour. This was seeded on an LBGC (LB / glucose / carbenicillin) agar plate and cultured at 25 ° C. to form colonies. Colonies were collected and rotated (210 rpm) for 2 hours in SBS medium containing carbenicillin. Next, helper phages were added and allowed to stand at 37 ° C. for 1 hour, and then kanamycin and chloramphenicol were added, followed by rotary culture (210 rpm) at 25 ° C. for 2 nights.

The culture solution was centrifuged (5000 × g, 30 minutes, 4 ° C.), 1/4 amount of 20% polyethylene glycol / 2.5 M NaCl solution was added to the supernatant, and the mixture was allowed to stand in ice for 1 hour. After centrifugation (15000 × g, 30 minutes, 4 ° C.), the precipitated phage was suspended in TBS and collected. To this was added TBS containing an equal amount of 3% BSA / TBS and 1/20 amount of 10% Tween 20, and kept at 37 ° C. for 1 hour, followed by centrifugation to remove insoluble components (18000 × g, 5 minutes, 4 ° C.) The supernatant was recovered as primary panning phage.

  For the recovered phage, the same panning operation as described above was repeated to recover the quaternary panning phage. However, a 24-well plate was used in the secondary panning operation, a 12-well plate was used in the tertiary panning operation, and a 6-well plate was used in the fourth panning operation. Further, in the washing operation after the third panning, in addition to washing with TBS-T and TBS, washing with 100 mM glycine hydrochloride buffer (pH 2.7) was also performed.

  According to the above method, panning was repeated using an artificial glycolipid (M3-DPPE) prepared from mannotriose, and a phage-displayed single-chain antibody that binds to M3-DPPE by quaternary panning was obtained.

(Example 5) Selection of solid phase material (part 3)
Using the silica gel plate, nitrocellulose membrane, nylon membrane and + charged nylon membrane as the solid phase, and using M3-DPPE, M5-DPPE, lactose (Lac) -DPPE and DPPE as the artificial glycolipid, the above phage The binding property of the display type single chain antibody was examined. As shown in FIG. 3, in the silica gel plate, binding of the phage-displayed single chain antibody to M3-DPPE and M5-DPPE was detected, but not on other solid phases. Further, non-specific adsorption was remarkable in the nylon membrane. From the examination (No. 1 to No. 3) in selecting the solid phase material, it was concluded that the silica gel plate is most suitable for this analysis method.

(Example 6) Affinity analysis of mannose-specific phage-displayed single-chain antibody to mannose oligomer About 5 clones of phage-displayed single-chain antibody obtained by panning with M3-DPPE, the affinity for various mannose oligomers was determined. It investigated using this analysis method. The binding of each clone was detected and quantified with an image analyzer. The result is shown in FIG.

  By using this analysis method, binding to M3-DPPE was observed in any clone. Moreover, the coupling | bonding with M5-DPPE was recognized, and the affinity (binding amount) with respect to both artificial glycolipids differed for every clone.

FIG. 1 is a diagram in which artificial glycolipids are spotted on a solid phase of various materials, and the immobilization of the artificial glycolipids is confirmed by lectin. This is an example showing that any material can immobilize artificial glycolipids. FIG. 2 is a diagram in which artificial glycolipids dissolved in various solvents are spotted on a PVDF membrane and detected with an orcinol sulfate reagent. In the PVDF membrane, the artificial glycolipid spot size is different depending on the type of solvent. FIG. 3 shows the results of immobilizing artificial glycolipids on solid phases of various materials and examining the binding of phage-displaying single-chain antibodies. Specific binding was observed only on silica gel plates. It is shown that it is suitable for this analysis method. FIG. 4 is an example comparing the affinity of M3-DPPE and M5-DPPE for five clones of phage-displayed single chain antibodies obtained by panning with M3-DPPE.

Claims (8)

In the method for analyzing the binding specificity of an antibody to a sugar chain, comprising contacting a phage-displayed antibody with a sugar chain immobilized on a carrier made of an inorganic substance, the carrier made of an inorganic substance is a silica gel plate. The method as described above, wherein the sugar chain immobilized on a carrier made of an inorganic substance is a glycolipid. Immobilized oligosaccharide to a carrier made of an inorganic material, an artificial glycolipid A method according to claim 1. The types of sugar components constituting the sugar chain are D-mannose (D-Man), L-fucose (L-Fuc), DN-acetylglucosamine (D-GlcNAc), D-glucose (D-Glc), The method according to claim 1 or 2 , which is D-galactose (D-Gal), DN-acetylgalactosamine (D-GalNAc), or sialic acid. The method according to any one of claims 1 to 3, wherein the phage-displayed antibody is a human-type single-chain phage library. A phage-displayed antibody uses a human cDNA library as a template to amplify a fragment containing the CDR1 and CDR2 regions of the VH or VL region of an immunoglobulin gene and a fragment containing the CDR3 region by the PCR method, mix them, and template As described above, the VH or VL region was amplified by the PCR method, and these amplified DNA fragments were each incorporated into a non-expression type phagemid vector to prepare a VH library and a VL library. These E. coli VH library and VL library Infecting a helper phage, converting it into a phage library, co-infecting these phage-type VH and VL libraries with Escherichia coli expressing Cre recombinase, and performing a recombination reaction between VH and VL vectors in E. coli. Infect this E. coli with helper phage Is obtained by making a phage expressing single chain full-length antibody, the method according to any one of claims 1 to 4. Phage-displayed antibody from human single-stranded phage library, those recovered in the index of binding to the artificial glycolipid having predetermined sugar chain structure, any one of claims 1 to 5 The method described in 1. The kit for performing the analysis method of any one of Claim 1 to 6 containing the support | carrier consisting of the inorganic substance which fix | immobilized the sugar chain. The kit according to claim 7 , wherein the carrier made of an inorganic substance to which a sugar chain is immobilized is a silica gel plate.

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