+

WO2010126115A1 - Procédé de criblage d'un anticorps monoclonal reconnaissant une structure tridimensionnelle de protéine membranaire - Google Patents

Procédé de criblage d'un anticorps monoclonal reconnaissant une structure tridimensionnelle de protéine membranaire Download PDF

Info

Publication number
WO2010126115A1
WO2010126115A1 PCT/JP2010/057631 JP2010057631W WO2010126115A1 WO 2010126115 A1 WO2010126115 A1 WO 2010126115A1 JP 2010057631 W JP2010057631 W JP 2010057631W WO 2010126115 A1 WO2010126115 A1 WO 2010126115A1
Authority
WO
WIPO (PCT)
Prior art keywords
membrane protein
antibody
liposome
screening
binding
Prior art date
Application number
PCT/JP2010/057631
Other languages
English (en)
Japanese (ja)
Inventor
日野智也
村田武士
荒川孝俊
野村紀通
小林拓也
岩田想
Original Assignee
独立行政法人科学技術振興機構
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 独立行政法人科学技術振興機構 filed Critical 独立行政法人科学技術振興機構
Priority to JP2011511458A priority Critical patent/JP5526448B2/ja
Publication of WO2010126115A1 publication Critical patent/WO2010126115A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a liposome / membrane protein complex used when screening an antibody that co-crystallizes with a membrane protein, a method for producing the liposome / membrane protein complex, a method for screening the antibody, and a kit for screening the antibody. .
  • Membrane proteins play an important role as proteins that support life activities such as transmission of information in cell membranes, transport of substances, production of energy, and formation of cytoskeleton.
  • life activities such as transmission of information in cell membranes, transport of substances, production of energy, and formation of cytoskeleton.
  • membrane proteins have been accelerated all over the world based on the obtained DNA information.
  • membrane protein research is highly important.
  • membrane proteins include single-transmembrane proteins, multiple-transmembrane proteins, superficial membrane proteins, proteins that form transmembrane channels, and the like. It may be lipid modified or sugar chain modified.
  • results of various genome projects indicate that nearly 30% of proteins encoded by higher organism genomes are integral membrane proteins having a transmembrane helix.
  • a group generally referred to as a receptor is distributed in the cell membrane, cytoplasm, and nucleus, and transmits information on various physiologically active substances from the outside to the cells and DNA. For example, it binds to physiologically active substances such as cytokines, neurotransmitters, hormones, and drugs to transmit information and support life activities.
  • physiologically active substances such as cytokines, neurotransmitters, hormones, and drugs to transmit information and support life activities.
  • pharmaceuticals that act directly on receptor proteins common names: clopidogrel, salmeterol / fluticasone, rituximab, epoetin alfa, valsartan, etc. boast high sales worldwide as blockbusters.
  • GPCRs G protein-coupled receptors
  • ADP receptor adenosine diphosphate receptor
  • GPCRs are general terms for receptors that perform signal transmission through a trimeric protein called G protein when signal information is transmitted into a cell. It is also involved in allergic reactions such as asthma and reactions in which HIV infects cells. GPCRs can be said to be the protein group that has received the most attention in current basic research and drug discovery research.
  • membrane proteins are a very important target, and many researchers around the world are working day and night on functional analysis, structural analysis, production, purification, and commercialization of membrane proteins.
  • solubilization making them soluble in aqueous solution and ready for use in experiments.
  • the inventors of the present application are studying a crystallization method, which is the most important step in the three-dimensional structure analysis of a membrane protein.
  • the X-ray crystal structure analysis method which is a standard method for structural analysis
  • protein molecules are aligned in a lattice formed by repeating a series of unit cells, and the resulting crystal is transmitted through X-rays.
  • the three-dimensional structure is analyzed from the diffraction pattern.
  • the crystals are generally fragile and extremely sensitive to environmental conditions. Therefore, it is necessary to adjust these environmental conditions to an optimum level for stable crystallization.
  • the crystallization of membrane proteins contains difficult elements not found in general soluble proteins, and there are few successful examples.
  • the compatibility with surfactants used for solubilization and the behavior of solubilized membrane proteins different from general soluble proteins complicate and make the membrane protein crystallization process difficult.
  • the obtained crystal has characteristics not found in the crystal of soluble protein.
  • the gap between proteins in the crystal is occupied not only by the solvent but also by the micelle of the surfactant.
  • the micelle itself does not have a structure with atomic resolution, and is considered a part of the solvent in the crystal.
  • the size of micelles is important, and micelle-sized surfactants that just fill the gaps between proteins stabilize the crystals.
  • the second feature is that the interaction between proteins, which is essential for obtaining good crystals, occurs only on polar surfaces not covered with micelles. It does not contribute greatly in the atomic resolution). As is clear from this, it is difficult to obtain good crystals from the hydrophobic protein entirely embedded in the membrane, and relatively good from the membrane protein having many subunits outside the membrane. Easy to obtain crystals. Further, when the size of the micelle of the surfactant is smaller, generally good crystals tend to be obtained.
  • Non-patent Document 1 For the purpose of improving the efficiency of crystallization of membrane proteins, the inventors of the present application make a database from the reported crystallization conditions of membrane proteins, and are suitable for crystallization of membrane proteins (transmembrane helix type) based on the database. A screening kit has been prepared (Non-patent Document 1).
  • Non-patent Document 2 bacterial cytochrome oxidase
  • Non-Patent Document 2 by binding an antibody to a membrane protein, a specific conformation of the membrane protein is stabilized, and the hydrophilic surface as a whole of the membrane protein / antibody complex is expanded to increase the crystallinity. It is improving. This is an innovative research result that succeeded in efficiently crystallizing a membrane protein that is very difficult to crystallize by using an antibody.
  • Non-patent Documents 3 and 4 the three-dimensional structure analysis of the potassium channel and the chloride ion channel was later performed by Mackinnon et al. Using the same method (Non-patent Documents 3 and 4). Although these membrane proteins had a structure of about 3.5 mm even without an antibody, they succeeded in improving the resolution to 2.5 mm or more by using an antibody. Furthermore, it is a disadvantage of this method that it takes a very long time to produce antibodies used for crystallization, but instead of using antibodies, research is also being conducted to create artificial binding proteins using phage display and similar techniques. (Non-patent document 5).
  • ELISA method Enzyme Linked ImmunoSorbent Assay method
  • Non-patent document 6 describes a case where an antibody that binds to a membrane protein is screened by a technique different from the ELISA method.
  • Non-Patent Document 6 uses a channel protein that penetrates the membrane three times. Specific antibody phages are screened by immobilizing a biotinylated liposome / GluRD protein complex in an avidin-coated tube and panning a mouse-derived phage antibody library immunized with a mixed solution containing the GluRD protein. It is described that the obtained antibody specifically recognizes and binds to the X domain near the N-terminal of the protein, which is located in a region away from the transmembrane region in the GluRD protein.
  • Patent Document 1 discloses a method in which a receptor is immobilized on a solid phase carrier and a binding state of a ligand that specifically binds to the receptor is examined.
  • a membrane fragment expressing epidermal growth factor receptor (EGFR) is immobilized on a solid phase carrier, and it is confirmed that EGF, which is a ligand of the EGFR, binds to the membrane fragment.
  • Patent Document 2 describes a method of immobilizing a protein on a solid phase carrier and examining the binding state of an antibody that specifically binds to the protein.
  • Patent Document 2 it is confirmed that a liposome / ribonucleoprotein complex is immobilized on a solid phase carrier and an anti-ribonucleoprotein antibody is bound to the complex.
  • the membrane protein in the case of screening an antibody using the ELISA method, when a purified membrane protein is used as an antigen, it becomes an operation in the presence of a surfactant, so that it cannot be immobilized efficiently.
  • the membrane protein is denatured by operations such as repeated washing with a buffer containing a surfactant, and as a result, the natural structure presented on the surface of a living cell is maintained. It is difficult to obtain an antibody that recognizes a membrane protein, particularly an antibody that recognizes a three-dimensional structure and has high affinity and very high specificity.
  • Non-Patent Document 6 for the purpose of searching for an epitope, a biotinylated liposome / GluRD protein complex is prepared and an antibody that binds to a membrane protein is screened.
  • the binding site of the screened antibody is limited to a hydrophilic region near the protein N-terminal and away from the transmembrane region, the versatility for general membrane proteins is low.
  • the protein N-terminal of the membrane protein is not so far from the transmembrane region, or a method for screening an antibody that binds to the loop portion of the membrane protein has not been clarified.
  • the protein N-terminal (or C-terminal) polypeptide is a relatively flexible region having no specific three-dimensional structure.
  • the hydrophilic region present at the protein N-terminus (or C-terminus) of membrane proteins generally has a large fluctuation in the relative position and orientation with respect to the transmembrane region in a solvent. Even if an antibody binds to such a region, it cannot form a regular crystal lattice that is regularly arranged in the crystal. Therefore, an antibody that binds near the protein N-terminus (or C-terminus) is not crystallized. It can be said that it is not suitable.
  • Non-Patent Document 6 a membrane protein that penetrates the membrane three times is used, but generally, the more the number of penetrations, the more difficult the handling in various experimental processes. In such a case, the method described in Non-Patent Document 6 cannot be applied.
  • the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a liposome / membrane protein complex used for screening an antibody that co-crystallizes with a membrane protein. Another object of the present invention is to provide a method for producing a liposome / membrane protein complex for use in screening an antibody that co-crystallizes with a membrane protein. Furthermore, another object of the present invention is to provide a screening method for antibodies that co-crystallize with membrane proteins. Another object of the present invention is to provide a screening kit comprising a liposome / membrane protein complex used for screening an antibody that co-crystallizes with a membrane protein.
  • a liposome / membrane protein complex used for screening an antibody that co-crystallizes with a membrane protein, the solid phase carrier, the liposome bound to the solid phase carrier, and the liposome.
  • a liposome / membrane protein complex wherein at least a part of the membrane protein is exposed on the outer surface of the liposome.
  • This configuration is an example to be described later, an antibody specifically recognizing the natural three-dimensional structure of the target membrane protein, an antibody having a high binding specificity to the target membrane protein, an antibody having a high affinity for the target membrane protein, An antibody that expands the entire hydrophilic region of the membrane protein and the antibody, an antibody that increases the overall thermal stability of the membrane protein and the antibody, or an antibody that specifically recognizes the three-dimensional structure of the loop portion of the membrane protein Including liposome / membrane protein complexes that have been demonstrated to be usable in screening. Therefore, it becomes possible to screen an antibody for co-crystallization with a membrane protein.
  • a method for producing a liposome complex used for screening an antibody that co-crystallizes with a membrane protein comprising: a) a lipid solution, a linker, the membrane protein, and a surfactant.
  • a method for producing a liposome complex is provided.
  • This production method is an example which will be described later, an antibody specifically recognizing the natural three-dimensional structure of the target membrane protein, an antibody having high binding specificity to the target membrane protein, and an antibody having high affinity to the target membrane protein Specific recognition of the three-dimensional structure of an antibody that expands the entire hydrophilic region of the membrane protein and antibody, an antibody that increases the overall thermal stability of the membrane protein and antibody, or the loop portion of the membrane protein It has been demonstrated that liposome / membrane protein complexes can be produced that have been demonstrated to be used in screening antibodies. Therefore, it becomes possible to screen an antibody for co-crystallization with a membrane protein.
  • a screening method for an antibody that co-crystallizes with a membrane protein wherein d) a test antibody is contacted with a liposome complex used for screening an antibody that co-crystallizes with a membrane protein.
  • an antibody that specifically recognizes the natural three-dimensional structure of the target membrane protein an antibody that has high binding specificity to the target membrane protein, and an affinity for the target membrane protein.
  • High antibody antibody that expands the whole hydrophilic region that combines membrane protein and antibody, antibody that increases the overall thermal stability of membrane protein and antibody, or specifically the three-dimensional structure of the loop portion of membrane protein It has been demonstrated that antibodies that recognize can be screened. Therefore, it becomes possible to screen an antibody for co-crystallization with a membrane protein.
  • a method for screening an antibody that co-crystallizes with a membrane protein i) a step of contacting an antibody library with the liposome complex according to any one of claims 1 to 8, and j A) a step of selecting the antibody bound in step i), k) a step of denaturing the membrane protein, and l) contacting the modified membrane protein denatured in step k) with the antibody selected in step j).
  • a screening method comprising the steps of: m) removing the antibody bound in step l).
  • a method for producing an antibody the step of screening an antibody for co-crystallization with a membrane protein by the screening method, and a method for co-crystallization with the screened membrane protein.
  • a method for producing an antibody for co-crystallization with a membrane protein comprising the step of producing a replica of the antibody.
  • a screening kit for an antibody that co-crystallizes with a membrane protein comprising a membrane protein, a lipid, a linker, and a solid phase carrier.
  • This configuration is an example to be described later, an antibody specifically recognizing the natural three-dimensional structure of the target membrane protein, an antibody having a high binding specificity to the target membrane protein, an antibody having a high affinity for the target membrane protein, An antibody that expands the entire hydrophilic region of the membrane protein and the antibody, an antibody that increases the overall thermal stability of the membrane protein and the antibody, or an antibody that specifically recognizes the three-dimensional structure of the loop portion of the membrane protein A component of a liposome / membrane protein complex that has been demonstrated to be screenable. Therefore, it becomes possible to screen an antibody for co-crystallization with a membrane protein.
  • the present invention also provides a liposome / membrane protein complex comprising a solid phase carrier, a liposome bound to the solid phase carrier, and a membrane protein embedded in the liposome, Provided is a liposome / membrane protein complex that is used when screening an antibody that recognizes a loop portion of a membrane protein, in which at least a part of the membrane protein is exposed on the outer surface of the liposome.
  • this liposome / membrane protein complex can be used for screening an antibody that recognizes a loop portion of a membrane protein in Examples described later. Therefore, if this liposome / membrane protein complex is used, an antibody that recognizes the loop portion of the membrane protein can be efficiently obtained.
  • a method for screening an antibody comprising a step of producing a test antibody from antibody-producing cells, and examining the binding of the test antibody to the liposome / membrane protein complex
  • a method for screening an antibody that recognizes a loop portion of a membrane protein comprising a step of selecting a liposome / membrane protein complex-binding antibody having binding property to the liposome / membrane protein complex.
  • this screening method can be used for screening an antibody that recognizes a loop portion of a membrane protein in Examples described later. Therefore, if this liposome / membrane protein complex is used, an antibody that recognizes the loop portion of the membrane protein can be efficiently obtained.
  • a method for producing an antibody comprising the steps of screening an antibody that recognizes a loop part of a membrane protein by the screening method, and an antibody that recognizes the loop part of the screened membrane protein.
  • the present invention also provides a liposome / membrane protein complex comprising a solid phase carrier, a liposome bound to the solid phase carrier, and a membrane protein embedded in the liposome,
  • screening one or more antibodies selected from the group consisting of an antibody having a dissociation constant of 1.0 ⁇ 10 ⁇ 8 M or less for a protein and an antibody that enhances the overall thermal stability of the membrane protein and the antibody.
  • the liposome / membrane protein complex used is provided.
  • This liposome / membrane protein complex is an antibody that has a binding property to the wild type of the membrane protein and has no binding property to the loop partial mutant of the membrane protein, and a dissociation to the membrane protein in Examples described later. It can be used to screen one or more antibodies selected from the group consisting of antibodies having a constant of 1.0 ⁇ 10 ⁇ 8 M or less and antibodies that increase the overall thermal stability of membrane proteins and antibodies. Proven. Therefore, the antibody can be efficiently obtained by using this liposome / membrane protein complex.
  • a method for screening an antibody comprising a step of producing a test antibody from antibody-producing cells, and examining the binding of the test antibody to the liposome / membrane protein complex, A step of selecting a liposome / membrane protein complex-binding antibody having a binding property to the liposome / membrane protein complex, and having a binding property to the wild type of the membrane protein, and a loop partial mutant of the membrane protein Selected from the group consisting of an antibody having no binding property to an antibody, an antibody having a dissociation constant with respect to a membrane protein of 1.0 ⁇ 10 ⁇ 8 M or less, and an antibody that enhances the overall thermal stability of the membrane protein and the antibody.
  • a method for screening one or more antibodies is provided.
  • This screening method is an example to be described later, and has an affinity for the wild-type membrane protein, an antibody that does not have the binding property to the loop partial mutant of the membrane protein, and a dissociation constant for the membrane protein of 1. It has been demonstrated that it can be used to screen one or more antibodies selected from the group consisting of antibodies of 0 ⁇ 10 ⁇ 8 M or less and antibodies that increase the overall thermal stability of membrane proteins and antibodies. . Therefore, the antibody can be efficiently obtained by using this liposome / membrane protein complex.
  • a method for producing an antibody which has a binding property to a wild-type membrane protein and does not have a binding property to a loop partial mutant of the membrane protein by the screening method.
  • Screening for one or more antibodies selected from the group consisting of antibodies, antibodies with a dissociation constant of 1.0 ⁇ 10 ⁇ 8 M or less for membrane proteins, and antibodies that increase the overall thermal stability of membrane proteins and antibodies And binding to the wild type of the membrane protein, and binding to the loop partial mutant of the membrane protein.
  • This production method is an example which will be described later, and has an affinity for the wild-type membrane protein, an antibody that does not bind to the loop partial mutant of the membrane protein, and a dissociation constant for the membrane protein of 1. Demonstration that it can be used to efficiently produce one or more antibodies selected from the group consisting of antibodies of 0 ⁇ 10 ⁇ 8 M or less and antibodies that increase the overall thermal stability of membrane proteins and antibodies. Has been. Therefore, the antibody can be efficiently obtained by using this production method.
  • the present invention also provides a liposome / membrane protein complex comprising a solid phase carrier, a liposome bound to the solid phase carrier, and a membrane protein embedded in the liposome, Provided is a liposome / membrane protein complex that is used in screening for a compound in which at least a part of the membrane protein is exposed on the outer surface of the liposome and specifically binds to the loop portion of the membrane protein. .
  • this liposome / membrane protein complex can be used for screening a compound that specifically binds to the loop portion of the membrane protein in Examples described later. Therefore, if this liposome / membrane protein complex is used, a compound that specifically binds to the loop portion of the membrane protein can be efficiently obtained.
  • a method for screening an antibody comprising a step of producing a test antibody from antibody-producing cells, and examining the binding of the test antibody to the liposome / membrane protein complex
  • a method for screening a compound that specifically binds to a loop portion of a membrane protein comprising the step of selecting a liposome / membrane protein complex-binding antibody having binding property to the liposome / membrane protein complex.
  • this screening method can be used in the examples described later to screen for compounds that specifically bind to the loop portion of the membrane protein. Therefore, if this liposome / membrane protein complex is used, a compound that specifically binds to the loop portion of the membrane protein can be efficiently obtained.
  • a method for producing an antibody the step of screening a compound that specifically binds to a loop portion of a membrane protein by the screening method, and the loop portion of the membrane protein that has been screened.
  • a method for producing a compound that specifically binds to a loop portion of a membrane protein comprising the step of producing a replica of the compound that specifically binds to.
  • this production method can be used in the examples described later to efficiently produce a compound that specifically binds to the loop portion of the membrane protein. Therefore, if this production method is used, a compound that specifically binds to the loop portion of the membrane protein can be efficiently obtained.
  • an adenosine receptor A2a binding antibody (hereinafter referred to as “A2a receptor binding antibody”) that recognizes the three-dimensional structure of adenosine receptor A2a (hereinafter also referred to as “A2a receptor”). May be referred to). It has been demonstrated that this A2a receptor-binding antibody can be used for co-crystallization with the A2a receptor in Examples described later. Therefore, if this A2a receptor-binding antibody is used, it can be suitably used for crystallizing the A2a receptor.
  • the present invention also provides a sugar transporter-binding antibody that recognizes the three-dimensional structure of a sugar transporter. It has been demonstrated that this sugar transporter-binding antibody can be used for co-crystallization with a sugar transporter in Examples described later. Therefore, if this sugar transporter-binding antibody is used, it can be suitably used for crystallizing the sugar transporter.
  • an antibody that specifically recognizes the natural three-dimensional structure of the target membrane protein an antibody that has high binding specificity to the target membrane protein, an antibody that has high affinity for the target membrane protein, a membrane protein and an antibody Antibody that expands the entire hydrophilic region combining the antibodies, an antibody that increases the overall thermal stability of the membrane protein and antibody, an antibody that recognizes the loop portion of the membrane protein, or the three-dimensional structure of the loop portion of the membrane protein
  • liposome / membrane protein complexes that have been demonstrated to be used in screening antibodies that specifically recognize. Therefore, an antibody that can be suitably used for co-crystallization with a membrane protein can be obtained extremely efficiently as compared with known techniques.
  • a screening method for antibodies that can be suitably used for co-crystallization with membrane proteins can be assembled.
  • a screening system using a liposome / membrane protein complex, a production method, or a compound obtained by the production method can be obtained.
  • FIG. 1 is a conceptual diagram of the liposome-ELISA method.
  • FIG. 2 is a graph showing the results of measuring the binding state of mouse-derived anti-FLAG-M2 antibody (Sigma) to A2a receptor-reconstituted liposomes by liposome-ELISA method.
  • FIG. 3 is a comparison diagram of the antigen-antibody reaction between the liposome-ELISA method and the modified dot blotting method using the A2a receptor and the hybridoma supernatant.
  • FIG. 4 is a gel filtration elution profile of A2a receptor and mouse-derived Fab fragment.
  • FIG. 5 is a diagram showing the results of measuring the effect of increasing the thermal stability of the A2a receptor by the mouse-derived Fab fragment.
  • FIG. 1 is a conceptual diagram of the liposome-ELISA method.
  • FIG. 2 is a graph showing the results of measuring the binding state of mouse-derived anti-FLAG-M2 antibody (Sigma) to A2a receptor-reconstituted liposome
  • FIG. 6 is a schematic diagram of a chimeric A2a receptor.
  • FIG. 7 shows the result of confirming the expression results of a group of chimeric A2a receptors and their mutants by fluorescence detection. The lower part shows the results of detecting the antigen-antibody reaction against a group of chimeric A2a receptors and their mutants by Western blot.
  • FIG. 8 is a graph of fluorescent gel filtration analysis of human sugar nucleic acid transporter and recombinant Fab fragment complex.
  • FIG. 9 is a photograph of a complex crystal of A2a receptor and Fab.
  • FIG. 10 shows the crystal structure of the A2a receptor and Fab.
  • FIG. 11 is a photograph of a complex crystal of rat sugar transporter and Fv.
  • Non-Patent Document 1 has already revealed that the efficiency of crystallization increases when a membrane protein is co-crystallized with an antibody.
  • antibodies used for co-crystallization do not have any characteristics as long as they bind to membrane proteins, and there are antibodies that can and cannot co-crystallize.
  • the present inventors have studied a method for screening an antibody for co-crystallization with a membrane protein.
  • the following conditions were assumed as antibodies suitable for co-crystallization with membrane proteins.
  • the membrane protein / antibody complex to be used is preferably highly pure. Therefore, an antibody with high binding specificity is suitable for the antibody to be used.
  • the membrane protein contained in the produced crystal preferably has a natural three-dimensional structure. Therefore, an antibody used for co-crystallization with a membrane protein is suitably an antibody having high binding specificity for the natural three-dimensional structure of the membrane protein.
  • the antibody used for co-crystallization with the membrane protein needs to maintain the binding with the membrane protein during the steps of purification and crystallization. Therefore, an antibody having high affinity for the membrane protein is suitable as the antibody used for co-crystallization with the membrane protein.
  • a hydrophilic surface is essential for protein crystallization, but most of membrane proteins are covered with a surfactant, so that it is extremely difficult to crystallize compared to water-soluble proteins.
  • the antibody used is an antibody that expands the entire hydrophilic region of the membrane protein and the antibody.
  • the antibody used is preferably an antibody that increases the overall thermal stability of the membrane protein and the antibody.
  • the antibody used for co-crystallization with the membrane protein is suitable for the antibody that recognizes and binds to the natural three-dimensional structure of the membrane protein.
  • an antibody that recognizes and binds to the three-dimensional structure of the loop portion of the membrane protein is suitable.
  • the protein N-terminal and C-terminal regions are generally relatively flexible regions having a large fluctuation in the relative position and orientation relative to the transmembrane region in the solvent. Even if an antibody binds to such a region, it cannot form a regular crystal lattice that is regularly arranged in the crystal. Therefore, an antibody that binds near the protein N-terminus or C-terminus tends not to be suitable for crystallization. Because it is thought that there is.
  • antibody screening methods for obtaining antibodies used for co-crystallization were studied. And a liposome / membrane protein complex used for screening an antibody co-crystallizing with a membrane protein, a method for producing a liposome / membrane protein complex used for screening an antibody co-crystallizing with a membrane protein, and a membrane A screening method for antibodies co-crystallized with proteins and a screening kit for antibodies co-crystallized with membrane proteins were clarified, and the present invention was completed.
  • GPCR G protein coupled receptor
  • Transporter protein is a general term for proteins that penetrate biological membranes and transport substances through the membrane.
  • the lipophilic low-molecular compound moves spontaneously (according to the concentration gradient) from the high concentration side to the low concentration side through the biological membrane. However, less lipophilic substances do not move as such. Further, the movement from the low concentration side to the high concentration side (against the concentration gradient) does not proceed spontaneously, and free energy must be supplied.
  • Membrane transporters are responsible for these involuntary transports. Transport by membrane transporters is divided into two as described above, and among these, the transfer of substances with low lipophilicity is called accelerated diffusion (a type of passive transport). Moreover, the movement from the low concentration side which requires energy to the high concentration side is called active transport.
  • the liposome-ELISA method applies the principle of a general ELISA method (Enzyme-Linked ImmunoSorbent Assay), immobilizes a liposome / membrane protein complex on a solid phase carrier, and reacts with the membrane protein. This is a method for screening a test compound. A conceptual diagram is shown in FIG.
  • the liposome / membrane protein complex is a liposome / membrane protein complex used for screening an antibody that co-crystallizes with a membrane protein, and binds to the solid phase carrier and the solid phase carrier. And a membrane protein embedded in the liposome, and at least a part of the membrane protein is exposed on the outer surface of the liposome. Then, a liposome-ELISA method using the liposome / membrane protein complex has an effect of screening a monoclonal antibody that specifically recognizes the natural three-dimensional structure of the membrane protein.
  • the membrane protein is thought to maintain the natural three-dimensional structure in the liposome, and many of the monoclonal antibodies obtained by the liposome-ELISA method recognize the natural three-dimensional structure of the membrane protein. It is because it is considered.
  • the detection operation accompanied with the denaturation of the membrane protein such as the denaturing dot blotting method is effective in screening the monoclonal antibody specifically recognizing the natural three-dimensional structure of the membrane protein with higher accuracy. .
  • the membrane protein is not limited as long as it is a protein that interacts with the membrane, and includes, for example, a receptor protein, a channel protein, a transporter protein, an adhesion molecule, or a membrane-bound enzyme.
  • a receptor protein or a transporter protein when used, an antibody that can be used for co-crystallization is obtained, so that the receptor protein or the transporter protein is preferable.
  • a receptor protein is more preferable because many drugs targeting the receptor protein have been developed and are important targets for medical and pharmaceutical purposes.
  • GPCR is preferred because it is the most abundant receptor protein and is an important target for medical and pharmaceutical purposes.
  • the membrane protein is not limited in the number of times it has penetrated the membrane, and includes a membrane protein that is 1 to 20 times transmembrane. From the examples described later, when a membrane protein that has passed through the membrane 7 or 10 times is used, an antibody that can be used for co-crystallization with the membrane protein is obtained.
  • the number of times is preferably 10 times or less, more preferably 7 times and 10 times.
  • the constituent material of the liposome is not particularly limited as long as it does not inhibit the formation of the lipid bilayer.
  • phospholipid protein, nucleic acid, sugar chain, biotinylated lipid, lectin, membrane stabilizer , Antioxidants, charged substances and the like.
  • the phospholipids any of various commonly used phospholipids can be used.
  • phospholipids such as cardiolipin, or hydrogenated products thereof.
  • glycolipids such as cephalin, cerebroside, ceramide, sphingomyelin, ganglioside, etc., and one or more of these can be used.
  • the phospholipid may be egg yolk, soybean or other natural lipid derived from animals and plants (eg, egg yolk lecithin, soybean lecithin, etc.), synthetic lipid or semi-synthetic lipid.
  • the constituent material of the liposome may include a linker that binds to the solid phase carrier.
  • the linker include biotin, avidin, streptavidin, enzyme, substrate, coenzyme, prosthetic group, antibody, antigen, nucleic acid, aptamer, polysaccharide, lectin, metal ion, metal chelator, receptor, and binding to them A compound or a compound fragment selected from the group consisting of functional compounds. In view of excellent binding ability, biotin or avidin is preferable.
  • the solid phase carrier is a solid support insoluble in the reaction medium for supporting the liposome / membrane protein complex immobilized by the method of the present invention.
  • the shape can be any shape such as a plate, bead, tube or fiber.
  • a material for the solid phase carrier glass, plastic, metal or the like can be used. Since the antibody which can be used for co-crystallizing with the membrane protein is obtained from the examples described later, the effect of the present invention is most exhibited in the case of plastic.
  • a thermoplastic resin with a small amount of fluorescence generation is preferable in order to suppress the background.
  • linear polyolefins such as polyethylene and polypropylene, cyclic polyolefins, fluorine-containing resins, and the like are preferably used, and saturated cyclic polyolefins that are particularly excellent in heat resistance, chemical resistance, low fluorescence, and moldability are more preferably used.
  • the saturated cyclic polyolefin refers to a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and an ⁇ -olefin.
  • the solid phase carrier may include a linker-binding compound that binds to the linker.
  • a linker-binding compound that binds to the linker.
  • biotin, avidin, streptavidin, enzyme, substrate, coenzyme, prosthetic group, antibody, antigen, nucleic acid, aptamer, polysaccharide, lectin, metal ion, metal chelator, receptor, compound having binding ability to them A compound selected from the group consisting of: In view of excellent binding ability, biotin or avidin is preferable.
  • antibody refers to a molecule that can specifically bind to a specific epitope on an antigen, and includes monoclonal antibodies.
  • antibodies can exist in various forms, such as Fv, Fab, F (ab ′) 2 , single chain antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies and the like. It is done.
  • the “natural three-dimensional structure of the membrane protein” includes the same three-dimensional structure as the three-dimensional structure in which the membrane protein is functioning normally in the living body, or substantially the same three-dimensional structure.
  • bond means a connection between substances.
  • the linkage may be either covalent or non-covalent, and includes, for example, ionic bonds, hydrogen bonds, hydrophobic interactions, or hydrophilic interactions.
  • embedded includes a state in which a part or all of another substance is inserted in one substance.
  • crystallization means that a protein is precipitated as crystals. Crystals obtained by crystallization can be analyzed for X-ray crystal structure analysis to analyze the three-dimensional structure of the protein from an X-ray diffraction image scattered by applying X-rays. It is generally preferable that the crystal used for the analysis of the three-dimensional structure of the protein has high purity [Ferre-D'Amare AR, Burley SK, Structure, 357-359 (1994)]. The result of analyzing the three-dimensional structure of a protein can be used, for example, in drug design (SBDD: Structure Based Drug Design) based on a protein three-dimensional structure.
  • SBDD Structure Based Drug Design
  • Protein crystallization methods include a static batch method, a free interface diffusion method, a microdialysis method, a vapor diffusion method, and the like.
  • the vapor diffusion method is a method in which a protein solution containing a precipitant is placed in a container containing a buffer solution containing a higher concentration of precipitant, and crystallization is promoted by a difference in saturated vapor pressure in an enclosed space. It is. Depending on the arrangement of the solution, it can be classified into hanging drop method, sitting drop method and sandwich drop method. Commercially available precipitating agents or crystallization kits can also be used.
  • co-crystallization means to crystallize a protein and the compound together in a state where the compound is bound to the protein or in a state where the compound is mixed in an aqueous solution containing the protein. Including. For example, it includes crystallizing the membrane protein together with an antibody against the membrane protein bound thereto.
  • the method for producing a liposome / membrane protein complex is a method for producing a liposome / membrane protein complex used for screening an antibody that co-crystallizes with a membrane protein, and includes a) a lipid solution and a linker.
  • a step of preparing an aqueous solution containing the membrane protein and the surfactant and b) adding a surfactant removing agent to the mixture, and adding the surfactant in the mixture to the surfactant Adsorbing to a surfactant remover to produce a liposome embedded with the linker and the membrane protein; and c) binding the liposome to the solid phase carrier via the linker, Producing a body.
  • membrane proteins are considered to maintain their natural three-dimensional structure in liposomes, and many of the compounds obtained by the liposome-ELISA method recognize the natural three-dimensional structure of membrane proteins. This is because it is considered. Furthermore, by performing a detection operation accompanied by membrane protein denaturation such as a denaturing dot blotting method, there is an effect that an antibody that specifically recognizes the natural three-dimensional structure of the membrane protein can be screened with high accuracy.
  • the surfactant can solubilize the membrane protein by contacting the membrane protein in an aqueous solution. Liposomes can also be formed. It is desirable that the surfactant is appropriately selected and used depending on the purpose of the experiment, and any conventionally known surfactant can be used. Commonly used surfactants are roughly classified into nonionic, anionic and zwitterionic depending on their electrical properties.
  • Nonionic surfactants include digitonin, polyoxyethylene alkyl ether (Brij type), polyoxyethylene sorbitan (Tween type), ⁇ -dodecyl maltoside, ⁇ -octyl glucoside, ⁇ -nonyl glucoside, ⁇ -heptylthio Examples thereof include glucoside, ⁇ -octylthioglucoside, sucrose monodecanoate, sucrose monododecanoate, octyltetraoxyethylene, octylpentaoxyethylene, and dodecyloctaoxyethylene.
  • the anionic surfactant include taurodeoxycholic acid.
  • Zwitterionic surfactants include N, N-dimethyldecylamine N-oxide, N, N-dimethyldodecylamine N-oxide, N, N-dimethyldodecylammoniopropane sulfonate, and (3-[(3-col Amidopropyl) -dimethylammonio] -1-propanesulfonate (CHAPS).
  • the “surfactant removing agent” includes a substance having a property of adsorbing the surfactant in the sample solution.
  • a method of gradually adsorbing and removing a surfactant by adding it to a sample solution over time is known.
  • the screening method using the liposome / membrane protein complex is a screening method for an antibody that co-crystallizes with a membrane protein, and is used when screening an antibody that co-crystallizes with a membrane protein.
  • the step of detecting includes a detection system such as a dot blotting method, a Western blotting method, an immunoblotting method, or an ELISA method.
  • a detection system such as a dot blotting method, a Western blotting method, an immunoblotting method, or an ELISA method.
  • the dot blotting method or the Western blotting method is preferable.
  • a dot blotting method with a simple operation is more preferable.
  • the test antibody includes a culture supernatant of a hybridoma.
  • the test antibody may be a polyclonal antibody or a monoclonal antibody.
  • the test antibody can exist in various forms, for example, Fv, Fab, F (ab ′) 2 , single chain antibody, chimeric antibody, humanized antibody, human antibody, bispecific antibody, etc. Is mentioned.
  • the test antibody may be a mixture of one or more compounds selected from the above test antibody group. In order to eliminate complicated steps such as antibody isolation and purification and increase efficiency, it is preferable to be a hybridoma culture.
  • the culture supernatant of hybridomas can be prepared by using a method as described in Kohler and Milstein, Nature, 256: 495 (1975).
  • mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to generate antibodies that specifically bind to the immunizing agent or to generate lymphocytes that can be generated.
  • Lymphocytes can also be immunized in vitro.
  • the immunizing agent typically comprises a membrane protein polypeptide or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used when non-human mammalian sources are desired.
  • lymphocytes are fused with immortalized cell lines using a suitable fusing agent such as polyethylene glycol to form hybridoma cells
  • a suitable fusing agent such as polyethylene glycol
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells from rodents, cows and humans. Usually, rat or mouse myeloma cell lines are used.
  • the hybridoma cells are preferably cultured in a suitable culture medium containing one or more substances that inhibit the survival or growth of unfused, immortalized cells.
  • the culture medium of the hybridoma typically contains hypoxatin, aminoptylline and thymidine (“HAT medium”). ), This substance prevents the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level antibody expression by selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • a more preferred immortalized cell line is a mouse myeloma line, which is available from, for example, the Salk Institute, Cell, Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia.
  • Human myeloma and mouse-human heteromyeloma cell lines for generating human monoclonal antibodies have also been described [Kozbor, J. et al. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal ti Antibodies Production Techniques M and Applications, Marcel Deker, Inc. , New York, (1987) pp. 1 51-63].
  • the culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against membrane proteins.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is preferably determined by in vitro binding assays such as immunoprecipitation or radioimmunoassay (RIA) or enzyme linked immunoassay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of a monoclonal antibody is described, for example, in Munson and Pollard, Anal. Biochem. , 107: 220 (1980).
  • the clones can be subcloned by limiting dilution and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown as ascites in vivo in a mammal.
  • test antibody can be a culture supernatant of a hybridoma that can be obtained during these steps.
  • the monoclonal antibody secreted by the subclone can be obtained from a culture medium or ascites fluid by a conventional immunoglobulin purification method such as protein A-Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography. Can be isolated or purified.
  • a conventional immunoglobulin purification method such as protein A-Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography.
  • Monoclonal antibodies can then be produced by recombinant DNA methods such as those described in US Pat. No. 4,816,567.
  • the DNA encoding the monoclonal antibody of the present embodiment is obtained using a conventional method (for example, using an oligonucleotide probe capable of specifically binding to the gene encoding the heavy and light chains of the mouse antibody), It can be easily isolated and sequenced.
  • the hybridoma cells of the present invention are a preferred source of such DNA.
  • the DNA can be incorporated into expression vectors that are transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins.
  • Monoclonal antibodies can be synthesized in recombinant host cells.
  • DNA can be obtained, for example, by replacing the coding sequences of human heavy and light chain constant domains in place of homologous mouse sequences [US Pat. No. 4,816,567; Morrison et al., Supra], or immunoglobulin coding.
  • the sequence can be modified by covalently linking part or all of the coding sequence of the non-immunoglobulin polypeptide.
  • Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention, or can be substituted for the variable domains of one antigen binding site of the antibodies of the invention, producing chimeric bivalent antibodies.
  • the antibody may be a monovalent antibody.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is generally cleaved at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted or deleted with other amino acid residues to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using standard techniques known in the art.
  • a humanized form of a non-human (eg mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (eg Fv, Fab, F (ab ′) 2 or other antigen binding subsequence of an antibody), It contains the minimum sequence derived from non-human immunoglobulin.
  • a humanized antibody is a CDR residue of a non-human species (donor antibody) in which the complementarity determining region (CDR) of the recipient has the desired specificity, affinity and ability, such as mouse, rat or rabbit.
  • Human immunoglobulin substituted by In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody has at least one, typically all or almost all CDR regions corresponding to those of non-human immunoglobulin and all or almost all FR regions of human immunoglobulin consensus sequences, typically Contains substantially all of the two variable domains.
  • a humanized antibody optimally comprises an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992)].
  • Fc immunoglobulin constant region
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • human antibodies can be obtained from phage display libraries [Hoogenboom and Winter, J. et al. Mol. Biol. 227: 381 (1991); Marks et al. Mol. Biol. , 222: 581 (1991)] and can be made using various methods known in the art. Techniques such as Cole et al. And Boerner et al. Can also be used for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies Cancer Al Cancer Al Alan R. Liss. P. 77 (1985); , 147 (1): 86-95 (1991)).
  • human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, eg, introducing endogenous immunoglobulin genes into mice with partial or complete inactivation. Upon administration, human antibody production is observed that is very similar to that found in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425. No.
  • antibodies may be affinity matured using the known selection and / or mutagenesis methods described above.
  • Preferred affinity matured antibodies are 5 times, more preferably 10 times, more preferably 20 or 30 times more affinities than that of the starting antibody from which the mature antibody was prepared (generally mouse, humanized or human).
  • Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. In this case, one of the binding specificities is for a membrane protein and the other is for any other antigen, preferably a cell surface protein or receptor or receptor subunit.
  • bispecific antibodies are well known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305: 537-539 (1983)]. Because of the random collection of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. . Purification of the correct molecule is usually accomplished by an affinity chromatography step. A similar procedure is described in WO 93/08829 published May 13, 1993, and Traunecker et al., EMBO J. et al. 10: 3655-3659 (1991).
  • An antibody variable domain having a desired binding specificity can be fused to an immunoglobulin constant domain sequence.
  • the fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred that at least one fusion has a first heavy chain constant region (CH1) containing the site necessary for light chain binding.
  • CH1 first heavy chain constant region
  • the interface between a pair of antibody molecules can be manipulated to maximize the proportion of heterodimers recovered from recombinant cell culture.
  • a suitable interface includes at least a portion of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan).
  • a complementary “cavity” of the same or similar size as the large side chain is created at the interface of the second antibody molecule where the large amino acid side chain is replaced with a smaller one (eg, alanine or threonine). This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) 2 bispecific antibodies). Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a procedure for proteolytically cleaving intact antibodies to produce F (ab ′) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The produced Fab fragment is then converted to a thionitrobenzoate (TNB) derivative.
  • TAB thionitrobenzoate
  • One of the Fab-TNB derivatives is then reconverted to Fab-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab-TNB derivative to form a bispecific antibody.
  • the produced bispecific antibody can be used as a drug for selective immobilization of an enzyme.
  • Fab fragments can be recovered directly from E. coli, which can be chemically coupled to form bispecific antibodies.
  • Shalaby et al. Exp. Med. 175: 217-225 (1992) describe the preparation of two fully humanized bispecific antibody F (ab ') molecules.
  • Each Fab fragment is secreted separately from E. coli and undergoes directed chemical coupling in vitro to form bispecific antibodies.
  • the bispecific antibody thus formed is capable of binding to cells overexpressing ErbB2 receptor and normal human T cells, and induces cytolytic activity of human cytotoxic lymphocytes against human breast tumor targets. Become.
  • bispecific antibodies have been produced using leucine zippers.
  • Leucine zipper peptides from Fos and Jun proteins are linked to the Fab portions of two different antibodies by gene fusion.
  • Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. Hollinger et al., Proc. Natl. Acad. Sci.
  • a fragment consists of a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is short enough to allow pairing between two domains on the same chain.
  • VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of the other fragment to form two antigen binding sites.
  • sFv single chain Fv
  • More than bivalent antibodies are also contemplated.
  • trispecific antibodies can be prepared. Tutt et al. Immunol. 147: 60 (1991).
  • bispecific antibodies can bind to two different epitopes of the membrane protein polypeptide provided herein.
  • the anti-membrane protein polypeptide arm is a trigger molecule on leukocytes such as a T cell receptor molecule (eg, CD2, CD3, CD28, or B7) so as to concentrate the cellular defense mechanism on a specific membrane protein polypeptide expressing cell.
  • leukocytes such as a T cell receptor molecule (eg, CD2, CD3, CD28, or B7) so as to concentrate the cellular defense mechanism on a specific membrane protein polypeptide expressing cell.
  • Fc ⁇ R Fc receptor for IgG
  • Bispecific antibodies can also be used to localize cytotoxic agents to cells that express specific membrane protein polypeptides.
  • bispecific antibodies of interest bind to the membrane protein polypeptide and can further bind to tissue factor (TF).
  • TF tissue factor
  • the test antibody may be optimized with respect to the target membrane protein in advance using various methods known in this field using an antibody library.
  • various methods known in this field for example, a phage display method can be used.
  • Use of an optimized test antibody has an advantage that a test antibody with high binding specificity and affinity can be obtained more efficiently.
  • the “phage display method” includes a technique for displaying the mutant polypeptide on the particle surface of a phage, for example, a filamentous phage, as a fusion protein to a coat protein.
  • a phage for example, a filamentous phage
  • a fusion protein to a coat protein a phage display technique.
  • phage display is that large libraries of randomized protein variants can be quickly and efficiently screened for those sequences that bind to target molecules with high affinity. Display on phage of peptide and protein libraries has been used to screen millions of polypeptides for those with specific binding properties.
  • Multivalent phage display methods have been used to display small random peptides and small proteins, typically by fusing them to the filamentous phage PIII or PVIII. Wells and Lowman, Curr. Opin. Struct. Biol.
  • phage display In monovalent phage display, a protein or peptide library is fused to a phage coat protein or portion thereof and expressed at low levels in the presence of wild type protein. The binding activity effect is reduced for multivalent phage, whereby the selection is based on the inherent ligand affinity and a phagemid vector is used that simplifies DNA manipulation. Lowman and Wells, Methods: A companion Methods in Enzymology, Vol. 3, 205-0216 (1991). Phage display also includes techniques for generating antibody-like molecules (C. Janeway, P. Travers, M. Walport, Shromchik, (2001) Immunobiology, 5th Edition, Garland Publishing, New York, 627-). 628).
  • phagemid includes a bacterial replication origin, for example, ColE1, and a plasmid vector having a copy of the intergenic region of bacteriophage.
  • the phagemid can be used for any known bacteriophage, including filamentous bacteriophages and lambda-based bacteriophages.
  • the plasmid will also generally contain a selectable marker for antibiotic resistance. Segments of DNA cloned into these vectors can be propagated as plasmids. When cells encapsulating these vectors are given all the genes necessary for the production of phage particles, the replication pattern of this plasmid changes to rolling circle replication, producing a single-stranded copy of the plasmid DNA. And packaging the phage particles.
  • Phagemids can form infectious or non-infectious phage particles.
  • the term includes a phage coat protein gene or fragment thereof that binds to a heterologous polypeptide gene as a gene fusion so that the heterologous polypeptide appears on the surface of the phage particle.
  • the membrane protein contained in the liposome / membrane protein complex may be cleaved so that the N-terminal region is shortened.
  • an antibody that binds to a loop portion of a membrane protein can be suitably used for co-crystallization with the membrane protein. This is because, when the N-terminal region protruding from the membrane or liposome is short, the possibility that the antibody binds to the N-terminal region is low, and the possibility that the antibody binds to the loop portion of the membrane protein is considered high.
  • the “step of denaturing the membrane protein” is not particularly limited as long as the membrane protein can be denatured.
  • a compound having an effect of denaturing a protein such as a surfactant or urea may be brought into contact, and a step of denaturing by applying heat or pressure may be included.
  • SDS sodium lauryl sulfate
  • Another embodiment of the present invention is an antibody production method for screening an antibody for co-crystallization with a membrane protein by the screening method described above, and for co-crystallization with the screened membrane protein.
  • a method for producing an antibody for co-crystallization with a membrane protein, comprising the step of producing a replica of the antibody of By using this production method, as demonstrated in the examples described later, it is possible to efficiently produce antibodies for co-crystallization with membrane proteins.
  • ⁇ Embodiment 4 Method for screening antibody that recognizes loop portion of membrane protein by liposome-ELISA method, etc.> Another embodiment of the present invention is a liposome / membrane protein complex used for screening an antibody that recognizes a loop portion of a membrane protein or a three-dimensional structure thereof (hereinafter also referred to as “loop portion recognition antibody”).
  • an antibody that specifically recognizes the three-dimensional structure of the loop portion of the membrane protein can be screened with high accuracy.
  • an antibody recognizes a loop part of a membrane protein it is preferable to recognize a three-dimensional structure rather than a primary structure. This is because an antibody that recognizes the three-dimensional structure of a membrane protein can be expected to have more excellent functions such as affinity.
  • a test antibody is produced from antibody-producing cells, and the binding property of the test antibody to the liposome / membrane protein complex is examined, and the liposome / membrane protein complex is obtained.
  • a method for screening a loop portion-recognizing antibody comprising a step of selecting a liposome / membrane protein complex-binding antibody having binding property to a body. By utilizing this screening method, it is possible to efficiently screen for loop portion recognition antibodies as demonstrated in the examples described later.
  • the antibody-producing cell includes a cell capable of producing an antibody against the membrane protein constituting the liposome / membrane protein complex.
  • the screening method for the loop portion recognition antibody includes a step of modifying the membrane protein or a replica thereof to prepare a modified membrane protein, and the test antibody or the liposome / membrane protein complex against the modified membrane protein. A step of examining the binding property of the body-bound antibody, and selecting an antibody having binding property to the liposome / membrane protein complex and not binding to the modified membrane protein. . If this screening method is used, antibodies that recognize the three-dimensional structure of the loop portion of the membrane protein can be screened with high accuracy, as demonstrated in Examples described later.
  • Another embodiment of the present invention is a method for producing an antibody, the step of screening a loop portion recognition antibody by the above screening method, the step of producing a replica of the screened loop portion recognition antibody, A method for producing a loop portion recognition antibody. If this production method is used, a loop part recognition antibody can be produced efficiently as demonstrated in the examples described later. For example, in Example (3-3-1), which will be described later, a replica of the loop-recognizing antibody selected by screening is produced by ascites in a mouse. Since this replica has the same or substantially the same characteristics as the antibody selected by screening, the loop part recognition antibody can be efficiently produced by this production method.
  • the replica may be a replica produced by using the antibody-producing cells used in the screening method for loop portion recognition antibody.
  • a copy of the selected loop portion-recognizing antibody can be produced from the cells already owned by the researcher, so it can be said that this is an efficient production method.
  • the cell used for ascites is one of the hybridomas used in the preceding step (2-1).
  • the replica is also referred to as a polynucleotide encoding the amino acid sequence of the loop portion recognition antibody (hereinafter referred to as “functional polynucleotide”) contained in the antibody-producing cells used in the screening method for the loop portion recognition antibody. Or a functional mutant of the same) or a replica produced using a cell into which the functional variant is introduced.
  • This production method is an efficient production method. Moreover, if this production method is used, it is possible to use desired cells, such as cells with high antibody production efficiency, as cells that produce antibodies.
  • the functional polynucleotide or the amino acid sequence of the loop portion recognition antibody can be obtained from the antibody-producing cells used in the screening method for the loop portion recognition antibody using a conventional technique such as PCR.
  • “functional variant” means a compound composed of amino acids or nucleotides in a replica.
  • the loop part recognition antibody as described above can be suitably used as an antibody for co-crystallization with a membrane protein.
  • the protein N-terminal and C-terminal regions are generally relatively flexible regions that are largely fluctuated in the relative position and orientation with respect to the transmembrane region in the solvent. Even if an antibody binds to such a region, it cannot form a regular crystal lattice regularly arranged in the crystal, and an antibody that binds near the protein N-terminus or C-terminus tends to be unsuitable for crystallization. it is conceivable that.
  • the loop portion recognition antibody can be suitably used as an antibody for co-crystallization with the antibody.
  • the above-mentioned loop-part recognition antibody affects the function of the membrane protein by binding to the loop part or its three-dimensional structure.
  • the loop part binding compound affects the function of the membrane protein
  • the above-mentioned loop part recognition antibody can be used as a pharmaceutical or a candidate product utilizing the effect.
  • the “membrane protein loop portion” refers to a region connecting two transmembrane regions existing in the membrane protein and a region not buried in the membrane.
  • the “antibody-producing cell” is not particularly limited as long as it has antibody-producing ability.
  • it may be a human or other mammals (eg, rat, mouse, rabbit, cow, monkey, pig, horse, sheep, goat, dog, cat, guinea pig, hamster, etc.).
  • mammalian cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (CHO cell), dhfr gene-deficient Chinese hamster cell CHO (CHO (dhfr) cell), mouse L cell, mouse AtT-20, mouse Examples include myeloma cells, rat GH3, human FL cells, and human HEK293 cells.
  • the antibody-producing cells may be microorganisms (Escherichia, Bacillus, yeast, etc.), bird cells, or insect cells.
  • introduction of a polynucleotide encoding an antibody into a cell and production of the antibody can be performed according to methods known in the art.
  • a calcium phosphate method, lipofection method, electroporation method, adenovirus method, retrovirus method, or microinjection can be used as a method for introducing a polynucleotide into a cell [Revised 4th edition Xinjiang Genetic Engineering Handbook, Sheep Tsuchiya (2003): 152-179.].
  • Examples of production methods using antibody cells include, for example, [Protein Experiment Handbook, Yodosha (2003): 128-142.], [Shimamoto et al., Biologicals. 2005 Sep; 33 (3): 169-174.
  • the polynucleotide can be used in a form generally called a vector or a plasmid.
  • a plasmid for example, a plasmid derived from E.
  • coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast eg, pSH19, pSH15
  • bacteriophages such as lambda phage
  • animal viruses such as retroviruses, vaccinia viruses, baculoviruses, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.
  • antibodies produced using cells can be purified using methods known in the art.
  • antibody purification methods include ammonium sulfate or ethanol precipitation, protein A, protein G, gel filtration chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl chromatography, It can be achieved by using apatite chromatography, lectin chromatography and the like (Protein Experiment Handbook, Yodosha (2003): 27-52.).
  • the “replica” means that there is a compound (hereinafter sometimes referred to as “parent compound”) that triggers the creation of a copy, and the specific properties of the parent compound are compared.
  • parent compound is an antibody, protein, nucleic acid, sugar chain, or low molecular weight compound
  • the corresponding replica is in the form of an antibody, protein, nucleic acid, sugar chain, or low molecular weight compound.
  • the parent antibody is an antibody that recognizes the loop portion of the membrane protein
  • the replica is also an antibody that recognizes the loop portion of the membrane protein.
  • the structure of the duplicate is not particularly limited as long as it is within the above definition.
  • a compound is a substance made of two or more elements.
  • the parent compound is an antibody (hereinafter also referred to as “parent antibody”) and the replica is an antibody
  • the specific properties of the parent antibody and the replica are compared or substantially the same.
  • the amino acid sequence of the replica is not limited.
  • the heavy chain CDR of the replica has 80% or more homology with respect to the heavy chain CDR of the parent antibody.
  • the heavy chain CDR of the replica may have an amino acid sequence in which one or several bases are deleted, substituted or added in the amino acid sequence of the heavy chain CDR of the parent antibody.
  • the heavy chain CDR of the replica is encoded by the base sequence of a nucleic acid that hybridizes under stringent conditions to a nucleic acid consisting of a base sequence complementary to the base sequence encoding the heavy chain CDR of the parent antibody. It may have an amino acid sequence. This is because the heavy chain CDR is the most important site for determining the specificity of an antibody for an antigen. In addition, there is a functional difference between antibodies having a certain similar structure or more as in the above conditions. This is because it is known that it is difficult.
  • the amino acid sequence of the antibody light chain CDR, heavy chain FR, light chain FR, etc. is also a region that can be involved in the characteristics of the antibody, although not as much as the above heavy chain CDR.
  • the corresponding region has a certain structure or more.
  • the structure of the antibody is represented here by an amino acid sequence, the same applies basically even when represented by a base sequence. It is known that a base sequence encoding an antibody or protein can take a wider range than an amino acid sequence due to degeneracy.
  • the above “80% or more” is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more. This is because the higher the homology is, the closer the parent compound and the duplicate have characteristics.
  • the “one or several” is preferably 50 or less, more preferably 30 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. More preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because the smaller the number of deletions, substitutions or additions, the closer the parent compound and the duplicate have characteristics.
  • an antibody is taken as an example of a replica. However, even if it is a nucleic acid, peptide, polypeptide, sugar chain, or low molecular weight compound, the replica and the parent compound must have a certain structure or more. Is preferred.
  • amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) having carboxylic acids and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), and aromatic-containing sides Amino acids having a chain (H, F, Y, W) can be mentioned (the parentheses indicate single letter symbols of amino acids).
  • the replica also includes a derivative of the parent compound within the above definition.
  • a “derivative” refers to a degree that does not significantly change the structure and properties of a matrix by introducing a functional group, oxidation, reduction, substitution of atoms or addition of atoms when an organic compound is considered as a matrix. Means a compound having been modified. The modification may be performed as an actual chemical reaction, but it may be performed on a desk.
  • the derivative includes a salt, solvate, isomer, prodrug, and the like (for example, ester) of the parent compound.
  • the derivatives include any other compound that is provided (directly or indirectly) with the parent compound or an active metabolite thereof or residue thereof when administered to a subject.
  • Such a derivative can be obtained by those skilled in the art without undue experimentation.
  • [Burger's Medicinal Chemistry And Drug Discovery, 5 th Edition, Vol 1: Principles and Practice] can refer to.
  • the derivative is preferably a parent salt or solvate.
  • the above derivatives are preferably pharmacologically acceptable derivatives.
  • the “salt” is not particularly limited, but is, for example, an anionic salt formed with an arbitrary acidic (for example, carboxyl) group or a cationic salt formed with an arbitrary basic (for example, amino) group.
  • Salts include inorganic and organic salts, including those described in [Berge, Bighley and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19].
  • a “solvate” is a compound formed by a solute and a solvent. As for the solvate, for example, [J. Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953)] can be referred to. If the solvent is water, the solvate formed is a hydrate.
  • This solvent is preferably one that does not interfere with the biological activity of the solute.
  • preferred solvents include, without limitation, water, ethanol, and acetic acid. The most preferred solvent is water.
  • “isomer” includes molecules having the same molecular formula but different structures. Includes enantiomers (enantiomers), geometric (cis / trans) isomers, or isomers having one or more asymmetric centers that are not mirror images of one another (diastereomers).
  • a “prodrug” is a compound that is a precursor and undergoes a chemical change by metabolic processes or various chemical reactions when the compound is administered to a subject, and the parent compound or a salt thereof or a salt thereof Includes compounds that yield solvates.
  • prodrugs for example, [T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, A.C.S. Symposium Series, Volume 14] can be referred to.
  • the derivative may have a structure of 80% or more when the similarity (tanimoto coefficient) of PubChem's Compound Search is applied to the parent compound.
  • the lower limit of the similarity included in the derivative is not particularly limited, but is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more. is there. This is because the higher the similarity is, the closer the parent compound and the duplicate have characteristics.
  • the tanimoto coefficient calculation formula is a similarity coefficient between the authoritative compounds most frequently used in the field of chemoinformatics. The tanimoto coefficient calculation formula can be referenced from the PubChem website (http://pubchem.ncbi.nlm.nih.gov/).
  • homology is the ratio of the number of identical amino acids in two or more amino acid sequences calculated according to a method known in the art. Before calculating the ratio, the amino acid sequences of the amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence if necessary to maximize the same ratio. Nor do any conservative substitutions be considered identical. Further, it means the ratio of the same number of amino acids to all amino acid residues including overlapping amino acids in an optimally aligned state. Alignment methods, ratio calculation methods, and related computer programs are well known in the art and use common sequence analysis programs (eg GENETYX, GeneChip Sequence Analysis, etc.) Can be measured. In addition, “homology” is obtained by calculating the ratio of the same base in two or more DNA strands or two or more RNA strands according to a method known in the art as described above. is there.
  • stringent conditions means, for example, (1) low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.005 at 50 ° C.
  • 1% sodium dodecyl sulfate 1% sodium dodecyl sulfate, (2) denaturing agents such as formamide during hybridization, eg 50% (vol / vol) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 42 ° C.
  • hybridization as applied to a polynucleotide means a property that allows pairing between nucleotides by hydrogen bonding between nucleotide bases.
  • Base pairs can occur in Watson-Crick base pairs, Hoogsteen base pairs, or any other sequence specific form.
  • recognition in an antigen-antibody reaction can be used in the meaning normally used in the field of antibody engineering. For example, it includes the act of contacting the antibody for binding.
  • “having no binding” can be used in the meaning normally used in the field of biotechnology. It also means that the binding is remarkably low, or the amount of binding is so low that it can be ignored in in vitro experiments.
  • the number of molecules that the test substance binds to the target substance is significantly less than that of the control compound having the binding ability to the target substance. Including cases.
  • the degree of “remarkably” depends on the method for investigating the number of molecules, but may be set to 0.5 times or less, 0.3 times or less, or 0.1 times or less, for example. Alternatively, it may be set to 0.05 times or less, or may be set to 0.01 times or less.
  • Examples of the method for investigating the number of molecules include Western blotting, mass spectrometry, and intermolecular interaction analysis. Or, when the test substance does not have binding properties to the target substance, whether the number of molecules that the test substance binds to the target substance is so small that a significant difference is observed compared to the control compound. You may judge with. For the determination of whether there is a significant difference, for example, when it can be assumed that the population follows a normal distribution, it is preferable if there is a significant difference in Student's t-test, which is a parametric test.
  • one-sided test should be p ⁇ 0.05, more preferably one-sided test should be p ⁇ 0.03, and most preferably one-sided test should be p ⁇ 0.01. Good.
  • the student t-test is not limited to a one-sided test, but may be a two-sided test. Further, when it cannot be assumed that the population follows a normal distribution, the presence or absence of a significant difference may be tested by performing a Mann-Whitney U test as a nonparametric test.
  • ⁇ Embodiment 5 Functional antibody screening method by liposome-ELISA method, etc.> Another embodiment of the present invention is a liposome / membrane protein complex comprising a solid phase carrier, a liposome bound to the solid phase carrier, and a membrane protein embedded in the liposome, At least a part of the membrane protein is exposed on the outer surface of the liposome, and is a liposome / membrane protein complex used for screening functional antibodies.
  • the above-mentioned functional antibody is an antibody having a binding property to the wild type of the membrane protein and having no binding property to the loop partial mutant of the membrane protein, and a dissociation constant for the membrane protein is 1.
  • This liposome / membrane protein complex can be suitably used for screening functional antibodies, as demonstrated in the examples described later.
  • a test antibody is produced from antibody-producing cells, and the binding property of the test antibody to the liposome / membrane protein complex is examined, and the liposome / membrane protein complex is obtained.
  • a method for screening a functional antibody comprising a step of selecting a liposome / membrane protein complex-binding antibody having binding property to a body. By using this screening method, functional antibodies can be efficiently screened as demonstrated in the examples described later.
  • the functional antibody screening method includes a step of modifying the membrane protein or a replica thereof to prepare a modified membrane protein, and the test antibody or the liposome / membrane protein complex against the modified membrane protein.
  • the method may further comprise a step of examining the binding property of the binding antibody and selecting an antibody having binding property to the liposome / membrane protein complex and not binding to the modified membrane protein.
  • Another embodiment of the present invention is a method for producing an antibody, the method comprising screening a functional antibody by the screening method described above, and producing a replica of the screened functional antibody.
  • This is a method for producing a functional antibody. If this production method is used, a functional antibody can be produced efficiently as demonstrated in the examples described later. For example, in Example (3-3-1) described later, a replica of a functional antibody selected by screening is produced by ascites in a mouse. Since this replica has the same or substantially the same characteristics as the antibody selected by screening, functional antibodies can be efficiently produced by this production method.
  • the replica may be a replica prepared using the antibody-producing cells used for preparing the test antibody in the functional antibody screening method.
  • this is an efficient production method because a copy of the selected functional antibody can be produced from cells already owned by the researcher.
  • the cell used for ascites is one of the hybridomas used in the preceding step (2-1).
  • the replica is a polynucleotide encoding an amino acid sequence of a functional antibody, which is contained in the antibody-producing cell used for preparing the test antibody in the functional antibody screening method, or a functional product thereof. It may be a replica produced using a cell into which the mutant has been introduced.
  • This production method is an efficient production method. Moreover, if this production method is used, it is possible to use desired cells, such as cells with high antibody production efficiency, as cells that produce antibodies.
  • the obtained functional antibody is considered to be an antibody having an effect specific to the antibody recognizing the loop portion as described above.
  • the affinity for the antigen can be obtained by a conventional method. It can be said that the antibody is higher than the antibody. In this case, since the obtained functional antibody has high affinity, it is considered that an excellent effect can be exhibited when used as a diagnostic agent or a pharmaceutical product. In addition, it is considered that it can be suitably used as an antibody for co-crystallization with a membrane protein.
  • the obtained functional antibody has the property of expanding the entire hydrophilic region combining the membrane protein and the antibody, it can be suitably used as an antibody for co-crystallization with the membrane protein. Conceivable. This is because membrane proteins are often covered with a surfactant on the hydrophilic surface necessary for crystallization, so it is important to expand the hydrophilic region in crystallization of membrane proteins. This is because it is considered.
  • the obtained functional antibody has the property of enhancing the overall thermal stability of the membrane protein and the antibody, it can be suitably used as an antibody for co-crystallization with the antibody. .
  • the membrane protein is denatured by heat in the crystallization work process and may not produce a good quality crystal, so it is considered that increasing the thermal stability is important in the crystallization of the membrane protein. .
  • loop partial mutation type means that the amino acid sequence of the loop part differs by one or more amino acids compared to the wild type and has a three-dimensional structure different from the wild type.
  • ⁇ Embodiment 6 Screening of compounds by liposome-ELISA method> Another embodiment of the present invention is a liposome / membrane protein complex used for screening a compound that co-crystallizes with a membrane protein, comprising a solid phase carrier and a liposome bound to the solid phase carrier. And a membrane protein embedded in the liposome, and at least a part of the membrane protein is a liposome / membrane protein complex exposed on the outer surface of the liposome.
  • membrane proteins are considered to maintain their natural three-dimensional structure in liposomes, and many of the compounds obtained by the liposome-ELISA method recognize the natural three-dimensional structure of membrane proteins. This is because it is considered. Further, by performing a detection operation involving denaturation of the membrane protein such as a denaturing dot blotting method, there is an effect that a compound that specifically recognizes the natural three-dimensional structure of the membrane protein can be screened with high accuracy.
  • the compound that co-crystallizes with the membrane protein may be, for example, a nucleic acid, a peptide, or a sugar as long as it can be used for co-crystallization with the membrane protein. Since an antibody that can be used for co-crystallization with a membrane protein is obtained from the examples described later, an antibody is preferable.
  • ⁇ Embodiment 7 Screening method for compound specifically binding to loop portion of membrane protein by liposome-ELISA method>
  • Another embodiment of the present invention is a liposome / membrane protein complex comprising a solid phase carrier, a liposome bound to the solid phase carrier, and a membrane protein embedded in the liposome,
  • a liposome / membrane protein complex comprising a solid phase carrier, a liposome bound to the solid phase carrier, and a membrane protein embedded in the liposome
  • an antibody that specifically binds to a loop portion of a membrane protein or its three-dimensional structure is actually obtained by using a liposome-ELISA method using an antibody as the type of compound. Furthermore, by performing a detection operation involving denaturation of membrane protein such as the denaturing dot blotting method, there is an effect that a compound that specifically binds to the three-dimensional structure of the loop portion can be screened with high accuracy.
  • the compound specifically binds to the loop portion of the membrane protein it is preferable to specifically bind to the three-dimensional structure rather than the primary structure. This is because a compound that specifically binds to the three-dimensional structure of the membrane protein can be expected to have better properties such as binding specificity to the membrane protein.
  • the binding property of the test compound to the above-mentioned liposome / membrane protein complex is examined, and the liposome / membrane protein complex binding property having binding property to the above-mentioned liposome / membrane protein complex.
  • a method for screening a loop part binding compound comprising a step of selecting a compound. By using this screening method, it is considered that the loop partial binding compound can be efficiently screened.
  • an antibody is used as the type of compound, and an antibody that specifically binds to the loop portion of the membrane protein or its three-dimensional structure is actually obtained by this screening method.
  • the test compound is not limited as long as it can specifically bind to the loop portion of the membrane protein.
  • the test compound is a nucleic acid, peptide, polypeptide, sugar, low molecular compound, or a compound library thereof. There may be.
  • the screening method for the loop partial binding compound includes a step of modifying the membrane protein or a replica thereof to prepare a modified membrane protein, and the test compound or the liposome / membrane protein complex against the modified membrane protein. A step of examining the binding property of the body-binding compound, and selecting a compound having the binding property to the liposome / membrane protein complex and not binding to the modified membrane protein. .
  • this screening method it is considered that a compound that specifically binds to the three-dimensional structure of the loop portion of the membrane protein can be screened with high accuracy.
  • an antibody is used as the type of compound, and an antibody that specifically binds to the three-dimensional structure of the loop portion of the membrane protein is actually obtained by this screening method.
  • Another embodiment of the present invention is a method for producing a compound, the step of screening a loop part binding compound by the above screening method, the step of producing a duplicate of the screened loop part binding compound,
  • a method for producing a loop portion binding compound comprising: If this production method is used, as demonstrated in the examples described later, it is possible to efficiently produce a loop partial binding compound.
  • Example (3-3-1) described later an antibody is used as the type of compound, and a replica of the antibody that recognizes the loop portion of the membrane protein selected by screening is ascitesed in mice. It is made by.
  • the characteristics of the low molecular weight compound can be analyzed by conventional techniques such as NMR and MS, and a duplicate can be created based on the analysis information. Alternatively, it can be extracted from the compound group based on the analysis information.
  • the above-mentioned loop part binding compound affects the function of membrane proteins by binding to the loop part or its three-dimensional structure.
  • the loop partial binding compound affects the function of the membrane protein, it is considered that the above-mentioned loop partial binding compound can be used as a pharmaceutical or a candidate product utilizing the effect.
  • A2a receptor binding antibody> Another embodiment of the present invention is an A2a receptor-binding antibody that recognizes the three-dimensional structure of the A2a receptor. Since this A2a receptor-binding antibody recognizes a three-dimensional structure, it can be suitably used as an antibody for co-crystallization with the A2a receptor. Further, since this A2a receptor-binding antibody recognizes a three-dimensional structure, it is considered to be an A2a receptor-binding antibody having excellent properties such as high affinity. Therefore, it is thought that there is an excellent effect when used in applications such as diagnostic agents or therapeutic agents.
  • the A2a receptor-binding antibody may be an A2a receptor-binding antibody that binds to the wild-type A2a receptor and does not bind to the denatured A2a receptor.
  • the denatured A2a receptor is an A2a receptor whose steric structure is different from that of the wild type, and can be prepared by a denaturing agent such as SDS.
  • Another embodiment of the invention is an A2a receptor binding antibody that recognizes the loop portion of the A2a receptor. Since this A2a receptor-binding antibody recognizes the loop portion, it can be suitably used as an antibody for co-crystallization with the A2a receptor.
  • the A2a receptor-binding antibody is preferably an antibody that recognizes the three-dimensional structure of the loop portion in order to improve the accuracy of cocrystallization and various functions.
  • the dissociation constant of the A2a receptor-binding antibody with respect to the A2a receptor is not particularly limited, but may be an antibody of 1.0 ⁇ 10 ⁇ 8 M or less.
  • This dissociation constant is preferably 1.0 ⁇ 10 ⁇ 8 M or less, more preferably 9.0 ⁇ 10 ⁇ 9 M or less, more preferably 8.0 ⁇ 10 ⁇ 9 M or less, and more It is preferably 7.0 ⁇ 10 ⁇ 9 M or less, more preferably 6.0 ⁇ 10 ⁇ 9 M or less, and further preferably 5.0 ⁇ 10 ⁇ 9 M or less. This is because the lower the dissociation constant, the more excellent effects can be expected when the A2a receptor-binding antibody is used in applications such as co-crystallization, a diagnostic agent, or a therapeutic agent.
  • Another embodiment of the present invention is an A2a receptor-binding antibody that has binding to the wild type of the A2a receptor and does not have binding to the loop partial displacement type of the A2a receptor. Since this A2a receptor-binding antibody recognizes the loop portion, it can be suitably used as an antibody for co-crystallization with the A2a receptor.
  • the A2a receptor-binding antibody is preferably an antibody that recognizes the three-dimensional structure of the loop portion in order to improve the accuracy of cocrystallization and various functions.
  • Another embodiment of the present invention is an A2a receptor-binding antibody that has binding to the wild-type A2a receptor and does not bind to the A2a receptor in which the loop portion is denatured. Since this A2a receptor-binding antibody recognizes the three-dimensional structure of the loop portion, it can be suitably used as an antibody for co-crystallization with the A2a receptor. Further, since this A2a receptor-binding antibody recognizes the three-dimensional structure, it is considered to be an A2a receptor-binding antibody having excellent properties such as high affinity. Therefore, it is thought that there is an excellent effect when used in applications such as diagnostic agents or therapeutic agents.
  • sugar transporter-binding antibody Another embodiment of the present invention is a sugar transporter-binding antibody that recognizes the three-dimensional structure of a sugar transporter. Since this sugar transporter-binding antibody recognizes a three-dimensional structure, it is considered that the sugar transporter-binding antibody can be suitably used as an antibody for co-crystallization with a sugar transporter. Further, since this sugar transporter-binding antibody recognizes a three-dimensional structure, it is considered to be a sugar transporter-binding antibody having excellent properties such as high affinity. Therefore, it is thought that there is an excellent effect when used in applications such as diagnostic agents or therapeutic agents.
  • the sugar transporter-binding antibody may be a sugar transporter-binding antibody that has binding to the wild type of the sugar transporter and does not have binding to the modified sugar transporter.
  • the modified sugar transporter is a sugar transporter having a three-dimensional structure different from that of the wild type, and can be adjusted by a denaturing agent such as SDS.
  • Another embodiment of the present invention is a sugar transporter-binding antibody that recognizes a loop portion of a sugar transporter. Since this sugar transporter-binding antibody recognizes the loop portion, it can be suitably used as an antibody for co-crystallization with the sugar transporter.
  • the sugar transporter-binding antibody is preferably an antibody that recognizes the three-dimensional structure of the loop portion in order to improve the accuracy of cocrystallization and various functions.
  • the dissociation constant of the sugar transporter-binding antibody with respect to the sugar transporter is not particularly limited, but may be an antibody of 1.0 ⁇ 10 ⁇ 8 M or less.
  • This dissociation constant is preferably 1.0 ⁇ 10 ⁇ 8 M or less, more preferably 9.0 ⁇ 10 ⁇ 9 M or less, more preferably 8.0 ⁇ 10 ⁇ 9 M or less, and more Preferably it is 7.0 ⁇ 10 ⁇ 9 M or less, more preferably 6.0 ⁇ 10 ⁇ 9 M or less, more preferably 5.0 ⁇ 10 ⁇ 9 M or less, more preferably 4.0. ⁇ 10 ⁇ 9 M or less, more preferably 3.0 ⁇ 10 ⁇ 9 M or less. This is because the lower the dissociation constant, the more excellent effects can be expected when the sugar transporter-binding antibody is used in applications such as co-crystallization, a diagnostic agent, or a therapeutic agent.
  • Another embodiment of the present invention is a sugar transporter-binding antibody that has a binding property to a wild type of a sugar transporter and does not have a binding property to a loop partial displacement type of the sugar transporter. Since this sugar transporter-binding antibody recognizes the loop portion, it can be suitably used as an antibody for co-crystallization with the sugar transporter.
  • the sugar transporter-binding antibody is preferably an antibody that recognizes the three-dimensional structure of the loop portion in order to improve the accuracy of cocrystallization and various functions.
  • Another embodiment of the present invention is a sugar transporter-binding antibody having binding ability to a wild type sugar transporter and not binding to a sugar transporter in which a loop portion is denatured. Since this sugar transporter-binding antibody recognizes the three-dimensional structure of the loop part, it can be suitably used as an antibody for co-crystallization with the sugar transporter. Further, since this sugar transporter-binding antibody recognizes a three-dimensional structure, it is considered to be a sugar transporter-binding antibody having excellent properties such as high affinity. Therefore, it is thought that there is an excellent effect when used in applications such as diagnostic agents or therapeutic agents.
  • An embodiment of the present invention is a liposome / membrane protein complex used for screening an antibody that co-crystallizes with a membrane protein, which is a solid phase carrier, a liposome bound to the solid phase carrier, And a membrane protein embedded in the liposome, wherein at least a part of the membrane protein is exposed on the outer surface of the liposome.
  • the membrane protein is thought to maintain the natural three-dimensional structure in the liposome, and many of the monoclonal antibodies obtained by the liposome-ELISA method recognize the natural three-dimensional structure of the membrane protein. It is because it is considered. Further, by performing an operation involving denaturation of the membrane protein such as a denaturing dot blotting method, there is an effect that a monoclonal antibody that specifically recognizes the natural three-dimensional structure of the membrane protein can be screened with high accuracy.
  • Another embodiment is a liposome / membrane protein complex used in screening an antibody that co-crystallizes with a membrane protein, the solid phase carrier, the liposome bound to the solid phase carrier, and the liposome A membrane protein embedded in the liposome and a linker embedded in the liposome, wherein at least a part of the membrane protein is exposed on the outer surface of the liposome, and the liposome is interposed via the linker.
  • Liposome / membrane protein complex bound to a solid support.
  • the membrane protein is thought to maintain the natural three-dimensional structure in the liposome, and many of the monoclonal antibodies obtained by the liposome-ELISA method recognize the natural three-dimensional structure of the membrane protein. It is because it is considered. Further, by performing an operation involving denaturation of the membrane protein such as a denaturing dot blotting method, there is an effect that a monoclonal antibody that specifically recognizes the natural three-dimensional structure of the membrane protein can be screened with high accuracy.
  • the membrane protein is a liposome / membrane protein complex, wherein the membrane protein is a membrane protein selected from the group consisting of a receptor protein, a channel protein, a transporter protein, an adhesion molecule, and a membrane-bound enzyme, or a salt thereof. It is. Since these membrane proteins are important targets for medical and pharmacological purposes, cocrystallization with these targets has the effect of advancing the development of therapeutic agents, diagnostic agents, and the like.
  • Another embodiment is a liposome / membrane protein complex, wherein the membrane protein is a multiple-transmembrane membrane protein.
  • the membrane protein is a multiple-transmembrane membrane protein.
  • Multiple membrane-spanning membrane proteins are important medically and pharmacological targets, so co-crystallization with these targets will have the effect of advancing the development of therapeutics and diagnostics .
  • Another embodiment is a liposome / membrane protein complex, wherein the membrane protein is a membrane protein that has penetrated the membrane seven times or more.
  • Membrane proteins that penetrate the membrane more than 7 times have many medically and pharmacological targets such as GPCRs and transporter proteins, and if they are co-crystallized with these targets, therapeutic drugs and diagnosis It has the effect of advancing the development of medicines.
  • Another embodiment is a liposome / membrane protein complex in which the membrane protein is a G protein coupled receptor. Since G protein-coupled receptors are important targets for medical and pharmacological purposes, cocrystallization with these targets has the effect of advancing the development of therapeutic agents, diagnostic agents, and the like.
  • Another embodiment is a liposome / membrane protein complex used in screening a compound that co-crystallizes with a membrane protein, the solid phase carrier, the liposome bound to the solid phase carrier, and the liposome A liposome / membrane protein complex in which at least a part of the membrane protein is exposed on the outer surface of the liposome.
  • Another embodiment is a liposome / membrane protein complex used for screening an antibody that specifically binds to a loop portion of a membrane protein, which is bound to the solid phase carrier and the solid phase carrier.
  • a liposome / membrane protein complex comprising a liposome and a membrane protein embedded in the liposome, wherein at least one loop portion of the membrane protein is exposed on the outer surface of the liposome.
  • the membrane protein is thought to maintain the natural three-dimensional structure in the liposome, and many of the monoclonal antibodies obtained by the liposome-ELISA method recognize the natural three-dimensional structure of the membrane protein. It is because it is considered. Moreover, it is because it demonstrates that it couple
  • Another embodiment is a method for producing a liposome / membrane protein complex for use in screening a compound that co-crystallizes with a membrane protein, comprising a lipid solution, a linker, the membrane protein, and a surfactant.
  • a step of mixing an aqueous solution to prepare a mixed solution, and adding a surfactant removing agent to the mixed solution, adsorbing the surfactant in the mixed solution to the surfactant removing agent, and the linker and the membrane A method for producing a liposome complex, comprising: producing a liposome in which a protein is embedded; and further, producing a liposome complex by binding the liposome to the solid phase carrier via the linker. It is.
  • the liposome-ELISA method By performing the liposome-ELISA method using the liposome / membrane protein complex produced by this method, it is possible to screen for a monoclonal antibody that specifically recognizes the natural three-dimensional structure of the membrane protein. This is because the membrane protein is thought to maintain the natural three-dimensional structure in the liposome, and many of the monoclonal antibodies obtained by the liposome-ELISA method recognize the natural three-dimensional structure of the membrane protein. It is because it is considered. Further, by performing an operation involving denaturation of the membrane protein such as a denaturing dot blotting method, there is an effect that a monoclonal antibody that specifically recognizes the natural three-dimensional structure of the membrane protein can be screened with high accuracy.
  • Another embodiment is a method for screening an antibody that co-crystallizes with a membrane protein, wherein the test antibody is contacted with a liposome / membrane protein complex used for screening an antibody that co-crystallizes with a membrane protein.
  • a step a step of detecting the antibody bound in the previous step, a step of denaturing the membrane protein, a step of contacting the test antibody with the denatured membrane protein denatured in the previous step, and binding in the previous step And a step of detecting an antibody.
  • Another embodiment is a screening kit for an antibody that co-crystallizes with a membrane protein, and includes a membrane protein, a lipid, a linker, and a solid phase carrier.
  • Another embodiment is a kit for screening an antibody that co-crystallizes with a membrane protein, the membrane protein, lipid, linker, solid phase carrier, protein denaturant, dot blotting method, western blotting method, immunoblotting method Or a reagent for use in any one of the ELISA methods.
  • a monoclonal antibody that specifically recognizes the natural three-dimensional structure of the membrane protein This is because the membrane protein is thought to maintain the natural three-dimensional structure in the liposome, and many of the monoclonal antibodies obtained by the liposome-ELISA method recognize the natural three-dimensional structure of the membrane protein. It is because it is considered.
  • an operation involving denaturation of the membrane protein such as a denaturing dot blotting method, there is an effect that a monoclonal antibody that specifically recognizes the natural three-dimensional structure of the membrane protein can be screened with high accuracy.
  • Example 1 Presentation of natural membrane protein using liposome and evaluation of liposome-ELISA method> (1-1) Reconstitution of A2a Receptor into Biotinylated Lipid-Containing Liposomes Human Adenosine Receptor A2a (A2a Receptor) with N-terminal FLAG-tag and C-terminal His10-tag as Pichia pastris
  • the expression system was purified using an affinity column using TALON resin.
  • TALON resin As a result of investigating the type of lipid used for liposome reconstitution and the reconstitution method, it was found that the surfactant removal method using Bio-Beads using phosphatidylcholine derived from hen's egg as the lipid was optimal.
  • the purified A2a receptor was added to a 1 mg / ml hen egg-derived phosphatidylcholine solution containing 5 ⁇ g / ml 16: 0 biotinyl CAP-PE solubilized with PBS containing 1% octylglucoside to a final concentration of 0.1 mg / ml. And left on ice for 30 minutes. Next, 30 mg of Bio-Beads was added to 1 ml, and the work of stirring at 4 ° C. for 1 hour was performed twice.
  • Bio-Beads 100 mg was added to 1 ml, and the mixture was stirred overnight, and Bio-Beads was removed to prepare biotinylated lipid-containing A2a receptor reconstituted liposomes. It was confirmed by measuring the binding activity of 3H-labeled Ligand that the reconstituted A2a receptor maintained its native structure.
  • the membrane protein retains the natural three-dimensional structure in the liposome, so that many of the monoclonal antibodies obtained by the liposome-ELISA method recognize the natural three-dimensional structure of the membrane protein. Conceivable.
  • the inventors of the present application have actually obtained more than 200 monoclonal antibodies for the human-derived adenosine receptor A2a using this method.
  • Example 2 Screening of monoclonal antibody specifically recognizing natural three-dimensional structure of membrane protein by liposome-ELISA method and modified dot blotting method>
  • (2-1) Primary screening of a monoclonal antibody that specifically recognizes the natural three-dimensional structure of a membrane protein using the liposome-ELISA method
  • the liposome-ELISA method is basically the same as in Example 1. .
  • the liposome solution is further diluted 2-fold with PBS, and instead of the mouse-derived anti-FLAG-M2 antibody (Sigma), the hybridoma culture supernatant containing the monoclonal antibody is diluted 50-fold with PBS containing 1% BSA. Used. Accordingly, the color development time of ABTS was set to 1 hour.
  • an antibody used for co-crystallization with a membrane protein is suitably an antibody having high binding specificity for the natural three-dimensional structure of the membrane protein. From the above results, it is considered that the monoclonal antibody obtained in this example can be suitably used for co-crystallization with a membrane protein because of its high binding specificity for the natural three-dimensional structure of the membrane protein. . Furthermore, it is considered that a monoclonal antibody that can be suitably used for co-crystallization with a membrane protein can be efficiently obtained by using this screening method.
  • Example 3 Measurement of binding affinity of a monoclonal antibody that specifically recognizes a natural three-dimensional structure of a membrane protein> (3-1) Dissociation constant measurement by Biacore measurement Antibody binding and dissociation rates were evaluated using Biacore T-100 based on the surface plasmon resonance principle. Immobilization of anti-mouse Fc antibody on CM5 sensor chip using amine coupling method using HBS-DDM buffer (10mmol / L Hepes pH7.4, 150mmol / L sodium chloride, 0.05% wt / vol dodecyl maltoside) did.
  • HBS-DDM buffer 10mmol / L Hepes pH7.4, 150mmol / L sodium chloride, 0.05% wt / vol dodecyl maltoside
  • An anti-receptor antibody-immobilized chip was prepared by allowing the hybridoma culture supernatant containing the monoclonal antibody obtained by the method of Example 2 to flow through this sensor chip.
  • the purified human-derived adenosine receptor A2a was diluted with HBS-DDM so as to have a concentration of 600 nmol / L, and flowed to the sensor chip to obtain a sensorgram.
  • the dissociation constant was estimated by analysis with BiaEvaluation software. As a result, the dissociation constant between A2a receptor and mAb1 IgG was 4.4 ⁇ 10 ⁇ 9 M.
  • the dissociation constant between A2a receptor and mAb1 IgG was 4.4 ⁇ 10 ⁇ 9 M, and an antibody having high affinity was successfully obtained.
  • the antibody used for co-crystallization with the membrane protein needs to have a high affinity in order to maintain the binding with the membrane protein during the steps of purification and crystallization.
  • the monoclonal antibody used in this example is a monoclonal antibody having a high affinity with the membrane protein. It is considered that it can be suitably used for crystallization.
  • using the screening method of Example 2 it is considered that a monoclonal antibody that can be suitably used for co-crystallization with a membrane protein can be efficiently obtained.
  • the antibody was fragmented by adding an activated papain solution and an antibody solution 1: 1 and reacting at 37 ° C. overnight. 0.3 mol / L iodoacetamide was added to the antibody / papain solution in an amount of 1/10 to inactivate papain. After dialysis overnight against 1 ⁇ phosphate buffer, a purified Fab fragment was obtained by collecting the flow-through of the cation exchange column.
  • the antibody used for co-crystallization with the membrane protein needs to maintain the binding with the membrane protein during the steps of purification and crystallization. Therefore, an antibody having high affinity for the membrane protein is suitable as the antibody used for co-crystallization with the membrane protein.
  • the monoclonal antibody used in this example (monoclonal antibody obtained by the method of Example 2) is a monoclonal antibody having a high binding durability with the membrane protein. It is thought that it can be suitably used for co-crystallization of Furthermore, using the screening method of Example 2, it is considered that a monoclonal antibody that can be suitably used for co-crystallization with a membrane protein can be efficiently obtained.
  • Example 4 Measurement of thermal stability of a complex of a monoclonal antibody and a membrane protein that specifically recognizes the natural three-dimensional structure of the membrane protein> (4-1) Measurement of thermal stability of complex of Fab fragment and A2a receptor Evaluation of change in thermal stability of A2a receptor accompanying the binding of the monoclonal antibody obtained by the method of Example 2 to the receptor Went. The method followed [A. I. Alexandrov et al. (2008) Structure 16, 351-359].
  • a receptor or receptor obtained by purifying a dye CPM N- [4- (7-diethylamino-4methyl-3-coumarinyl) phenyl] maleimide
  • CPM N- [4- (7-diethylamino-4methyl-3-coumarinyl) phenyl] maleimide
  • FIG. 5 shows A2a receptor alone or a complex of A2a receptor and Fab fragment Fab1 of an antibody clone that recognizes a flexible region, and a complex of Fab fragment Fab2 of a monoclonal antibody clone obtained by the method of Example 2.
  • the heat load denaturation process in the body is shown.
  • the time “Half Life; t1 / 2” until the fluorescence value half that of the maximum fluorescence value is shown is used as the evaluation criterion for thermal stability.
  • membrane proteins are unstable, and many proteins are denatured at the stage of purification and crystallization from biological membranes.
  • membrane receptors and transporters derived from mammals which are medically important, is remarkable.
  • a hydrophilic surface is essential for protein crystallization, but most of membrane proteins are covered with a surfactant and are extremely difficult to crystallize compared to water-soluble proteins.
  • the above-mentioned antibody-membrane protein complex that recognizes the natural three-dimensional structure of the membrane protein is suitable for co-crystallization in that the thermal stability is increased and the hydrophilic region is enlarged. It is believed that there is.
  • the monoclonal antibody used in this example (monoclonal antibody obtained by the method of Example 2) can be suitably used for co-crystallization with a membrane protein.
  • the screening method of Example 2 it is thought that the monoclonal antibody which can be used conveniently for a co-crystallization with a membrane protein is obtained efficiently.
  • Example 5 Determination of a membrane protein binding site of a monoclonal antibody that specifically recognizes a natural three-dimensional structure of a membrane protein>
  • the antibody binding site for the A2a receptor of the monoclonal antibody obtained by the method of Example 2 was investigated.
  • a chimera mutant in which the primary sequence corresponding to the transmembrane region of the multi-transmembrane protein, particularly the 7-transmembrane receptor is substituted is prepared, and Western blot analysis is performed on the expressed membrane fraction.
  • the antibody binding site is determined by the disappearance of the signal accompanying the mutation operation.
  • the yeast plasmid pRS426GAL1-GFP linearized with the restriction enzyme SmaI and the gene sequence of the A2a receptor amplified by the PCR method or a mutant thereof are sequenced.
  • the budding yeast strain FGY217 was transformed by the lithium acetate method.
  • the transformed host strain solution was applied to 2% glucose-containing uracil-deficient minimal medium prepared as a 2% agar plate and allowed to stand at 30 ° C. for 3 days to form colonies. Single colonies were dispensed into two centrifuge tubes containing 10 mL of 0.1% glucose-containing uracil-deficient medium, one of which was collected after shaking culture at 30 ° C.
  • the collected cells are sheared using glass beads (trade name: Glass beads, acid-washed, manufactured by Sigma), and a plasmid solution is obtained from the obtained cell extract using a plasmid miniprep kit (Qiagen). did. It was confirmed that the mutation was appropriately performed by analyzing the DNA sequence of the plasmid.
  • Trisglycine 12% polyacrylamide gel (Invitrogen) was used for electrophoresis at 100 V for 120 minutes to develop the composition.
  • the developed gel was photographed with a fluorescence image analyzer (trade name: LAS-1000, Fuji Film) to confirm a single band derived from the GFP fusion mutant protein.
  • Example 2 was used as a primary antibody.
  • the antigen-antibody reaction was performed by allowing the HRP-conjugated anti-IgG antibody (trade name: goat anti-mouse IgG HRP conjugate: manufactured by Santa cruz) to act as the monoclonal antibody and the secondary antibody obtained by the above method.
  • a secondary antibody was detected using a chemiluminescent reagent (trade name: Immobilon Western Detection Reagents, manufactured by Millipore), and it was determined that the portion of the mutant corresponding to the band that was not detected was the epitope region. .
  • FIG. 7 shows the result of confirming the expression results of the A2a receptor non-mutant and a group of the mutants by fluorescence detection. Further, the lower part of FIG. 7 shows the result of detecting the antigen-antibody reaction against the A2a receptor non-mutant and a group of the mutants by Western blotting.
  • the N-terminal region of a membrane protein is a relatively flexible region in which the three-dimensional structure is unstable in a solvent. Therefore, an antibody that recognizes and binds to the N-terminal region of a membrane protein is very likely to recognize an amino acid sequence rather than a three-dimensional structure.
  • the antibody used in this example (monoclonal antibody obtained by the method of Example 2) recognizes the loop portion, there is a high possibility that it recognizes the three-dimensional structure. Recognizing a three-dimensional structure means that the binding specificity of the antibody is higher than that of an antibody that recognizes only the amino acid sequence.
  • an antibody used for co-crystallization with a membrane protein is suitably an antibody having high binding specificity for the natural three-dimensional structure of the membrane protein. From the above results, the antibody used in this example (monoclonal antibody obtained by the method of Example 2) has high binding specificity to the natural three-dimensional structure of the membrane protein. It is considered that it can be suitably used for cocrystallization.
  • the hydrophilic region present at the protein N-terminus (or C-terminus) of membrane proteins generally has large fluctuations in the relative position and orientation relative to the transmembrane region in a solvent. Even if an antibody binds to such a region, it cannot form a fixed crystal lattice regularly arranged in the crystal, and an antibody that binds near the protein N-terminus (or C-terminus) is not suitable for crystallization. It can be said that. Therefore, an antibody having high binding specificity for the loop portion of the membrane protein is suitable for the antibody used for co-crystallization with the membrane protein.
  • the antibody used in this example (monoclonal antibody obtained by the method of Example 2) has high binding specificity to the loop portion of the membrane protein, and thus co-crystallization with the membrane protein. It is thought that it can be used suitably for. Furthermore, using the screening method of Example 2, it is considered that a monoclonal antibody that can be suitably used for co-crystallization with a membrane protein can be efficiently obtained.
  • a Fab antibody phage library was constructed by incorporating the phage display vector pComb3XSS.
  • the size of the obtained library was approximately 10 7 to 10 8 clones.
  • sequence analysis of the V H and V L regions of 10 clones randomly extracted from each of the obtained libraries it was confirmed that the clones constituting the library had no overlap in amino acid sequence.
  • coli XL-1 Blue strain and add 100 ⁇ g / mL ampicillin and 10 ⁇ g / mL tetracycline.
  • the mixture was smeared on the contained LB agar medium and cultured overnight at 37 ° C.
  • the colonies that grew on the plate were recovered by suspending them in LB medium, and phage rescue was performed by infecting with VCSM13 helper phage, followed by 100? G / mL ampicillin, 10? G / mL tetracycline, and 70? G / mL.
  • the phage subpopulation was amplified and produced in the culture supernatant by culturing overnight at 30 ° C. in SB medium containing kanamycin.
  • the phage is precipitated by adding 4% (w / v) PEG6000 and 3% (w / v) NaCl to the E. coli culture supernatant, suspended again in TBS buffer, and about 10 13 to 10 14 phage next time.
  • Fab fragments were produced from the antibody phage obtained in the primary screening.
  • the filamentous phage gene III product (gp3) is not fused
  • an HA tag is added to the C-terminus of the H chain of the produced Fab.
  • E. coli glycerol stocks of positive Fab clones were inoculated into SB medium (50 mL) containing 100 g / mL ampicillin and cultured overnight at 37 ° C. with shaking.
  • This preculture was inoculated into SB medium (5 L) containing 100 g / mL ampicillin, and then main culture was started by shaking at 37 ° C.
  • the turbidity at 600 nm (OD 600 nm ) of the culture was monitored over time, and IPTG was added so that the final concentration was 1 mM when the value reached 0.5 to 0.8.
  • shaking culture was performed at 30 ° C. for 16 to 18 hours, and the cells were collected by centrifugation (6,000 g, 30 minutes, 4 ° C.).
  • the periplasmic fraction extract was prepared by lysozyme treatment and osmotic shock.
  • periplasmic extraction buffer An equal amount of periplasmic extraction buffer was added to the cell pellet, suspended, and allowed to stand on ice for 30 minutes, and then the supernatant was collected by centrifugation (6000 g, 30 minutes, 4 ° C.) to obtain a periplasmic extract.
  • the purification of Fab was performed by ammonium sulfate salting out, dialysis, Ni-NTA affinity column, and Protein G affinity column.
  • the liposome ELISA method was basically the same as (1-1) and (1-2) in Example 1, except that the A2a receptor is replaced with a human sugar nucleic acid transporter, and the mouse-derived anti-FLAG-M2
  • the antibody Sigma was used in place of the crude E. coli extract containing the recombinant Fab antibody fragment. Then, a recombinant Fab antibody fragment that binds to the biotinylated liposome / human sugar nucleic acid transporter complex was obtained.
  • Biotin-conjugated anti-HA tag IgG is dissolved in TBS-D buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (w / v) DDM] to a concentration of 50 ⁇ g / mL.
  • the sample was immobilized on a Biacore SA chip by flowing through a flow cell at a flow rate of 10 ⁇ L / min for 120 seconds.
  • 12 mg / mL BSA and 12 mg / mL CM-dextran were added to the Fab fragment expressed in E. coli, and then flowed in a flow cell at a flow rate of 10 ⁇ L / min for 300 seconds to capture the Fab on the chip.
  • a purified human sugar nucleic acid transporter preparation (0, 4, 8, 16, or 32 ⁇ g / mL concentration) dissolved in TBS-D buffer as an analyte was passed through the flow cell for 120 seconds at a flow rate of 30 ⁇ L / min. Binding stability was assessed by measuring the 10 minute dissociation response immediately after. In the control flow cell, the same experiment was performed without capturing the Fab fragment on the chip, and the binding / dissociation kinetics of the Fab fragment and the membrane protein were analyzed by taking the difference in the interaction response between them.
  • the captured Fab fragment was dissociated by flowing a 10 mM NaOH solution through the flow cell at a flow rate of 30 ⁇ L / min for 30 seconds.
  • the obtained complex sample (300 ⁇ L) was equilibrated with TBS-D buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (w / v) DDM] Superdex200 10/300 And eluted at a flow rate of 0.4 mL / min.
  • TBS-D buffer 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (w / v) DDM]
  • Superdex200 10/300 And eluted at a flow rate of 0.4 mL / min.
  • the same gel filtration was performed on a sample of a purified sample of 10 ⁇ g of human sugar nucleic acid transporter / GFP fusion protein alone.
  • the antibody used for co-crystallization with the membrane protein needs to maintain the binding with the membrane protein during the steps of purification and crystallization. That is, from the above results, the monoclonal antibody used in this analysis (recombinant Fab antibody fragment obtained in (6-4) of Example 6) is a monoclonal antibody having a high binding durability with a membrane protein. Therefore, it is considered that it can be suitably used for cocrystallization with a membrane protein. And if the screening method of Example 6 is used, it is thought that the antibody fragment which can be used conveniently for a co-crystallization with a membrane protein is obtained efficiently.
  • Example 7 Co-crystallization of membrane protein> (7-1) Co-crystallization of A2a receptor Gel filtration was performed in the same manner as in (3-3-2) above to obtain a fraction of A2a receptor and Fab complex. This fraction was concentrated with Amicon Ultra-4 fractionated molecular weight of 100 k (millipore) or the like to obtain a protein concentration of about 10 mg / ml. This protein solution was crystallized by a vapor diffusion method according to a conventional method using PEG400 (Sigma) or the like as a precipitant. As a result, crystals of A2a receptor and Fab complex as shown in FIG. 9 were obtained.
  • the obtained antibody is an antibody that specifically recognizes the natural three-dimensional structure of the target membrane protein, an antibody with high binding specificity to the target membrane protein, an antibody with high affinity for the target membrane protein, or a membrane protein And an antibody that expands the entire hydrophilic region, and an antibody that increases the overall thermal stability of the membrane protein and the antibody, or an antibody that specifically recognizes the loop portion of the membrane protein. Proven.
  • membrane proteins have been successfully co-crystallized using the obtained antibodies. Therefore, if the above-mentioned liposome / membrane protein complex is used, it is considered that an antibody that can be suitably used for co-crystallization with the membrane protein can be obtained very efficiently as compared with known techniques. For the same reason, a screening method or a production method of an antibody having the above characteristics has been clarified.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de criblage d'un anticorps qui doit être co-cristallisé avec une protéine. L'invention concerne plus spécifiquement un complexe de protéine membranaire-liposome qui est utilisé pour cribler un anticorps à co-cristalliser avec une protéine membranaire, comprenant : un support de phase solide; un liposome se liant au support de phase solide; et protéine membranaire incorporée dans le liposome, au moins une partie de la protéine membranaire étant exposée sur la surface extérieure du liposome.
PCT/JP2010/057631 2009-04-30 2010-04-28 Procédé de criblage d'un anticorps monoclonal reconnaissant une structure tridimensionnelle de protéine membranaire WO2010126115A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2011511458A JP5526448B2 (ja) 2009-04-30 2010-04-28 膜蛋白質の立体構造を認識するモノクローナル抗体のスクリーニング方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009-110994 2009-04-30
JP2009110994 2009-04-30

Publications (1)

Publication Number Publication Date
WO2010126115A1 true WO2010126115A1 (fr) 2010-11-04

Family

ID=43032259

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/057631 WO2010126115A1 (fr) 2009-04-30 2010-04-28 Procédé de criblage d'un anticorps monoclonal reconnaissant une structure tridimensionnelle de protéine membranaire

Country Status (2)

Country Link
JP (1) JP5526448B2 (fr)
WO (1) WO2010126115A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013183660A (ja) * 2012-03-06 2013-09-19 Japan Science & Technology Agency アゴニストの親和性を亢進する抗ヒトアデノシンＡ２ａ受容体モノクローナル抗体
WO2017082214A1 (fr) * 2015-11-09 2017-05-18 国立大学法人京都工芸繊維大学 Procédé de criblage d'anticorps à chaîne unique et anticorps à chaîne unique
WO2024071374A1 (fr) * 2022-09-30 2024-04-04 富士フイルム株式会社 Procédé de criblage pour cellule qui produit une substance objective, procédé de production d'acide nucléique et procédé de production de substance objective

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03137932A (ja) * 1988-08-29 1991-06-12 Canon Inc プロテオリポソームの形成方法
WO2006066368A1 (fr) * 2004-12-23 2006-06-29 Vrije Universiteit Brussel Procede de criblage et de selection de ligands

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008280266A (ja) * 2007-05-09 2008-11-20 Sekisui Chem Co Ltd モノクローナル抗体

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03137932A (ja) * 1988-08-29 1991-06-12 Canon Inc プロテオリポソームの形成方法
WO2006066368A1 (fr) * 2004-12-23 2006-06-29 Vrije Universiteit Brussel Procede de criblage et de selection de ligands

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BINZ H.K ET AL.: "High-affinity binders selected from designed ankyrin repeat protein libraries.", NAT. BIOTECHNOL., vol. 22, 2004, pages 575 - 582, XP002343919, DOI: doi:10.1038/nbt962 *
DUTZLER R. ET AL.: "Gating the selectivity filter in C1C chloride channels.", SCIENCE, vol. 300, 2003, pages 108 - 112 *
JESPERSEN L.K. ET AL.: "Use of proteoliposomes to generate phage antibodies against native AMPA receptor.", EUR. J. BIOCHEM., vol. 267, 2000, pages 1382 - 1389 *
OSTERMEIER C. ET AL.: "Fv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase.", NAT. STRUCT. BIOL., vol. 2, 1995, pages 842 - 846, XP008027288, DOI: doi:10.1038/nsb1095-842 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013183660A (ja) * 2012-03-06 2013-09-19 Japan Science & Technology Agency アゴニストの親和性を亢進する抗ヒトアデノシンＡ２ａ受容体モノクローナル抗体
WO2017082214A1 (fr) * 2015-11-09 2017-05-18 国立大学法人京都工芸繊維大学 Procédé de criblage d'anticorps à chaîne unique et anticorps à chaîne unique
JPWO2017082214A1 (ja) * 2015-11-09 2018-10-18 国立大学法人京都工芸繊維大学 単鎖抗体のスクリーニング方法及び単鎖抗体
WO2024071374A1 (fr) * 2022-09-30 2024-04-04 富士フイルム株式会社 Procédé de criblage pour cellule qui produit une substance objective, procédé de production d'acide nucléique et procédé de production de substance objective

Also Published As

Publication number Publication date
JP5526448B2 (ja) 2014-06-18
JPWO2010126115A1 (ja) 2012-11-01

Similar Documents

Publication Publication Date Title
EP3850363B1 (fr) Procédé de criblage à haut rendement de l'affinité de peptide-mhc pour des ligands de récepteurs de cellules t utilisant des complexes mhc stabilisés et sans peptide
JP6164535B2 (ja) Ｇｐｃｒ：ｇタンパク質複合体に対して作製された結合ドメインおよびそれに由来する使用
Pardon et al. A general protocol for the generation of Nanobodies for structural biology
JP2024028750A (ja) 共有抗原を標的とする抗原結合タンパク質
JP2022105574A (ja) 多重特異性分子
JP6053672B2 (ja) アフィニティに基づく用途のためのエピトープタグ
US20240018268A1 (en) Anti-idiotypic antibodies against anti-cd19 antibodies
JP6827485B2 (ja) ムスカリン性アセチルコリン受容体の結合剤およびそれらの使用
Nevoltris et al. Conformational nanobodies reveal tethered epidermal growth factor receptor involved in EGFR/ErbB2 predimers
Park et al. Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection
US20190255186A1 (en) Single variable domain t-cell receptors
JP6687525B2 (ja) オピオイド受容体結合剤およびその使用
EP3161166A1 (fr) Compositions et procédés pour la production rapide de répertoires polyvalents de nanocorps
JP2021533785A (ja) 共有抗原を標的指向する抗原結合タンパク質
Wu et al. Structure and analysis of nanobody binding to the human ASIC1a ion channel
TW201811828A (zh) 抗原結合分子和使用彼之方法
Denham et al. A generic cell surface ligand system for studying cell–cell recognition
JP5526448B2 (ja) 膜蛋白質の立体構造を認識するモノクローナル抗体のスクリーニング方法
KR20190129062A (ko) 항체 선택 방법
Estelles et al. Exosome nanovesicles displaying G protein-coupled receptors for drug discovery
CN111093696B (zh) 抗iii型纤连蛋白结构域的抗原结合区及其使用方法
JP2024511155A (ja) 多価タンパク質およびスクリーニング法
JP2009278927A (ja) Ｔ細胞受容体を模倣する抗体断片及びその製造方法
WO2021235509A1 (fr) Molécule de liaison à un antigène
Bednenko et al. Therapeutic antibodies targeting potassium ion channels

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10769813

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2011511458

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10769813

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载