JP2013180966A - Antibody reagent for human albumin measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a molecule having an improved bonding force to a human albumin by making a multivalent molecule capable of obtaining a multivalent antibody and a bispecific antibody.SOLUTION: The affinity to a human albumin can be improved by a multivalent molecule in which a first molecule that specifically binds to the human albumin and a second molecule that specifically binds to a human albumin molecular surface different from the first molecule are assumed to be a barnase variant and a burster which are made to a binding module.

Description

  The present invention relates to an antibody reagent for analyzing a specific component in a document by an immunoassay, and more specifically to a method for preparing an antibody reagent for analyzing human albumin in blood or urine by an immunoassay method. .

  An antibody in which each antigen binding site binds to a different antigen is called a bispecific antibody. For example, bispecific antibodies that recognize cancer cell antigens and effector cell antigens are expected to enhance antitumor action by effectively contacting these cells and are currently undergoing clinical evaluation. In recent years, research on bispecific antibodies having an antibody molecule as a binding site has been activated, and simple production methods thereof have been developed one after another. As an example, the production method described in Patent Document 1 expresses a fusion protein in which scFv (single chain antibody) is linked to each of burnase and burster, and high affinity of burnase and burster (Kd = 10 ^). -14) is used to produce a multivalent antibody and a bispecific antibody.

  Furthermore, as a conventional human albumin binding protein for testing, an antibody molecule having a molecular weight of about 150 kDa or a single chain Fv is used (Patent Documents 1 and 2). However, the conventional method depends on the avidity effect obtained by mixing a plurality of monovalent binding antibodies.

JP 2002-335978 A JP 2009-145329 A

  The above-mentioned barnase has RNA degrading activity, and when the burnase scFv fusion protein was expressed, the enzyme activity was disturbed by co-expressing its inhibitor, Burster. However, as described above, the affinity of barnase and burster is extremely high. To remove co-expressed burster, both proteins are denatured with 6M guanidine hydrochloride, and then activated by unwinding. A burnase scFv fusion protein was obtained. Therefore, there has been a problem that enormous time and man-hours are required to obtain the target multivalent antibody or bispecific antibody.

  Moreover, in the case of an avidity effect by mixing a plurality of antibodies, there is a problem that a step of adjusting the blending ratio of the antibodies to be mixed is necessary.

  In order to solve the above-mentioned conventional problems, the method for producing a multivalent binding molecule of the present invention is a first method in which at least one binding site that specifically binds to human albumin and a mutant of RNase burnase are linked. A second molecule obtained by linking a protein burster that specifically binds burnase with at least one binding portion that specifically binds to the surface of the albumin molecule different from the first molecule. And a step of linking the first molecule and the second molecule through a protein burster that specifically binds to a mutant of the RNase burnase.

  The method for producing a multivalent binding molecule of the present invention is characterized in that a variant of barnase does not have RNase activity.

  The method for producing a multivalent binding molecule of the present invention is characterized in that it comprises only a region where a mutant of RNase burnase binds to a protein burster that specifically binds to burnase.

  According to the present invention, the RNase burnase mutant has no RNase activity and can be expressed alone, so that the step of co-expressing the inhibitor Burster can be reduced. .

Diagram showing gene organization of barnase mutant and first anti-albumin scFv antibody fusion protein expression Diagram showing gene structure of Burster and second anti-human albumin scFv antibody fusion protein expression Diagram showing the structure of the barnase mutant and the first anti-human albumin scFv antibody fusion protein Diagram showing the structure of a burster and second anti-human albumin scFv antibody fusion protein Diagram showing configuration of multivalent binding molecule Diagram showing kinetic analysis results for wild-type and multivalent molecules

(Embodiment 1)
Embodiments of the present invention will be described below with reference to the drawings. The embodiment shown here is merely an example, and is not necessarily limited to this embodiment.

The following shows an example in which a multivalent binding molecule capable of binding to different epitopes is prepared for human albumin antigen. An example of selecting scFv is shown as the molecular form of the antigen binding site. An example of using a burster with an amino acid mutation so as to lose RNase activity is shown.
1) Gene isolation of anti-human albumin antibody and production of scFv gene.
2) Step of constructing Burster and anti-human albumin scFv antibody fusion protein expression plasmid.
3) Construction of Burster and anti-human albumin scFv antibody fusion protein expression plasmid.
4) A step of expressing a burnase mutant and an anti-albumin scFv antibody fusion protein.
5) A step of expressing Burster and anti-human albumin scFv antibody fusion protein.
6) Production and purification of multivalent binding molecules.
7) A step of performing kinetic analysis of the polyvalent molecule obtained in step 6) and evaluating an improvement in binding constant.

(1) Steps for isolating anti-human albumin antibody gene and preparing scFv gene For obtaining anti-albumin antibody, for example, HSA is immunized to BALB / C mice, and spleen cells and myeloma are usually used using polyethylene glycol. After the cells are fused by the method and the reactivity to the antigen is confirmed, the anti-monoclonal antibody can be prepared by cloning by a limiting dilution method. There is also a method of obtaining an antibody gene that can specifically bind to albumin by a screening method using a DNA library, such as a phage display method or a yeast display method. For selection of clones for different epitopes, for example, by using a sandwich ELISA method or SPR for a plurality of obtained antibodies, it is experimentally evaluated whether or not a plurality of antibodies can simultaneously bind to one antigen. Can be selected.

  A method for producing a gene sequence of scFv (single chain antibody) by extracting a gene encoding an antibody variable region from the obtained monoclonal antibody as described above is shown below. First, monoclonal antibodies are cultured to about 1 × 10 6 in a medium such as RPMI1640. Next, total RNA can be extracted with TRIZOL (manufactured by Invitrogen), mRNA can be extracted with a commercially available mRNA extraction kit (manufactured by Takara Bio), and cDNA can be prepared according to a conventional method. By using the obtained cDNA as a template and performing PCR reaction using, for example, an oligonucleotide primer of Mouse ScFv Module / Recombinant Phage Antibody System (Amersham) or a primer for amplifying an antibody variable region of an existing subclass. DNA fragments encoding the V region of the H chain and L chain of the antibody can be amplified. The resulting DNA fragment encoding the H and L chain V regions is linked to a commercially available cloning vector by restriction enzyme treatment, and the gene encoding the L chain V region and the gene encoding the H chain V region A scFv gene sequence can be constructed by providing a linker sequence such as SEQ ID NO: 1 between the sequences.

  In this way, a first scFv gene and a second scFv gene that can simultaneously bind to different epitopes on two types of human albumin can be obtained.

(2) Step of constructing Burster and anti-human albumin scFv antibody fusion protein expression plasmid A gene for Burster mutant expression can be prepared and obtained using an existing artificial gene synthesis technique. An appropriate amino acid such as SEQ ID NO: 1 or the amino acid sequence of the antibody hinge region is selected as a linker between the first scFv gene obtained as described above and the Burster gene sequence. And linking using conventional methods. Further, for purification after expression of the fusion protein, a gene sequence such as a histidine tag may be inserted on the 3 ′ side of the burnase gene sequence. In addition, it is preferable to insert a signal sequence such as pelB on the 5 ′ side of the scFv gene as necessary because the target protein is carried to the periplasm. Depending on the scFv characteristics, the signal sequence may not be included. Examples of the promoter used in the expression vector include T7, tac, lac, trc promoter, etc., but it is preferable to select appropriately and appropriately depending on the characteristics of the scFv to be expressed. The vector preferably has a gene resistant to ampicillin, kanamycin, tetracycline, chloramphenicol and the like. Hereinafter, an example in which the expression vector has an ampicillin resistance gene is shown.

(3) Construction of Burster and Anti-Human Albumin scFv Antibody Fusion Protein Expression Plasmid A gene for Burster expression can be prepared and obtained using an existing artificial gene synthesis technique. An appropriate amino acid such as SEQ ID NO: 1 or the amino acid sequence of the antibody hinge region between the second scFv gene that recognizes an epitope different from the first scFv obtained as described above and the Burster gene sequence Is selected as a linker and ligated using a conventional method on a vector used for expression. Further, for purification after expression of the fusion protein, a gene sequence such as a histidine tag may be inserted on the 3 ′ side of the burnase gene sequence. If necessary, scFv
It is preferable to insert a signal sequence such as pelB on the 5 ′ side of the gene because the target protein is carried to the periplasm. Depending on the scFv characteristics, the signal sequence may not be included. Examples of the promoter used in the expression vector include T7, tac, lac, trc promoter, etc., but it is preferable to select appropriately and appropriately depending on the characteristics of the scFv to be expressed. The vector preferably has a gene resistant to ampicillin, kanamycin, tetracycline, chloramphenicol and the like. Hereinafter, an example in which the expression vector has an ampicillin resistance gene is shown.

  The type and number of amino acids of the linker that links the burnase mutant and the first scFv and the linker that binds the burster and the second scFv depend on the epitope positions of the first scFv and the second scFv. What is necessary is just to select suitably.

(4) Step of expressing barnase mutant and anti-human albumin scFv antibody fusion protein The plasmid for expression of the barnase mutant and anti-albumin scFv antibody fusion protein obtained as described above is transformed into a host for protein E. coli expression. Convert. As a host for transformation, those derived from BL21 are preferred, but any other host capable of expression can be appropriately and appropriately selected. The transformed solution is seeded on an LB plate medium containing 50 μg / ml ampicillin and cultured at 37 ° C. for 16 hours. Next, the formed single colony is picked up and cultured at 37 ° C. and 250 rpm for 5 hours using 5 mL of 2YT medium containing 50 μg / ml ampicillin. Next, 5 mL of the culture medium containing Escherichia coli after culture is added to 1 L of LB medium containing 50 μg / ml ampicillin, and the OD at a central wavelength of 600 nm of the medium becomes 0.4 to 0.9 at 37 ° C. and 250 rpm. Incubate until. Next, about 0.1 mM to 1 mM IPTG in the culture solution is added, and further cultured at 37 ° C. and 250 rpm for about 4 to 20 hours. Depending on the properties of the fusion protein, it is preferable to set the culture temperature after addition of IPTG to 16 to 30 ° C. The cultured solution is centrifuged at 5000 rpm and 4 ° C. for 10 minutes. When a signal sequence for periplasm such as pelB is incorporated into the expression vector, the supernatant after centrifugation is removed, and 30 mL of 30 mM Tris-HCl pH 8.0, 20% sucrose and 60 μl of 0.5 M EDTApH 8.0 are added. Stir slowly for 10 minutes at room temperature using a stirrer. Next, the solution is centrifuged at 10,000 g for 10 minutes at 4 ° C., and the supernatant is removed. Next, 5 mM magnesium sulfate cooled on 30 mL of ice is added and resuspended, and the mixture is slowly stirred on ice for 10 minutes. The solution after stirring is centrifuged at 10000 g for 10 minutes at 4 ° C. The obtained supernatant is passed through gel filtration chromatography, ion exchange chromatography, an affinity column preliminarily immobilized with albumin, and the like, and the target fusion protein can be purified according to a conventional method. The structure of the obtained protein is shown in FIG. If the expression in the periplasm is low or not, resuspend the precipitate after centrifugation in 30 mM Tris-HCl pH 8.0, 0.1 M NaCl, etc., and disrupt E. coli with ultrasound. To obtain soluble proteins in the cytoplasm. Further, the insoluble protein may be rewound using an existing dilution method or dialysis method. In some cases, purification is required from the culture solution in which E. coli is grown. The purification method, procedure, reagent composition, and the like are preferably selected appropriately and appropriately depending on the characteristics of the protein.

(5) Step of expressing Burster and anti-human albumin scFv antibody fusion protein Transformation of Burster and anti-human albumin scFv antibody fusion protein plasmid obtained as described above into a host for protein E. coli expression To do. As a host for transformation, those derived from BL21 are preferred, but any other host capable of expression can be appropriately and appropriately selected. The transformed solution is seeded on an LB plate medium containing 50 μg / ml ampicillin and cultured at 37 ° C. for 16 hours. Next, the formed single colony is picked up and cultured at 37 ° C. and 250 rpm for 5 hours using 5 mL of 2YT medium containing 50 μg / ml ampicillin. Next, 5 mL of the culture medium containing Escherichia coli after culture is added to 1 L of LB medium containing 50 μg / ml ampicillin, and the OD at a central wavelength of 600 nm of the medium becomes 0.4 to 0.9 at 37 ° C. and 250 rpm. Incubate until. Next, about 0.1 mM to 1 mM IPTG in the culture solution is added, and further cultured at 37 ° C. and 250 rpm for about 4 to 20 hours. Depending on the properties of the fusion protein, it is preferable to set the culture temperature after addition of IPTG to 16 to 30 ° C. The cultured solution is centrifuged at 5000 rpm and 4 ° C. for 10 minutes. When a signal sequence for periplasm such as pelB is incorporated into the expression vector, the supernatant after centrifugation is removed, and 30 mL of 30 mM Tris-HCl pH 8.0, 20% sucrose and 60 μl of 0.5 M EDTApH 8.0 are added. Stir slowly for 10 minutes at room temperature using a stirrer. Next, the solution is centrifuged at 10,000 g for 10 minutes at 4 ° C., and the supernatant is removed. Next, 5 mM magnesium sulfate cooled on 30 mL of ice is added and resuspended, and the mixture is slowly stirred on ice for 10 minutes. The solution after stirring is centrifuged at 10000 g for 10 minutes at 4 ° C. The obtained supernatant is passed through gel filtration chromatography, ion exchange chromatography, an affinity column preliminarily immobilized with human albumin, and the like, and the target fusion protein can be purified according to a conventional method. The structure of the obtained protein is shown in FIG. If the expression in the periplasm is low or not, resuspend the precipitate after centrifugation in 30 mM Tris-HCl pH 8.0, 0.1 M NaCl, etc., and disrupt E. coli with ultrasound. To obtain soluble proteins in the cytoplasm. Further, the insoluble protein may be rewound using an existing dilution method or dialysis method. In some cases, purification is required from the culture solution in which E. coli is grown. The purification method, procedure, reagent composition, and the like are preferably selected appropriately and appropriately depending on the characteristics of the protein.

(6) Step of producing and purifying a multivalent binding molecule As described above, the obtained barnase mutant, the first anti-human albumin scFv antibody fusion protein, Burster, and the second anti-human albumin svFv antibody fusion protein For example, a multivalent binding molecule as shown in FIG. 5 can be obtained by mixing them at a molar ratio of 1: 1 and separating and purifying them by a desired purification method such as gel filtration chromatography.
(7) A step of performing kinetic analysis of the polyvalent molecule obtained in (6) and evaluating the improvement of the binding rate constant.

  Next, kinetic analysis of the two types of scFv antibodies was performed using BiacoreT100 (GE Healthcare). CM5 (GE Healthcare) was used as the measurement sensor chip. About 1000 RU of human albumin (SIGMA) was immobilized on the flow cell 2 on the CM5 chip by the amide coupling method. The flow cell 1 was activated with EDC / NHS and then treated with ethanolamine to obtain a reference cell.

Next, the two scFv antibodies and multivalent molecules obtained in the above steps were replaced with HBS-EP buffer (GE Healthcare) using a PD-10 (GE Healthcare) column. Each solution was diluted with HBS-EP buffer (GE Healthcare) to obtain an analyte solution.
HBS-EP buffer (GE Healthcare) was used as a running buffer, and a binding experiment was performed at 25 ° C., a flow rate of 60 μl / min, a binding time of 120 seconds, and a dissociation time of 1200 seconds. Moreover, the glycine solution (GE health care) of pH 2.0 was used for the regeneration solution, and it was made to flow through the flow cell for 30 seconds. As a result of analyzing the obtained sensorgram with Biacore T100 evaluation software (GE Healthcare), the kinetic results shown in FIG. 6 were obtained. The multivalent molecule clearly showed improved binding to the wild type.

  The method for producing a multivalent molecule using the barnase mutant and the burster according to the present invention is useful as a method for producing a multivalent binding molecule capable of specifically binding to an antigen contained in a body fluid.

DESCRIPTION OF SYMBOLS 1 1st anti-human albumin scFv antibody 2 Barnase variant 3 Linker 4 2nd anti-human albumin scFv antibody 5 Burster 6 Linker

Claims (3)

A first molecule in which at least one binding site that specifically binds to human albumin and a mutant of RNase burnase are linked,
A human comprising a second molecule in which at least one binding part that specifically binds to the surface of a human albumin molecule different from the first molecule is linked to a protein burster that specifically binds to the enzyme active site of burnase. Albumin binding molecule.   The molecule that binds to human albumin according to claim 1, wherein the binding molecule according to claim 1 has an amino acid sequence derived from an antibody molecule.   The molecule capable of binding to human albumin according to claim 1 or 2, wherein the mutant of RNase burnase does not have RNase activity.

Images