JP3461804B2 - Method for producing Fab 'antibody with homogenized isoelectric point and Fab' antibody with homogenized fluorescent label - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a Fab ′ antibody for isoelectric focusing, and a Fab ′ antibody for homogenizing fluorescent labeling.

BACKGROUND ART Antibodies are protein molecules called immunoglobulins (immunoglobulins), and their structural characteristics make Ig
It is divided into classes such as M, IgG, IgA, IgD and IgE. In the case of humans, IgG and IgA have subclasses IgG1 to 4 and IgA1 to 2. In the case of human, for example, the IgG1 antibody has two short polypeptide chains with a molecular weight of about 24 kDa called L chain (light chain, light chain) and H chain (heavy chain, as shown in the schematic diagram of FIG. 7). Molecular weight of about 55 kDa called heavy chain)
Has a Y-shaped paired structure.

The L chain has a shape in which two domains each consisting of about 110 amino acid residues are connected, and a variable region (variable region, VL region), a constant region (constant region, CL region) are respectively formed from the N-terminal side. being called. On the other hand, the H chain has a shape in which four domains each consisting of about 110 amino acid residues are connected, and the region at the most N-terminal side is called a variable region (VH region), followed by CH1 region, CH2 region There are constant regions called CH3 regions. Moreover, a hinge region exists between the CH1 region and the CH2 region. The polypeptide chain that constitutes the two regions, the VH region and the CH1 region, is called the Fd chain. In addition, the two H chains are linked by an S-S bond, and the vicinity of the C terminus of the L chain and the H chain
The vicinity of the C-terminus of the CH1 region of the chain is also linked by an SS bond.

IgG1 is decomposed by papain and cleaved at the N-terminal side of the S-S bond connecting two H chains, and two fragments consisting of a VH region, a CH1 region and an L chain, and two CH2 regions and CH3. One piece of region is produced. The former contains a portion that binds to the antigen and Fab (fragmen
t, antigen-binding), and the latter is capable of crystallization and is called Fc (fragment, crystalline). The fragment obtained by adding the hinge region or a part thereof to Fab is called Fab ′. When IgG1 was digested with pepsin, the F-chain was cleaved at the C-terminal side of the S-S bond portion of the H chain and Fab ′ was bound by the S-S bond.
(Ab ') ₂ is obtained.

The antibody having the above-mentioned structure is contained in the antiserum obtained by immunizing an animal, which is a mixture (polyclonal antibody) of antibodies derived from various types of clones. Kohler G, Milstein C et al. Established a method for producing a single clone-derived antibody (monoclonal antibody) from a hybridoma by applying the cell fusion technology in 1975.
Since then, the monoclonal antibody has been applied to various fields such as detection and identification of trace components in the living body, clinical examination, diagnosis and treatment of diseases.

To detect or identify a trace component of a living body using a monoclonal antibody, a method of forming a complex between the trace component and the monoclonal antibody and analyzing the complex by electrophoresis is used. When the isoelectric focusing method is used as the electrophoresis method, the analysis can be performed with extremely high resolution. here,
Isoelectric focusing is the method in which the effective charge of a sample is zero.
This is an electrophoretic method that utilizes the phenomenon that the sample does not migrate at H (isoelectric point, pI). The sample is concentrated at a position equal to its isoelectric point in the pH gradient formed on the carrier for migration, and the sample becomes stationary. To do. In recent years, isoelectric focusing has been performed with an inner diameter of 50.
It was devised to perform in a capillary (capillary) made of fused silica having a length of ˜100 μm and a length of 30 to 100 cm (capillary isoelectric focusing method), and it is possible to separate even a very fine sample.

As a method of detecting the sample separated by the electrophoresis method, by irradiating the sample with ultraviolet light or visible light,
There is a method (ultraviolet-visible detection method) of detecting from the change in the amount of light due to the absorption of such light in the sample, but the sample is labeled with a fluorescent substance in advance and the separated sample components are irradiated with light. A detection method (fluorescence detection method) that quantifies the concentration by detecting emitted fluorescence is excellent from the viewpoint of high detection sensitivity.

DISCLOSURE OF THE INVENTION When using a fluorescence detection method which is a highly sensitive detection method, the target substance to be labeled with a fluorescent substance is either an antigen or an antibody, but the antigen or antibody should be fluorescently labeled by a general labeling method. Is not preferable because of the following reasons.

That is, most of the antigens and antibodies are composed of proteins, and the number of amino groups at the N-terminal of the protein and the amino groups of the lysine side chains and their dissociation state are major factors that determine the isoelectric point of the protein (continuation). Biochemistry experiment course 2,
According to the general labeling method in which a fluorescent substance is chemically bonded using an amino group, the chemistry of the protein is considered to be significantly different from that of the protein itself.

In addition, since there are many amino acids that can react with a fluorescent labeling substance in a protein, the number and position of the fluorescent labeling substance to be bound becomes unspecified, and as a result, there are multiple isoelectric points even though they are one protein. Results in a mixture. Therefore, it becomes difficult to perform accurate analysis by isoelectric focusing. Further, there is a problem that the chemical stability of the protein itself is deteriorated because the tertiary structure of the protein is changed by labeling with the fluorescent substance.

Furthermore, since antigens have a mixture where the isoelectric points are originally non-uniform even without fluorescent labeling, even if the sample is found to have multiple isoelectric points as a result of isoelectric focusing, However, it is difficult to know whether the non-uniformity of the isoelectric point of the antigen itself originates or the non-uniformity of the isoelectric point of the antigen itself is caused by the fluorescent labeling.

It is also known that even an antibody produced by a hybridoma and having a uniform molecular weight has a non-uniform isoelectric point (Bouman H et al. Z Immunitatsforsch E
xp Klin Immunol. 1975 Oct, 150 (4): 370-7), when this antibody was fluorescently labeled and subjected to isoelectric focusing, it was concentrated at a certain position on the carrier for migration and was not stationary Unable to analyze. This phenomenon is called microheterogeneity.

The cause of this isoelectric point heterogeneity is deamidation of proteins (Robinson AB et al., Proc Natl Acad Sci USA.
1970 Jul; 66 (3): 753-7), N-terminal pyroglutamylation [S
tott DI et al., Biochem J 1972 Aug; 128 (5): 1221-7), addition of sugar chains (Cohenford MA et al., Immunol Commun 1983; 12 (2):
189-200), myristoylation (Pillai S et al., Proc Natl Ac
ad Sci USA 1987 Nov, 84 (21): 7654-8) and the like have been proposed, but the megaism of isoelectric point heterogeneity of antibody or individual protein has not been identified yet.

From the above, when performing analysis by electrophoresis using the antigen-antibody reaction, the antibody must be uniform in isoelectric point, in the case of fluorescently labeling the antibody with a uniform isoelectric point,
It can be said that the fluorescent labeling method using an amino group as described above is not preferable.

As a method for obtaining an antibody with a uniform isoelectric point, Shimura K
And the method of Karger BL are known (Anal Chem 199
4 Jan. 1; 66 (1): 9-15, or Tokuhei Hei 8-506182
(See Japanese Patent Publication). The methods disclosed in these documents are schematically shown in FIGS. That is, the IgG antibody produced by the hybridoma (FIG. 8A) is cleaved with a proteolytic enzyme (pepsin), and the obtained F (ab ′) ₂ antibody (FIG. 8B) is separated. This is treated with a reducing agent such as mercaptoethylamine to form three linked disulfide bonds (S
(-S bond) is reduced to obtain Fab ′ antibody (FIG. 8C).
The Fab ′ antibody is oxidized to form a reactive thiol group (SH
Only one group (group) is left (FIG. 8D), and a fluorescent dye is bound to this thiol group (FIG. 8E). Isoelectric focusing is performed using the obtained fluorescence-labeled Fab ′ antibody, and the fluorescence-labeled Fab ′ antibody having a uniform isoelectric point is taken out from the migration carrier (FIG. 8F). However, this method has a problem in that there are many steps until obtaining a fluorescence-labeled Fab ′ antibody having a uniform isoelectric point, and that each step is complicated.
Further, the antibody taken out from the electrophoretic carrier has a problem that it is uniform in isoelectric point immediately after being taken out but becomes non-uniform over time.

The present invention has been made in view of the above-mentioned problems of the prior art, and it is possible to obtain an isoelectric point-uniformized Fab ′ antibody having a high isoelectric point stability over time without requiring a complicated step. It is an object of the present invention to provide a method for producing a Fab ′ antibody having a uniform isoelectric point. Further, a fluorescently labeled isoelectric point-uniformized Fab obtained by the method for producing the isoelectric point-homogenized Fab ′ antibody
The purpose is to provide a b ′ antibody.

As a result of intensive studies, the present inventors have found that the cause of the nonuniformity of isoelectric points is caused by the CH1 region of Fab ′ antibody, and as a result of further research, the amide group-containing amino acid in the CH1 region was found. It has been found that hydrolysis (deamidation) of residues, ie asparagine residues and / or glutamine residues, is responsible for the non-uniform isoelectric point. And
Based on this finding, by replacing at least one of the amide group-containing amino acid residues in the CH1 region with an amide group-containing amino acid residue excluding cysteine by genetic engineering, a complicated process is not required, and isoelectricity is eliminated. It was found that a Fab ′ antibody having a uniform isoelectric point and having high stability over time can be obtained. In addition, by binding a fluorescent dye to the cysteine residue of an isoelectric point uniformized Fab ′ antibody having a cysteine residue that is not involved in binding to the L chain in a portion adjacent to the C-terminus of the CH1 region, It was found that a Fab-antibody having a uniform fluorescent point and isoelectric point, which has a high stability over time of the electric point, can be obtained, and the present invention has been completed.

That is, the present invention relates to the VH region of Fab ′ antibody and CH
A first step of providing an Fd chain gene encoding 1 region, and in the Fd chain gene, at least one of the codons encoding the amide group-containing amino acid residue of the CH1 region is a non-amide group except cysteine. Site-specific mutation to a codon encoding a contained amino acid residue to give a modified F
a second step of obtaining a d-chain gene, the modified Fd-chain gene and the L-chain gene encoding the L-chain of the Fab ′ antibody are linked in an expressible state to obtain a modified Fab ′ antibody-expressing gene Third step, and obtaining the isoelectric point-uniformized Fab ′ antibody by transforming a host cell with the modified Fab ′ antibody expression gene and culturing the obtained transformant
The method for producing an isoelectric point-uniformized Fab ′ antibody, which comprises the step of

The present invention also relates to an Fd chain gene encoding the VH region and CH1 region of the Fab ′ antibody, and the L ′ of the Fab ′ antibody.
A first step of providing a light chain gene encoding a chain;
The second step of ligating the Fd chain gene and the L chain gene in an expressible state to obtain a Fab ′ antibody expression gene;
At least one of the codons encoding the amide group-containing amino acid residue of the region is site-specifically mutated to a codon encoding the amide group-free amino acid residue except cysteine to obtain a modified Fab ′ antibody expression gene. Process of
A host cell is transformed with the modified Fab ′ antibody-expressing gene, and the resulting transformant is cultured to obtain an isoelectric point-uniformized Fab ′ antibody. The present invention provides a method for producing an antibody.

Furthermore, the present invention provides a first step of providing a CH1 gene encoding a region containing a CH1 region of a first Fab ′ antibody, and a codon encoding an amide group-containing amino acid residue of the CH1 region in the CH1 gene. Of at least one of the modified CH1 by site-directed mutation to a codon encoding an amino acid residue containing no amide group except cysteine.
A second step of obtaining a gene, a third step of cleaving the modified CH1 gene with a restriction enzyme to obtain a gene fragment containing a gene encoding the CH1 region, and V of the second Fab ′ antibody
A fourth step of providing a VH gene encoding an H region and an L chain gene encoding an L chain of the second Fab ′ antibody, and capable of expressing the gene fragment, the VH gene and the L chain gene And isoelectric point homogenization by ligating in the above state to obtain a modified Fab ′ antibody-expressing gene and transforming a host cell with the modified Fab ′ antibody-expressing gene and culturing the resulting transformant. The present invention provides a method for producing an isoelectric point-uniformized Fab ′ antibody, which comprises a sixth step of obtaining a purified Fab ′ antibody.

In the present invention, the amide group-free amino acid residue is preferably an aspartic acid residue, a glutamic acid residue, a glycine residue, or a serine residue.
The ab 'antibody is the H chain No. 162 according to Kabat numbering.
Fab ′ antibody having an asparagine residue at position No.
The amide group-containing amino acid residue is preferably the asparagine residue.

The site-specific mutation is carried out by polymerase chain reaction using a CH1 region amplification primer that substitutes at least one amide group-containing amino acid residue in the CH1 region with an amide group-free amino acid residue except cysteine. Preferably.

Furthermore, prior to the first step, a primer for introducing an amino acid sequence containing a cysteine residue not involved in binding to the L chain into a portion adjacent to the C-terminus of the CH1 region was used, CDNA complementary to the mRNA of
It further comprises a step of carrying out a polymerase chain reaction using as a template, and it is preferable that the isoelectric point uniformized Fab ′ antibody has a cysteine residue that is not involved in binding to the L chain.

In the method for producing an isoelectric point-uniformized Fab ′ antibody of the present invention, a step of binding a fluorophore dye to a cysteine residue that is not involved in binding with an L chain in the isoelectric point-uniformized Fab ′ antibody is further included. It is preferable to include.

Further, the bond between the cysteine residue and the fluorophore dye is preferably at least one bond selected from the group consisting of a thioester bond, a dithioester bond, and a thioether bond, and the fluorophore dye is rhodamine, At least one fluorophore dye selected from the group consisting of fluorescein, cyanine, indocyanine, indocarbocyanine, pyronin, lucifer yellow, quinacrine, squaric acid, coumarin, fluoroanthenylmaleimide and anthracene is preferable.

Furthermore, the present invention further comprises a step of binding a fluorophore dye to a cysteine residue that is not involved in binding to the L chain in the isoelectric point-uniformized Fab ′ antibody, wherein the isoelectric point-uniformized Fab ′ is Provided is a fluorescently labeled isoelectric point-uniformized Fab ′ antibody obtained by a method for producing an antibody.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a constitutional diagram of an isoelectric point-uniformized Fab ′ antibody expression gene.

FIG. 1B is a diagram showing a pCANTAB5E plasmid vector into which an isoelectric point-uniformized Fab ′ antibody expression gene has been introduced.

FIG. 2 is a diagram showing migration time and fluorescence intensity when fluorescence detection capillary isoelectric focusing was performed using an anti-human alpha 1 antitrypsin Fab ′ antibody without modification.

FIG. 3 is a diagram showing migration time and fluorescence intensity when performing fluorescence detection capillary isoelectric focusing using a modified anti-human alpha1 antitrypsin Fab ′ antibody (H-N162D modified Fab ′ antibody). Is.

FIG. 4 shows the migration time and the fluorescence intensity when fluorescence-detecting capillary isoelectric focusing was performed using the unmodified anti-human alpha1 antitrypsin Fab ′ antibody forcibly hydrolyzed. FIG.

FIG. 5 shows that after the modified anti-human alpha1 antitrypsin Fab ′ antibody (H-N162 modified DFab ′ antibody) was forcibly hydrolyzed, fluorescence detection capillary isoelectric focusing was performed using this. It is a figure which shows the moving time and the fluorescence intensity at the time of making.

FIG. 6A is a diagram schematically showing Escherichia coli into which a gene expressing the isoelectric point-uniformized Fab ′ antibody of the present invention has been incorporated.

FIG. 6B is a diagram schematically showing an isoelectric point-uniformized Fab ′ antibody of the present invention generated by antibody induction with IPTG.

FIG. 6C shows a fluorescently labeled isoelectric point uniformizing Fa of the present invention.
It is a figure which shows a b'antibody typically.

FIG. 7 is a diagram schematically showing a human IgG1 antibody.

FIG. 8A is a diagram schematically showing an IgG antibody produced by a hybridoma.

FIG. 8B is a diagram schematically showing an F (ab ′) ₂ antibody obtained by cleaving a hybridoma-produced IgG antibody with a proteolytic enzyme.

FIG. 8C is a diagram schematically showing Fab ′ antibodies obtained by treating F (ab ′) ₂ antibodies with a reducing agent to reduce disulfide bonds.

FIG. 8D is a diagram schematically showing a Fab ′ antibody in which only one reactive thiol group remains by oxidation.

FIG. 8E is a diagram schematically showing a fluorescently labeled Fab ′ antibody.

FIG. 8F is a diagram schematically showing an electrophoretic image obtained when a fluorescently labeled Fab ′ antibody is subjected to isoelectric focusing.

BEST MODE FOR CARRYING OUT THE INVENTION A method for producing an isoelectric point-uniformized Fab ′ antibody of the present invention is F
Fd encoding the VH region and the CH1 region of the ab 'antibody
A first step of providing a chain gene; and in the Fd chain gene, encoding at least one codon encoding an amide group-containing amino acid residue of the CH1 region, and encoding an amide group-free amino acid residue except cysteine. The second step of site-specific mutation of the codon to obtain a modified Fd chain gene, the modified Fd chain gene, and L of the Fab ′ antibody
A third step of linking an L chain gene encoding a chain in an expressible state to obtain a modified Fab 'antibody-expressed gene, and
And a fourth step of obtaining an isoelectric point-uniformized Fab 'antibody by transforming a host cell with the modified Fab' antibody-expressing gene and culturing the obtained transformant.

There is no particular limitation on the method of providing the Fd chain gene encoding the VH region and CH1 region of the Fab ′ antibody in the first step, and it can be obtained, for example, by the method described below. That is, after immunizing an animal with an antigen, for example, Antibodies: A Laboratory Manual,
Chapter 6, Cold Spring Harbor Laboratory, Cold Spr
ing Harbor, NY, 1988 according to the method described in, monoclonal antibody producing cells (hybridomas) were prepared, and from the monoclonal antibody producing cells, for example, BioMag mRN
All mRNA was extracted according to the protocol of A purification kit (PerSeptive), and this mRNA was used to
A single-stranded cDNA is synthesized (for example, cDNA synthesis system plus manufactured by Amersham Pharmacia) can be preferably used. Then, the Fd chain gene can be obtained by carrying out a polymerase chain reaction (PCR) using this single-stranded cDNA as a template and a DNA primer for isolating the Fd chain gene. The DNA primer for isolating the Fd chain gene is, for example, a nucleobase sequence (Sequences of Proteins of Immunological Interion) of the variable region (V region) and the constant region (C region) classified by Kabat et al.
est 5th ed., Public Health Service, NIH, Washingto
n DC, 1991) can be referred to.

As the DNA primer, it is preferable to use a primer for introducing an amino acid sequence containing a system residue that is not involved in binding to the L chain into a portion (hinge region) adjacent to the C-terminus of the CH1 region. Hoogenboom HR et al. (Nucleic Acid
s Res 1991 Aug 11; 19 (15): 4133-7), Kang AS et al.
(Methods (San Diego) (1991), 2 (2), 111-18) and the like can be referred to. The number of amino acid residues in the amino acid sequence introduced into the portion adjacent to the C-terminal of the CH1 region is not particularly limited as long as it is 1 or more, but the number is preferably 1 to 30. Moreover, the number of cysteine residues in the amino acid sequence is not particularly limited, but the number is preferably 1 to 3, and more preferably 1. F obtained using such a primer
By using d-chain gene, isoelectric point uniformized Fab ′
A cysteine residue that does not participate in binding to the L chain can be introduced into the portion adjacent to the C-terminus of the CH1 region of the antibody.

In preparing the monoclonal antibody-producing cells in the present invention, the type of antigen immunizing an animal and the type of animal immunizing with an antigen are not particularly limited. As the antibody gene, for example, one derived from mouse, rat or rabbit can be used. The class and subclass of the antibody are not particularly limited, but it is preferable to use the sequence that constitutes the IgG antibody because it accounts for a large proportion of the whole antibody.

In the second step, in the Fd chain gene, the C
At least one of the codons encoding an amide group-containing amino acid residue in the H1 region is site-specifically mutated to a codon encoding an amide group-free amino acid residue except cysteine, but this mutation method is not particularly limited. Absent. For example, site-specific mutagenesis, which is often used as a method for mutating the nucleotide sequence of a gene,
sis) is applicable. For methods of site-directed mutagenesis, see, eg, Sambrook et al, Molecular Cl
oning: A Laboratory Manual 2nd Edition, 15.2- 15.
113, Cold Spring Harbor Press, Cold Spring Harbor,
You can refer to NY, 1989.

In the present invention, the above site-specific mutation is CH1.
It is preferable to carry out the polymerase chain reaction using a CH1 region amplification primer that substitutes at least one amide group-containing amino acid residue in the region with an amide group-free amino acid residue excluding cysteine. This primer for CH1 region amplification is a primer having a base sequence complementary to a base sequence encoding a region containing at least one amide group-containing amino acid residue in the CH1 region of the Fd chain, wherein the amide group in the base sequence is At least one codon complementary to the codon encoding the contained amino acid is a primer in which a codon complementary to the codon encoding the amino acid containing no amide group except cysteine is substituted.

The above amide group-containing amino acid residue means an amino acid residue having an amide group in its side chain, and examples of such an amino acid residue include an asparagine residue and a glutamine residue. In the second step, at least one of the codons encoding these amino acid residues is site-specifically mutated to a codon encoding an amide group-free amino acid residue except cysteine. Here, the amide group-free amino acid residue means an amino acid residue having no amide group in the side chain. As the amide group-free amino acid residue excluding cysteine, both a natural amino acid residue and a non-natural amino acid residue are applicable. Natural amino acid residues include glycine residues, alanine residues, valine residues,
Leucine residue, isoleucine residue, serine residue, threonine residue, methionine residue, aspartic acid residue, glutamic acid residue, lysine residue, arginine residue, phenylalanine residue, tyrosine residue, brolin residue, histidine Residues, and tryptophan residues. Examples of the non-natural amino acid residue include amino acid in which the side chain of natural amino acid is substituted with an aromatic ring or the like, residues of artificial amino acid such as chemically synthesized amino acid, and the like.

In the present invention, at least one of the amide group-containing amino acid residues is mutated to an amide group-free amino acid residue excluding cysteine, which is obtained when a cysteine residue is used as the amide group-free amino acid residue. Although the Fab 'antibody has a uniform isoelectric point, a large number of cysteine residues that are not involved in binding to the L chain are introduced, and when a fluorophore dye is bound via the cysteine residue, fluorescence is emitted. The number and position of the dye groups are unspecified, resulting in a mixture showing multiple isoelectric points. Therefore, in the present invention, a cysteine residue is not used as an amide group-free amino acid residue.

In the present invention, since the isoelectric point homogenizing Fab ′ antibody obtained is excellent in homogeneity of the isoelectric point, the above-mentioned amide group-containing non-containing amino acid residue is an aspartic acid residue, a glutamic acid residue, or a glycine residue. It is preferably a group or a serine residue. The Fab ′ antibody used is a Fab ′ antibody having an asparagine residue at the H-chain number 162 according to Kabat numbering, and the asparagine residue is site-specific to an amide group-free amino acid residue except cysteine. It is preferable to mutate. In the present invention, it is more preferable to mutate the asparagine residue of the H chain No. 162 according to Kabat numbering to an aspartic acid residue. The Fab ′ antibody having an asparagine residue at the 162nd H chain by Kabat numbering is mouse I
Examples thereof include Fab antibody derived from gG antibody and Fab 'antibody derived from human IgG antibody. There are various subclasses of mouse IgG antibody and human IgG antibody, and even if the subclasses are different, F derived from these antibodies
The ab 'antibody has an asparagine residue at H-chain number 162 according to Kabat numbering. Here, the amino acid residue of the H chain No. 162 by Kabat numbering is Fa
This means the 162nd amino acid residue in the H chain (Fd chain) of the b'antibody, and this 162nd number is from Sequences of Proteins of Immunological Interes by Elvin A Kabat.
t (Paperback 5th edition (September 1992)) means the position of an amino acid residue specified based on the method described therein. It should be noted that the H chain No. 162 according to the Kabat numbering of the Fab ′ antibody exists in the CH1 region of the Fd chain.

The modified Fd chain gene obtained in the above-mentioned second step is ligated in the third step so that it can be expressed with the L chain gene encoding the L chain of the Fab ′ antibody. As a result, a modified Fab 'antibody-expressed gene is obtained.

The L chain gene encoding the L chain of Fab ′ antibody is
It can be obtained by the same method as the method of obtaining the Fd chain gene in the step. That is, after immunizing an animal with an antigen, a monoclonal antibody-producing cell (hybridoma)
From the monoclonal antibody-producing cells.
RNA is extracted and single-stranded cDNA is used by using this mRNA.
A may be synthesized, and PCR may be performed using this cDNA as a template and a DNA primer for isolating the L chain gene. In the present invention, the L chain gene is Fd in the first step.
It can also be obtained at the same time as the chain gene is isolated. In this case, in the first step, DN for Fd chain gene isolation
PCR may be carried out by using the A primer and the DNA primer for isolating the L chain gene together and using the cDNA as a template.

In the present invention, in addition to site-directed mutation of at least one codon encoding an amide group-containing amino acid residue in the CH1 region of the Fd chain gene to a codon encoding an amide group-free amino acid residue except cysteine, , In the L chain gene obtained as described above,
At least one codon encoding an amide group-containing amino acid residue (asparagine residue and / or glutamine residue) in the CL region is site-specific mutated to a codon encoding an amide group-free amino acid residue except cysteine. May be. This site-specific mutation can be performed before or after ligation with the Fab ′ antibody in an expressible state. In this case, the Fab ′ antibody used is L chain No. 157, L chain No. 161, L chain No. 1 by Kabat numbering.
Fab ′ antibody having an asparagine residue in at least one of position 90 (both in the CL region) is preferable, and at least one of the asparagine residues is
Site-directed mutagenesis to amino acid residues containing no amide group except cysteine is preferred. Among them, the asparagine residue of the L chain No. 161 by Kabat numbering is
It is preferable to mutate the amide group-containing amino acid residue except cysteine site-specifically, and it is further preferable to mutate the asparagine residue of the L chain No. 161 to an aspartic acid residue. As for the method of site-directed mutagenesis, as described above, Sambrook et al., Molecular Cloning: AL
aboratory Manual 2nd Edition, 15.2- 15.113, Cold S
You can refer to pring Harbor Press, Cold Spring Harbor, NY, 1989. Further, as in the case of the CH1 region, the site-specific mutation of the CL region is for CL region amplification in which at least one amide group-containing amino acid residue of the CL region is replaced with an amide group-free amino acid residue except cysteine. It is preferably carried out by polymerase chain reaction using primers.

The L chain gene thus obtained and the above-mentioned modified Fd
By linking the chain gene with a linker base sequence or the like, a modified Fab ′ antibody expression gene capable of expressing both genes can be obtained. More specifically, the L chain gene, the modified Fd chain gene and the linker base sequence are purified by, for example, low melting point agarose gel electrophoresis, and these are digested with an appropriate restriction enzyme to obtain the modified Fd chain gene, the linker base sequence, By ligating so that the L chain genes are arranged in order, a modified Fab ′ antibody expression gene in which the L chain gene and the modified Fd chain gene are linked in an expressible state can be obtained. The linker base sequence can be obtained, for example, using a plasmid vector for protein expression as a template and a DNA primer for isolating the linker base sequence.

In the fourth step following the third step, the modified F
The host cell was transformed with the gene expressing the ab 'antibody, and the resulting transformant was cultured to obtain an isoelectric point-uniformized Fa.
Obtain the b'antibody.

That is, the modified Fab ′ antibody expression gene obtained in the third step is ligated to an appropriate vector, which is introduced into a host cell for transformation. As the vector, various known vectors such as those derived from plasmids, those derived from phages and cosmids can be used. Examples of host cells include prokaryotic cells, that is, Escherichia coli (SOLR, J
M109, XL1-BlueMRF ', BL21 (DE3), HB2151), Bacillus subtilis, Bacillus brevis, eukaryotic cells, that is, yeast, animal-derived cells (HB101, CHO cells, COS)
Cells, COP-5, C127, 3T3 cells, etc.). As the host cell, the isoelectric point uniformized Fab ′ of the present invention is used.
It is preferable to use a non-proteolytic enzyme-producing bacterium because the antibody is less likely to be degraded.

As a method for introducing the vector into a host cell, a known method including a microinjection method, an electroporation method and the like can be applied. The method for culturing the transformant is not particularly limited, and a medium suitable for culturing the transformant may be selected. Further, as a method for extracting the isoelectric point-uniformized Fab ′ antibody produced by culturing the transformant, a method for homogenizing cells, a method for lysing a cell membrane using a surfactant or enzyme such as SDS, and There is sonication. Examples of the method for purifying the extracted isoelectric point-uniformized Fab ′ antibody include, for example, a centrifugation method using ultracentrifugation and a density gradient centrifugation, a column separation method using an affinity column, and a gel using a polyacrylamide gel. Examples include a separation method.

In the method described above, site-directed mutagenesis of the Fd chain gene was performed and then ligation with the L chain gene was performed. However, site-directed mutagenesis of the Fd chain gene was performed after ligating the Fd chain gene and the L chain gene. It is also possible to do

That is, first, in a first step, an Fd chain gene encoding the VH region and CH1 region of Fab ′ antibody,
The step of providing the L chain gene encoding the L chain of the Fab ′ antibody is performed, and in the subsequent second step, the Fd chain gene and the L chain gene are linked in an expressible state, and Fab ′ Obtain the antibody-expressed gene. Here, the method for obtaining the Fd chain gene and the L chain gene is not particularly limited,
For example, as described above, total mRNA is extracted from a monoclonal antibody-producing cell (hybridoma), single-stranded cDNA is synthesized using this mRNA, and this cDNA is prepared.
PCR may be performed using A as a template and a DNA primer for Fd chain gene isolation and a primer for L chain gene isolation. When obtaining the Fd chain gene, it is preferable to use a primer for introducing an amino acid sequence containing a cysteine residue that is not involved in binding to the L chain into a portion adjacent to the C-terminus of the CH1 region. Fd thus obtained
The method for ligating the chain gene and the L chain gene in an expressible state is not particularly limited. For example, a linker base sequence is obtained by using a DNA vector for isolating the linker base sequence using a plasmid vector for protein expression as a template. ,
The linker base sequence may be ligated to the Fd chain gene and the L chain gene.

In the third step, in the Fab ′ antibody-expressing gene, at least one of the codons encoding the amide group-containing amino acid residue of the CH1 region is located at the codon encoding the amide group-free amino acid residue except cysteine. A specific Fab 'antibody-expressed gene is obtained by performing a specific mutation.

The method of this site-directed mutation is not particularly limited, and for the purpose of amplifying a CH1 region in which at least one amide group-containing amino acid residue in the CH1 region is replaced with an amide group-free amino acid residue excluding cysteine, as described above. It is preferably carried out by polymerase chain reaction using primers. Examples of the amide group-containing amino acid residue include an asparagine residue and a glutamic acid residue, and examples of the amide group-containing non-containing amino acid residue include a natural amino acid residue excluding cysteine and an unnatural amino acid residue as described above. To be As the amino acid residue containing no amide group, an aspartic acid residue, a glutamic acid residue, a glycine residue, or a serine residue is particularly preferable. In addition, Fab 'to be used
It is preferable that the antibody is a Fab ′ antibody having an asparagine residue at the H-chain No. 162 according to the Kabat numbering, and this asparagine residue is site-specifically mutated to an amide group-free amino acid residue except cysteine. As the amide group-free amino acid residue, an aspartic acid residue is preferable.

Here, before or after ligating the L chain gene encoding the L chain of the Fab ′ antibody with the Fd gene, the amide group-containing amino acid residue (asparagine residue and / or glutamine residue) of the CL region in the L chain gene is At least one of the codons encoding the group) may be site-directed to a codon encoding an amino acid residue containing no amide group except cysteine. In this case, the Fab ′ antibody is a Fab ′ antibody having an asparagine residue in at least one of L chain No. 157, L chain No. 161, and L chain No. 190 according to Kabat's numbering. At least one of the groups is preferably site-directed to an amide group-free amino acid residue except cysteine. Among them, the first L chain by Kabat numbering
The asparagine residue at position 61 is preferably site-specifically mutated to an amide group-free amino acid residue except cysteine, and more preferably the asparagine residue at position 161 of the L chain is mutated to an aspartic acid residue.

In the fourth step, the modified Fab ′ is modified as described above.
An isoelectric point-uniformized Fab ′ antibody can be obtained by transforming a host cell with the antibody-expressing gene and culturing the obtained transformant. Type of host cell used at this time, type of vector introduced into host cell, method of introducing vector into host cell, and method of extracting isoelectric point uniformized Fab ′ antibody produced by culture of transformant The same applies to the above.

As the method for producing the isoelectric point-uniformized Fab ′ antibody, the following method can be applied in addition to the above method. That is, the heterogeneity of the isoelectric point of the Fab ′ antibody is caused by the hydrolysis (deamidation) of the amide group-containing amino acid residue in the CH1 region.
First, a CH1 gene in which an amide group-containing amino acid residue in the CH1 region is mutated to an amide group-free amino acid residue excluding cysteine is prepared, and this gene is homologous or different. Genes for obtaining various Fab ′ antibodies having a uniform isoelectric point were obtained by ligating with the VH gene and L chain gene of the antibody in an expressible state, and using this, an isoelectric point-uniformized Fab was obtained. '
Antibodies can be produced. Here, since the above CH1 gene can be inserted like a cassette between the VH gene, the L chain gene and other nucleotide sequences required for expression, various types of isoelectric point uniformized Fab ′ antibodies can be used. Can be easily obtained.

This method will be described in more detail below. That is, first, as a first step, a step of providing a CH1 gene encoding a region including the CH1 region of the first Fab ′ antibody is performed. As described above, for this CH1 gene, for example, as described above, total mRNA is extracted from a monoclonal antibody-producing cell (hybridoma) cell, single-stranded cDNA is synthesized using this mRNA, and this cDNA is used as a template for the CH1 region. PCR with covering primers
Can be obtained by performing. CH1 gene is C
Any code may be used as long as it encodes a region including the H1 region, and C
Even if it encodes only the H1 region, the CH1 region and VH
It may be a code for the area. Further, it may encode the CH1 region and the hinge region, or may encode the CH1 region, the VH region and the hinge region. In addition, VH
At least a part of the region and the hinge region may be coded. In addition, as a primer used for PCR, it is preferable to use a primer for introducing an amino acid sequence containing a cysteine residue that is not involved in binding to the L chain into a portion adjacent to the C-terminus of the CH1 region. The preferred number of amino acid residues and cysteine residues in this amino acid sequence is the same as above.

In the second step following the first step, CH1
In the gene, at least one of the codons encoding an amide group-containing amino acid residue in the CH1 region is site-specifically mutated to a codon encoding an amide group-free amino acid residue except cysteine to obtain a modified CH1 gene.

The method of this site-directed mutation is not particularly limited, and for the purpose of amplifying a CH1 region in which at least one amide group-containing amino acid residue in the CH1 region is replaced with an amide group-free amino acid residue excluding cysteine, as described above. It is preferably carried out by polymerase chain reaction using primers. Further, similar to the above, examples of the amide group-containing amino acid residue include an asparagine residue and a glutamic acid residue, and examples of the amide group-containing non-containing amino acid residue include natural amino acid residues other than cysteine and unnatural amino acid residues. Groups. As the amino acid residue containing no amide group, an aspartic acid residue, a glutamic acid residue, a glycine residue, or a serine residue is particularly preferable. In addition, the Fab ′ antibody to be used is the 16th H chain by Kabat numbering.
It is a Fab ′ antibody having an asparagine residue at position 2, and it is preferable to site-specifically mutate this asparagine residue to an amide group-free amino acid residue excluding cysteine. , Aspartic acid residues are preferred.

Next, in the third step, the modified CH1 gene is cleaved with a restriction enzyme to obtain a gene fragment containing the gene encoding the CH1 region. The restriction enzyme used here is not particularly limited, but examples of the restriction enzyme include Ba
Examples include mHI and BgII.

In the fourth step, the VH gene encoding the VH region of the second Fab ′ antibody and the L of the second Fab ′ antibody
And a light chain gene encoding the chain. Here, the VH gene and the L chain gene of the second Fab ′ antibody are determined using, for example, the mRNA of the antibody from which the second Fab ′ antibody is derived.
Synthesized double-stranded cDNA and used this cDNA as a template for VH
It can be obtained by performing PCR using the DNA primer for chain gene isolation and the DNA primer for L chain gene isolation. The second Fab ′ antibody may be the same or different in class or subclass from that of the first Fab ′ antibody. For example, the second Fab '
The antibody may be a mouse IgA antibody and the first Fab ′ antibody may be a mouse IgG antibody. Furthermore, the second Fa
The animal species from which the b'antibody is derived may be different or the same as that of the first Fab 'antibody. For example, second
Fab ′ antibody is a human IgG antibody, and the Fab
The b'antibody may be a mouse IgG antibody.

Further, in the L chain gene encoding the L chain of the second Fab ′ antibody, at least one codon encoding the amide group-containing amino acid residue (asparagine residue and / or glutamine residue) of the CL region is replaced with cysteine. May be site-specifically mutated to a codon encoding an amide group-free amino acid residue other than. In this case, the Fab ′ antibody is a Fab ′ antibody having an asparagine residue in at least one of L chain No. 157, L chain No. 161, and L chain No. 190 according to Kabat's numbering. At least one of the groups is preferably site-directed to an amide group-free amino acid residue except cysteine. Among them, the first L chain by Kabat numbering
The asparagine residue at position 61 is preferably site-specifically mutated to an amide group-free amino acid residue except cysteine, and more preferably the asparagine residue at position 161 of the L chain is mutated to an aspartic acid residue.

In the fifth step following the fourth step, the gene fragment, the VH gene and the L chain gene are ligated in an expressible state to obtain a modified Fab ′ antibody expression gene.
The method of ligation in an expressible state is not particularly limited, and for example, a protein expression plasmid vector is used as a template to obtain a linker base sequence using a DNA primer for isolation of the linker base sequence, and the linker base sequence and the Fd chain are The gene and the L chain gene may be ligated so that they can be expressed.

In the sixth step, host cells are transformed with the modified Fab ′ antibody-expressing gene, and the resulting transformant is cultured to obtain an isoelectric point-uniformized Fab ′ antibody. Type of host cell used at this time, type of vector introduced into host cell, method of introducing vector into host cell, and isoelectric point homogenizing F produced by culturing transformant
The method for extracting the ab 'antibody is the same as above.

The isoelectric point-uniformized Fab ′ antibody obtained by any of the above-mentioned three production methods has at least one amide group-containing amino acid residue in the CH1 region converted to an amide group-free amino acid residue except cysteine. However, since such conversion of the amino acid residue in the CH1 region does not affect the antigen-antibody reaction, the isoelectric point-uniformized Fab ′ antibody is produced by, for example, isoelectric focusing. It can be applied to the detection and identification of trace components in the body. At this time, the isoelectric point-uniformized Fab ′ antibody used is preferably labeled with a labeling agent. The type of labeling agent is not particularly limited,
It is preferred to use fluorophore dyes.

In producing the isoelectric point-uniformized Fab ′ antibody, CH
Using a primer for introducing an amino acid sequence containing a cysteine residue that is not involved in binding to the L chain into a region adjacent to the C terminus of the 1 region, a cDNA complementary to mRNA derived from antibody-producing cells is used as a template. When the chain reaction is performed, the obtained isoelectric point-uniformized Fab ′ antibody is C
Since the portion adjacent to the C-terminal of the H1 region has a cysteine residue that is not involved in binding to the L chain, a fluorophore dye can be bound to the SH group of this cysteine residue.

As the fluorophore dye, at least one fluorophore selected from the group consisting of rhodamine, fluorescein, cyanine, indocyanine, indocarbocyanine, pyronin, lucifer yellow, quinacrine, squaric acid, coumarin, fluoroanthenylmaleimide, and anthracene. A dye is preferable, and rhodamine and / or cyanine are particularly preferable. Rhodamine is more preferably used as the fluorophore dye. Rhodamine is 556n in methanol
It has a maximum absorption at m (molar extinction coefficient 93,000) and emits fluorescence having a maximum at 576 nm. For Rhodamine, Handbook of Fluoresc
ent Probes and Research C
chemicals, 5th Edition MOLE
Reference may be made to CULAR PROBES, INC., 1992.

Further, in the present invention, an aromatic heterocyclic compound represented by anthracene, naphthalene, phenanthrene, quinoline, birene, perylene, etc. or a polycyclic aromatic hydrocarbon can be used as a fluorophore dye. For such a fluorophore dye, reference can be made to, for example, fluorescent phosphorescence analysis, Taiji Nishikawa, Keizo Hiraki, Kyoritsu Shuppan, 1989.

When the above fluorophore dye is reacted with the isoelectric point-uniformized Fab ′ antibody, a cysteine residue that is not involved in the binding between the functional group in the fluorophore dye and the L chain in the isoelectric point-uniformized Fab ′ antibody The SH group of the group may be directly reacted, or the fluorophore dye and the SH group may be reacted via a polyfunctional compound. Here, the polyfunctional compound is a functional group reactive with a functional group in the fluorophore dye (for example, a halogenated methyl group, an active ester group, an acid chloride group, an acid anhydride group, a maleimide group, etc.). , And SH groups each have at least one reactive functional group. In any of the above cases, the bond generated by the reaction between the fluorophore dye and the Fab ′ antibody for homogenizing the isoelectric point is at least one bond selected from the group consisting of a thioester bond, a dithioester bond, and a thioether bond. Is preferred.

By the method described above, the isoelectric point-uniformizing Fab '
An antibody can be obtained, but the homogeneity of the isoelectric point is obtained by converting at least one of the amide group-containing amino acid residues in the CH1 region into an amide group-free amino acid residue excluding cysteine. Therefore, as long as this conversion maintains the isoelectric point uniformity, Fab ′
A part of the antibody may be further mutated by genetic engineering.
For example, the value of the isoelectric point can be changed while maintaining the homogeneity of the isoelectric point by introducing a charged amino acid into the C-terminal side of the L chain of the Fab ′ antibody by genetic engineering.

As described above, the isoelectric point uniformizing Fab ′ of the present invention is used.
Since the method for producing the antibody is a method for producing a Fab ′ antibody having a uniform isoelectric point by using a genetic engineering technique, it is disclosed in
Compared with the prior art disclosed in Japanese Patent Publication No. 506182, the steps until obtaining an isoelectric point-uniformized Fab ′ antibody are simple. That is, the method disclosed in Japanese Patent Publication No. 8-506182 discloses a step of cleaving an antibody produced by a hybridoma with a proteolytic enzyme to obtain an F (ab ′) ₂ antibody.
b ′) _{2 a} step of treating the antibody with a reducing agent to obtain a Fab ′ antibody, a step of oxidizing the Fab ′ antibody to leave only one reactive thiol group, and a fluorescent label bound to the thiol group with a fluorescent dye. Obtaining Fab ′ antibody, said fluorescently labeled Fab
Step of performing isoelectric focusing using b'antibody, fluorescent labeled Fab 'having a uniform isoelectric point from the electrophoretic carrier for isoelectric focusing
In contrast to the step of removing the antibody and the like, in the case of obtaining a fluorescence-labeled isoelectric point-uniformized Fab ′ antibody by the production method of the present invention, a Fab ′ antibody-expressing gene is obtained from cells producing the antibody The process is not complicated because it is sufficient to mutate the'antibody-expressed gene in a site-specific manner, culture the host cell transformed by the mutation, and fluorescently label the resulting isoelectric point-uniformized Fab 'antibody.

In addition, the fluorescence-labeled isoelectric point-uniformized Fab ′ antibody obtained by the conventional technique disclosed in Japanese Patent Publication No. 8-506182 is hydrolyzed even if the isoelectric point is uniform immediately after being taken out from the electrophoretic carrier. The isoelectric point becomes heterogeneous in a relatively short time by the method, whereas the fluorescence-labeled isoelectric point-equalized Fab ′ antibody obtained by the method of the present invention has a portion (CH1 region) that is susceptible to hydrolysis reaction and the like. The amide group-containing amino acid residue of 1) is genetically mutated to a compound (amino acid residue containing no amide group) that is difficult to undergo a hydrolysis reaction, so that the isoelectric point is highly stable over time.

(Examples) Hereinafter, preferred examples of the present invention will be described in more detail, but the present invention is not limited to these examples. In the examples, those not particularly described regarding genetic engineering methods are Sambrook et al., Molecular
Cloning: A Laboratory Manual 2nd Edition, Cold S
pring Harbor Press, Cold Spring Harbor, NY, 1989. In addition, reagents not specifically mentioned were those purchased from Takara Shuzo Co., Ltd. or Wako Pure Chemical Industries, Ltd.

  The names of abbreviations used in this example are shown below.

PCR: Polymerase chain reaction (gene amplification method) BAP: bacterial alkaline phosphatase IPTG: isopropyl-β-D-thiogalactopyranoside PBS: phosphate buffered saline BSA: bovine serum albumin (1) Establishment of hybridoma human alpha 1 antitrypsin (calbiochem-)
(Novabiochem) was used as an immunizing antigen to prepare a hybridoma producing an anti-human alpha 1 antitrypsin antibody by the method described below.

After BALB / c mice were immunized four times with the above immunogen, spleen cells were collected and subjected to cell fusion using cultured mouse bone marrow cells [X63Ag8] and polyethylene glycol for cloning.
The binding activity of the antibody in the culture supernatant of the obtained clones to the aforementioned immunogen was measured by the enzyme-antibody method, and for clones for which the reaction was considered to be positive, further confirmed using the indirect fluorescence method, Nine types of hybridomas producing alpha 1 antitrypsin antibody were established. The antibodies produced by these hybridomas bind to human alpha 1 antitrypsin. To prepare the Fd chain gene and L chain (κ chain) gene of the isoelectric point-uniformized Fab ′ antibody described below, cells producing these anti-human alpha1 antitrypsin antibodies having anti-alpha1 antitrypsin activity were prepared. used.

(2) Isolation of anti-human alpha 1 antitrypsin Fab ′ antibody expression gene IgG1 against human alpha 1 antitrypsin
Anti-human alpha 1 from hybridoma producing antibody
The antitrypsin Fab ′ antibody expression gene was isolated as follows.

That is, anti-human alpha 1 antitrypsin Fa
BioMag mRNA purification from cells producing b'antibody
All RNs according to the protocol of kit (PerSeptive)
A was extracted, and single-stranded cDNA was synthesized using cDNA synthesis system plus (manufactured by Amersham Pharmacia). Kabat et al. (Sequences of Proteins of Immunologi
cal Interest 5th ed., Public Health Service, NIH, W
(A. Washington DC, 1991), based on the nucleobase sequences of the variable region (V region) and the constant region (C region) classified by Fash chain gene isolation DNA primer and L chain gene isolation DNA primer Using the above single-stranded cD
Polymerase chain reaction (PCR) was performed using NA as a template. Here, Hoogenboom HR et al.
(Nucleic Acids Res 1991 Aug 11; 19 (15): 4133-7), and Kang AS et al. (Methods (San Diego) (1991), 2 (2), 111.
-18) was referred to.

An antibody that binds to human alpha 1 antitrypsin is expressed as a Fab ′ antibody, and therefore, the heavy chain (H
DNA so that both the chain) and the light chain (L chain) contain the constant region
The primer was designed. That is, as a DNA primer for Fd chain gene isolation, a 5 ′ side primer (F5-1 primer shown below) and a 3 ′ side primer (F3 primer shown below) were designed, and D chain for L chain gene isolation was designed.
As NA primers, a 5'side primer (Kapper5 primer shown below) and a 3'side primer (K3-1 primer shown below) were designed.

F5-, which is a 5'-side primer for Fd chain gene isolation
One primer has the sequence (SEQ ID NO: 1) shown below, and the F3 primer, which is the 3'side primer for Fd chain gene isolation, has the sequence (SEQ ID NO: 2) shown below. I was there. In the following sequences, 5'and 3'are 5'side and 3'of the primer, respectively.
Represents a side, S represents C or G, M represents A or C, R represents A or G, and W represents A or T.

F5-1 primer (SEQ ID NO: 1): 5 ′ SAGGTSMARCTGCAGSAGTCWGG 3 ′ F3 primer (SEQ ID NO: 2): 5 ′ GCGTCATCTAGAACAACCACAATCCCTGGGCACA 3 ′ The F3 primer has a disulfide bond with the L chain at a portion adjacent to the C-terminal of the CH1 region. Designed to be able to introduce a nucleotide sequence containing a cysteine residue that is not involved in
Also, since it is linked to the L chain via a linker, Xba I
Designed to be added by the site.

Kapp which is a 5'-side primer for L chain gene isolation
The er5 primer has the sequence (SEQ ID NO: 3) shown below, and the K3-1 primer, which is the 3'side primer for L chain gene isolation, has the sequence (SEQ ID NO: 4) shown below. Was used. In addition, W is A or T, S is C or G, B is a base other than A, N is A, T,
G, C, and M are A or C, D is a base other than C, Y is C or T, and H is a base other than G, respectively.

Kapper 5 primer (SEQ ID NO: 3): 5'CCAGWTSYGAGCTCSWBNTSACNCAGNMDYCH 3 'K3-1 primer (SEQ ID NO: 4): 5'ACACTCATTCCTGTTGAAGCT 3' PCR conditions are 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C 1
30 cycles were performed in minutes. After PCR, the obtained Fd chain,
The DNA fragment of the linker base sequence and L chain (κ chain) was purified by low melting point agarose gel electrophoresis, and the Fd chain was analyzed with XbaI.
XbaI and SacI for the linker base sequence and SacI for the L chain (κ chain)
Digested with. Each D is arranged so that the Fd chain, the linker base sequence, and the L chain (κ chain) are arranged in this order with the linker base sequence sandwiched therebetween.
The NA fragment was ligated. The ligation product was extracted with phenol / chloroform / isoamyl alcohol (25/24/1). This was dissolved in TE buffer and used as a template to prepare a SfiI site on the 5'side of the Fd chain and 3'of the L chain.
PCR was performed again with a primer designed to add a Not I site to the side. PCR is 94 ° C for 1 minute, 5
25 cycles were performed at 5 ° C. for 1 minute and 72 ° C. for 2.5 minutes.

After purification of the obtained PCR amplification product, SfiI (20U per re
Action) at 50 ℃ for 4 hours, Not I (40U per reactio
n) and digested at 37 ° C. for 4 hours. This was cloned into pCANTAB5E plasmid vector (manufactured by Amersham Pharmacia) (see FIGS. 1A and 1B). The pCANTAB5E plasmid vector contains a signal peptide that expresses the gene incorporated in the vector and secretes the gene-derived protein outside the beryplasm of Escherichia coli. As for the procedure and method for constructing a vector, those commonly used in the field of genetic engineering can be used.

(3) Transformation for expressing anti-human alpha1 antitrypsin Fab 'antibody An anti-human alpha1 antitrypsin Fab' antibody expression plasmid obtained by cloning an anti-human alpha1 antitrypsin Fab 'antibody expression gene into a pCANTAB5E plasmid vector is commercially available. E. coli HB2151 (manufactured by Amersham Pharmacia) was transformed. Expression Module / Recombinant for the transformation of E. coli HB2151 into competent cells
The protocol of Pharge Antbody System (manufactured by Amersham Pharmacia) was followed. Transformed E. coli HB215
1 was plated on SOBAG medium and incubated overnight at 30 ° C. HRP / Anti-E tag C in the resulting colony
colony lift assay according to the onjugate (Amersham Pharmacia) protocol
Then, Fab ′ antibody-expressing bacteria that cause an antigen-antibody reaction against human alpha 1 antitrypsin were screened.

Select multiple Fab 'antibody-expressing bacteria by screening and select Expression Module / Recombinant Pharge Antbody S
According to the protocol of ystem (manufactured by Amersham Pharmacia), 2YT-AG medium was inoculated and cultured at 30 ° C. with shaking overnight. The shake-cultured culture solution was added to 10 times the amount of 2YT-AG medium, and shake-cultured at 30 ° C. until A600 reached 0.5. The cells were collected by centrifugation at room temperature and the supernatant was removed. The cells were suspended in the same amount of 2YT-AI (without glucose, 100 μg / ml ampicillin, containing 1 mM IPTG) medium and cultured at 30 ° C. overnight with shaking to induce antibodies. Then, the bacteria were precipitated by centrifugation, the supernatant was taken, 100 μl of the culture supernatant containing the anti-human alpha1 antitrypsin Fab ′ antibody induced by IPTG was used, and the microtiter plate was used as an antigen adsorption plate to Alpha-1 antitrypsin (manufactured by Calbiochem-Novabiochem) was immobilized, and ELISA was performed to screen anti-human alpha1 antitrypsin Fab ′ antibody-expressing bacteria according to the protocol of HRP / Anti-E tag Conjugate (manufactured by Amersham Pharmacia).

(4) Transformation for Nucleotide Sequence Determination and Determination of Nucleotide Sequence of Antibody Gene From the anti-human alpha 1 antitrypsin Fab ′ antibody expressing bacterium obtained by screening, plasmid DN
A was extracted and commercially available E. coli XL10-GOL for nucleotide sequencing
D (Stratagene) was transformed. Anti-human alpha 1
XL10-GOLD transformed with antitrypsin Fab 'antibody expression gene is Epicurian Coli XL10-Gold ultracompete
According to nt Cells (Stratagene), DNA (about 10 ng) of a vector expressing an antibody gene was mixed and left in ice for 30 minutes. Then, after heat-treating at 42 ° C for 30 seconds, NZY medium (NZ amine 10 g, yeast extract 5 g,
Sodium chloride 5 g, magnesium chloride 12.5 mM, magnesium sulfate 12.5 mM, glucose 20 mM :, pH 7.
(5: 1 per liter) 900 μL was added, and the mixture was cultured at 37 ° C. for about 1 hour with shaking. LB agar medium containing 50 μg / ml ampicillin (10 g tryptone, 5 g yeast extract,
5 g of sodium chloride and 15 g of agar [pH 7] (per 1 liter) were spread and the transformed strain containing the transformed human anti-alpha1 antitrypsin Fab ′ antibody expression gene was selected.

From the selected resistant strains, pCANTAB5R plasmid containing the anti-human alpha1 antitrypsin F'ab antibody expression gene was extracted, and anti-human alpha1 antitrypsin was prepared by the chain terminator method using dideoxyoxynucleotide (Perkin Elmer). When the nucleotide sequence of each part of the Fab ′ antibody-expressed gene was determined, it was found to have an oven-reading frame (ORF) capable of expression.
In addition, the isolated anti-human alpha1 antitrypsin Fab ′ antibody expression gene contained an Fd chain gene (VH region and CH1 region gene) and an L chain gene (VL region and CL region gene). .

(5) Induction and purification of E. coli-produced anti-human alpha 1 antitrypsin Fab 'antibody E. coli-produced anti-human alpha 1 antitrypsin Fab
In order to use the b'antibody in the experiments described below, the screened cell lines were used to expand the culture scale to induce the antibody, and then purified. That is, RPAS Purification Modu
According to the protocol of le (Amersham Pharmacia), the induction and purification of the anti-human alpha 1 antitribucin Fab ′ antibody was carried out by the following procedure.

A single colony was picked from the Escherichia coli HB2151 cell line containing the screened anti-human alpha 1 antitrypsin Fab 'antibody expression gene, and then Expression Module / Recomb
According to the protocol of inant Pharge Antbody System (manufactured by Amersham Pharmacia), the 2YT-AG medium was inoculated and cultured with shaking at 30 ° C. overnight. The shaking-cultured culture solution was inoculated into a 10-fold amount of 2YT-AG medium, and shake-cultured at 30 ° C. overnight. The shake-cultured culture solution was added to 10 times the amount of 2YT-AG medium, and shake-cultured at 30 ° C. until A600 reached 0.5. The cells were collected by centrifugation at room temperature and the supernatant was removed. Suspend the bacteria in the same amount of 2YT-AI (without glucose) medium and
The cells were shaken and cultured overnight. The bacteria were precipitated by centrifugation, the supernatant was taken, filtered with a 0.45 um filter (manufactured by Millipore), and the pH was adjusted to 7 to obtain a culture supernatant.

The culture supernatant containing the anti-human alpha 1 antitrypsin Fab ′ antibody induced by IPTG was bound to the column at a flow rate of 5 ml / min using an anti-E-tag affinity column, and the attached washing buffer (Binding buffer) 25 ml (5m
l / min flow rate) to wash away the culture supernatant that does not contain antibody, and attach the elution buffer (Elution buffer) 10m
Anti-human alpha 1 antitrypsin F produced by E. coli at l
Ab 'antibody was eluted (flow rate of 5 ml / min). The eluted anti-human alpha 1 antitrypsin Fab ′ antibody was immediately neutralized by adding a neutralization buffer (Neutralization buffer) in an amount of 1/10 of the elution buffer. As described above, purification was performed by affinity chromatography using the anti-E-tag antibody as a ligand. The neutralized anti-human alpha 1 antitrypsin Fab 'antibody was used as microcon [fraction molecular weight 3
PBS for 0000) (Millipore)
It was dissolved in 1 ml of buffer and stored at -80 ° C.

(6) Fluorescent labeling of anti-human alpha1 antitrypsin Fab 'antibody produced in Escherichia coli Tetramethylrhodamine-5-iodoacetamide, which is a fluorescent dye (fluorescent labeling agent), was prepared as follows. That is, 50 mg of tetramethylrhodamine-5-iodoacetamide (Molecular Probes) 1 mg
%, Dissolved in 0.6 ml acetonitrile, 10,000 r
The precipitate was removed by centrifugation at pm for 5 minutes. Reverse-phase chromatographic column (Tosoh ODS-80T) equilibrated with 25% acetonitril-0.1% trifluoroacetic acid solution
s, diameter 4.6 mm, length 25 cm), eluted with a linear concentration gradient of 25-55% acetonitrile over 30 minutes, and detected by monitoring the absorption at 280 nm. The largest peak was collected, and its concentration was determined by measuring the absorbance by setting the molar absorption coefficient at 543 nm to 87,000. This was used as purified tetramethylrhodamine-5-iodoacetamide for fluorescent labeling of the purified anti-human alpha1 antitrypsin Fab ′ antibody.

Purified anti-human alpha 1 antitrypsin Fa
The b'antibody was fluorescently labeled as follows. That is, anti-human alpha 1 antitrypsin Fab 'antibody concentrate (100 μl) was diluted with 10 times volume of 0.1 M phosphate buffer, 5 mM EDTA (pH 7.0), and microcon (for fraction molecular weight 30,000) (Millipore). (Manufactured by the company) was centrifuged and the buffer was exchanged. This operation was repeated twice.
Anti-human alpha 1 antitrypsin Fab ′ antibody 20
20 μl of 100 mM mercaptoethylamine (manufactured by Nacalai Tesque) was added to 0 μl, stirred, and incubated at 37 ° C. for 30 minutes. Microcon (fraction molecular weight
(For 30000) (manufactured by Millipore), concentrated again to 20 μl, and 200 μl of 0.1 M phosphate buffer, 5 mM EDTA
Ultrafiltration was performed with the input (pH 7.5).

25 μmol of tetramethylrhodamine-5-iodoacetamide (manufactured by Molecular Probes) was added in an amount of 5 μl.
Dissolve in N, N-dimethylformamide (manufactured by Sigma),
75 μl of 0.1 M phosphate buffer 5 mM (containing EDTA (p
H7.5)) was added, 5 μl of 1 mM mercaptoethylamine (manufactured by Nacalai Tesque) was added, and the mixture was incubated at 37 ° C. for 10 minutes. This was mixed with an anti-human alpha 1 antitrypsin Fab ′ antibody treated with mercaptoethylamine and reacted overnight in the dark. The reaction product is Sepha
dex G-25 (Amersham Pharmacia), unreacted fluorescent dye and fluorescently labeled anti-human alpha 1
An antitrypsin Fab ′ antibody (which is labeled with one molecule of a fluorescent dye and therefore sometimes referred to as a single molecule fluorescence-labeled anti-human alpha 1 antitrypsin Fab ′ antibody) was isolated and used in the following experiments. . Single molecule fluorescent labeled anti-human alpha 1 antitrypsin Fab '
The concentration of the antibody is 87,0 as the molar extinction coefficient at 543 nm.
00 was determined by absorbance measurement.

(7) Evaluation by Isoelectric Focusing with Fluorescence Detection The obtained single molecule fluorescently labeled anti-human alpha 1 antitrypsin Fab ′ antibody was separated and detected using a capillary electrophoresis device P / ACE5510 manufactured by Beckman. As a capillary, the inner wall is covalently coated with polyacrylamide, inner diameter 0.05 mm, outer diameter 0.375 mm, total length 2
Fluorescence detection by laser excitation was performed at a position 20 cm from the anode side using a 7 cm fused silica capillary (manufactured by GL Sciences Inc.). Pharmaly Capillary
te3-10 (Pharmacia Biotech, 40-fold dilution of the stock solution), hydroxypropylmethylcellulose (Sigma, hereafter HPMC, abbreviated to 0.125% final concentration), N, N, N ', N'-tetramethylethylenediamine (TEMED, manufactured by Pharmacia Biotech, final concentration 0.6%), and then filled with amphoteric carrier liquid containing 2 × 10E-8 M single molecule fluorescent-labeled anti-human alpha 1 antitrypsin Fab ′ antibody from the anode Injection was performed for 30 seconds in high pressure mode.

20 mM phosphoric acid containing HPMC (final concentration 0.1%) was used as the anolyte, and 20 mM NaOH was used as the catholyte at 13.5 KV (500
(V / cm) voltage was applied for 10 minutes, and then the anolyte was injected into the anodic side in low pressure mode while maintaining the same voltage, and single-molecule fluorescent-labeled anti-human alpha 1 anti was focused on the pH gradient. Trypsin Fab 'antibody was detected. The excitation of the fluorescent substance is performed by an argon ion laser (Beckman Laser, Laser
Module 488) Using a wavelength of 488 nm, a 488 nm notch filter (manufactured by Beckman) and a bandpass filter for rhodamine (manufactured by Asahi spectroscopy, custom-made product) were set in the filter housing unit for detection. The obtained results are shown in FIG. As can be seen from FIG. 2, the isoelectric points of the single molecule fluorescently labeled anti-human alpha 1 antitrypsin Fab ′ antibody produced in E. coli were heterogeneous.

(8) Partial Degradation by CNBR The following experiment was conducted to investigate which region of the anti-human alpha1 antitrypsin Fab ′ antibody fluorescently labeled on the Fd chain has isoelectric point heterogeneity. That is, the C-terminal of the Fd chain was fluorescently labeled with tetramethylrhodamine-5-iodoacetamide (manufactured by Molecular Probes), and the above-mentioned single molecule fluorescently labeled anti-human alpha 1 was labeled.
The antitrypsin Fab ′ antibody was concentrated by centrifugation using a microcon (fraction molecular weight of 30,000) (manufactured by Millipore). Concentrated anti-human alpha 1 antitrypsin Fab 'antibody was added to 6M guanidine HCl, 0.1M Tris-HCl, 5
mM EDTA (pH 8.1) was added. Add 1M DTT to the concentrate 50
Reduced at 4 ° C for 4 hours. 1M monoiodoacetamide 4μ
1 was added, and the mixture was reacted at room temperature in the dark for 30 minutes. 5% of this
Flow through a Sephadex G25 (Amersham Pharmacia) column equilibrated with formic acid, collect the fluorescent fractions and dry,
Reduced CM anti-human alpha 1 antitrypsin Fab '
It was an antibody. In order to decompose the reduced CM anti-human alpha 1 antitrypsin Fab ′ antibody, it was dissolved in 1% CNBr (70% formic acid) and reacted at room temperature. Dilute it with water,
Anti-human alpha 1 antitrypsin Fab 'antibody CNBr
As a degradation product, separation and detection were carried out and evaluated by the same method as in (7) using a capillary electrophoresis device P / ACE5510 manufactured by Beckman.

As a result of capillary electrophoresis, among the partial degradation products (fragments) of the Fd chain of the single-molecule fluorescently labeled anti-human alpha 1 antitrypsin Fab ′ antibody, the shortest fragment (fragment containing the CH1 region) had heterogeneous isoelectric point. Was left, it was found that the non-uniformity of the isoelectric point was caused by the CH1 region.

(9) Correction of isoelectric point heterogeneity by site-directed mutation method From the result of (8), it was found that the isoelectric point heterogeneity was caused by the CH1 region. In order to investigate whether the residue has the most influence on the heterogeneity of the isoelectric point, CH1 as described below is examined.
The antibody was modified by designing a DNA primer that causes a site-specific mutation in the region. In the examples below, for example, the first H chain by Kabat numbering
In the case of representing that N (asparagine) at No. 62 is converted (or converted) to D (aspartic acid), “H-
The description "N162D" may be used. That is,
The Fab ′ antibody in which N (asparagine) of the H chain No. 162 according to Kabat numbering is converted to D (aspartic acid) is represented as “H-N162D modified Fab ′ antibody”, and the gene expressing this antibody is represented by “ It may be expressed as "H-N162D modified Fab 'antibody-expressed gene".

As the DNA primer, F5-1 primer (SEQ ID NO: 5 in the sequence listing) and H-N162D-BamHI primer (SEQ ID NO: 6 in the sequence listing) having the following base sequences were used. In the following sequences, 5'and 3'are 5'side and 3'of the primer, respectively.
Represents a side, S represents C or G, M represents A or C, R represents A or G, and W represents A or T.

F5-1 primer (SEQ ID NO: 5): 5'SAGGTSMARCTGCAGSAGTCWGG 3'H-N162D-BamHI primer (SEQ ID NO: 6): 5'GCTGGACAGGGATCCAGAGTCCCAGGTCACTGT 3'Anti-human alpha 1 obtained in (2) using these primers By performing PCR using the antitrypsin Fab ′ antibody expression gene as a template, the N (asparagine) of the H chain No. 162 according to the Kabat numbering was D
Prepare a gene fragment converted to (aspartic acid),
This was digested with BamHI. This digestion product contains the anti-human alpha 1 antitrypsin Fab ′ antibody-expressing gene BamH.
The gene fragment recovered from the I digestion product by low melting point agarose electrophoresis was ligated.

To ligate the ligated product to the same pCANTAB5E plasmid vector used in (2),
F5-2 primer and K3 having the following base sequences
-2 PCR was performed using the primers, digested with Sfi I and Not I restriction enzymes, and ligated to the pCANTAB5E plasmid vector to prepare H-N162D modified Fa.
An expression vector containing the b'antibody expression gene was prepared.

F5-2 primer (SEQ ID NO: 7): 5′CATGTGAACTGACTGGGCCCAGCCGGCCATGGCCGAGGTCCAG
CTGCAGCAGTCAGG 3'K3-2 primer (SEQ ID NO: 8): 5'CCACGATTCTGCGGCCGCACACTCATTCCTGTTGAAGCTCTTT
GTAAT 3 ′ The above expression vector was transformed into Escherichia coli HB2151.
Antibodies were induced in the same manner as above, and screening was performed by ELISA. A plasmid was extracted from a bacterium showing a positive reaction and transformed into Escherichia coli XL10. The amount of plasmid used for the sequencing reaction was extracted from the transformed XL10 bacterium, and the nucleotide sequence of the H-N162 modified Fab 'antibody expression gene was confirmed. Pyrobe, which has a high fidelity, is used as a polymerase in the production of variants.
st polymerase (Takara Shuzo) was used. Escherichia coli HB transformed with the above expression vector after confirming the nucleotide sequence
Expanding the culture scale of 2151, H-N162D modified F
An ab 'antibody was produced and the antibody was purified by an affinity column to obtain a purified H-N162D-modified Fab' antibody.

H-N162D modified F confirmed by the above method
The CH1 region of the ab 'antibody and the sequence of the portion adjacent to the C-terminus of the CH1 region (SEQ ID NO: 9 in the sequence listing) are shown below.

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWOSGSLSSGV
HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVARPASSTKVDKKIVPB
DCGCSR In this sequence, the C-terminal (right side) VPRDCGCSR sequence is an amino acid sequence containing a cysteine residue (C) that is not involved in binding to the L chain introduced into the portion adjacent to the C-terminus of the CH1 region. . The cysteine residue that is not involved in binding to the L chain is the cysteine residue on the C-terminal side in the VPRDCGCSR sequence. The portion excluding the VPRDCGCSR sequence is the sequence of the CH1 region.

The sequence of the similar portion in the Fab ′ antibody before modification with H-N162D (SEQ ID NO: 1 in the sequence listing)
0) is as follows.

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPKPVTVTWNSGSLSSGV
HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPB
DCGCSR A comparison of the above two sequences shows that the Fab ′ antibody before modification with H-N162D shows that the asparagine residue of H chain number 162 (underlined N) by Kabat numbering is aspartic acid after modification. (D underlined)
You can see that it is. In addition, in this example, the sequence (VPRDCGCSR) of the portion adjacent to the C-terminus of the CH1 region in the H-N162D modified Fab ′ antibody was H
-CH in Fab 'antibody before N162D modification
It is identical to the sequence (VPRDCGCSR) adjacent to the C-terminus of region 1.

(10) Separation / detection of fluorescence-labeled isoelectric point-uniformized Fab ′ antibody by fluorescence detection capillary isoelectric focusing electrophoresis The H-N162D-modified Fab ′ antibody obtained in (9) is used according to the method described in (6). Similarly, tetramethylrhodamine-5-iodoacetamide (manufactured by Molecular Probes) was fluorescently labeled to obtain a single molecule fluorescently labeled Fab 'antibody. This single-molecule fluorescently labeled Fab ′ antibody is (7)
Beckman Capillary Electrophoresis Device
Separation and detection were performed using P / ACE5510. The result is shown in FIG.

As can be seen from FIG. 3, H-N single-molecule fluorescently labeled
When the 162D-modified Fab ′ antibody was subjected to capillary electrophoresis, one large peak appeared at a migration time of around 29 minutes, and no peak substantially appeared at other portions.
This means that the H-N162D modified Fab 'antibody has a uniform isoelectric point. On the other hand, F that has not been modified
Ab 'antibody is separated by the same capillary electrophoresis.
In FIG. 2 showing the detection results, many peaks were observed near the moving time of 25 minutes and 29 minutes,
It can be seen that the Fab 'antibodies that have not been modified have non-uniform isoelectric points.

(11) Forced Hydrolysis of Fab ′ Antibodies Unmodified anti-human alpha1 antitrypsin Fab ′ antibodies are liable to undergo the usual hydrolysis reaction (deamidation) of their amide group-containing amino acid residues. The isoelectric point becomes nonuniform over time even under storage conditions.
The amide group-containing amino acid residue was hydrolyzed by subjecting the unmodified anti-human alpha1 antitrypsin Fab 'antibody to forced hydrolysis.
The same hydrolysis was also performed on the modified anti-human alpha 1 antitrypsin Fab ′ antibody.

That is, the unmodified anti-human alpha 1 antitrypsin Fab ′ antibody was thawed in 0.1 M phosphate buffer (containing 5 mM EDTA (pH 7.5)) and incubated at 37 ° C. for 1 hour.
The product was forcibly hydrolyzed by keeping it warm for 2 hours. The antibody thus obtained was fluorescently labeled with tetramethylrhodamine-5-iodoacetamide (Molecular Probes) in the same manner as in (6) to obtain a fluorescently labeled Fab ′ antibody. Regarding this antibody, in the same manner as in (7), Beckman Capillary Electrophoresis Device P / AC
Separation and detection were performed using E5510. The obtained results are shown in FIG.

On the other hand, the modified anti-human alpha 1 antitrypsin Fab ′ antibody (H-N162D) obtained in (9)
The modified Fab ′ antibody) is forcibly hydrolyzed in the same manner as above, and H-N162D modified Fab after hydrolysis is
The b ′ antibody was fluorescently labeled with tetramethylrhodamine-5-iodoacetamide (Molecular Probes) in the same manner as in (6), and Beckman capillary electrophoresis was performed in the same manner as in (7). Device P / ACE5510
Was used for separation and detection. The result is shown in FIG.

In FIG. 4, the horizontal axis represents the migration time and the vertical axis represents the fluorescence intensity. A large peak indicating the original isoelectric point (the leftmost peak in FIG. 4) appears in the shorter migration time. . In addition, a large number of large peaks appearing on the longer side of this peak due to the nonuniform component of the isoelectric point associated with hydrolysis (deamidation). Similar to FIG. 4, FIG. 5 shows the moving time on the horizontal axis and the fluorescence intensity on the vertical axis. In FIG. 5, a large peak showing the original isoelectric point (the leftmost peak in FIG. 5) appears in the shorter traveling time.
However, the number of peaks due to the non-uniform component of the isoelectric point that appeared on the longer side of this peak was small, and the size of the peak was less than half that in FIG.

From the above results, the amide group-containing amino acid residue in the CH1 region of the anti-human alpha1 antitrypsin Fab ′ antibody is susceptible to hydrolysis, which may cause the isoelectric point heterogeneity of the Fab ′ antibody. I was confirmed.

It was also found that the modified Fab ′ antibody (H-N162D modified Fab ′ antibody) has higher hydrolysis stability than the unmodified Fab ′ antibody. Therefore, at least one of the amide group-containing amino acid residues in the CH1 region of the Fd chain (in particular, the asparagine residue at the H chain No. 162 according to Kabat numbering)
By substituting an amino acid residue containing no amide group except cysteine, it is possible to make the isoelectric point uniform, reduce the decomposition rate by making it less susceptible to hydrolysis, and improve the stability. I knew I could do it.

One mode of the method for producing the isoelectric point-uniformized Fab ′ antibody (fluorescently labeled) of the present invention described above is schematically shown in FIGS. FIG. 6A shows Escherichia coli in which a gene expressing the isoelectric point-uniformized Fab ′ antibody of the present invention has been incorporated, and IPTG (isopropyl-β-D-thiogalactopyrano) was compared to this E. coli.
Antibodies are induced by making side act. Figure 6B
Is the isoelectric point homogenizing F of the present invention generated by this antibody induction.
Ab 'antibody is shown. This isoelectric point-uniformized Fab ′ antibody is
After purification with an affinity column or the like, it is fluorescently labeled.
FIG. 6C shows this fluorescence-labeled isoelectric point-uniformized Fab ′ antibody.

8A to 8F schematically show a method for producing an isoelectric point-uniformized Fab ′ antibody (fluorescence-labeled) according to the conventional technique disclosed in Japanese Patent Publication No. 8-506182. According to this method, first, an IgG antibody produced by a hybridoma (see FIG. 8).
A) is cleaved with a proteolytic enzyme (pepsin),
F (ab ′) ₂ antibody (FIG. 8B) is obtained. This is treated with a reducing agent such as mercaptoethylamine to reduce disulfide bonds to obtain Fab ′ antibody (FIG. 8C). This Fab '
The antibody is oxidized to leave only one reactive thiol group (SH group) (FIG. 8D), and a fluorescent dye is bound to this thiol group (FIG. 8E). Isoelectric focusing is performed using the obtained fluorescence-labeled Fab ′ antibody, and the fluorescence-labeled Fab ′ antibody having a uniform isoelectric point is taken out from the migration carrier (FIG. 8F).

As can be seen by comparing FIGS. 6A-C and 8A-F,
The method of FIGS.
While the steps until obtaining the b ′ antibody are numerous and complicated, the method of the present invention (FIGS. 6A to 6C) does not require such complicated steps.

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to obtain an isoelectric point-uniformized Fab ′ antibody having a high isoelectric point stability over time without the need for complicated steps. , Isoelectric point equalization Fa
It is possible to provide a method for producing a b ′ antibody. Further, it becomes possible to provide a fluorescence-labeled isoelectric point-uniformized Fab ′ antibody obtained by the method for producing the isoelectric point-uniformized Fab ′ antibody.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kiyohito Shimura, Hiraguchi City, Hamakita City, Shizuoka Prefecture, 5000 Hiraguchi Biophotonics Research Institute Co., Ltd. In the laboratory (56) References Tokuhyo Hyodai 8-506182 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C07K 1/13 C07K 16/18 C12P 21/08 BIOSIS / WPI (DIALOG)

Claims (11)

(57) [Claims] 1. A first step of providing an Fd chain gene encoding a VH region and a CH1 region of a Fab ′ antibody, and a codon encoding an amide group-containing amino acid residue of the CH1 region in the Fd chain gene. A site-specific mutation of at least one of the codons encoding an amide group-free amino acid residue other than cysteine to obtain a modified Fd chain gene, the modified Fd chain gene, and the Fab ′ A third step of obtaining a modified Fab 'antibody expression gene by ligating with an L chain gene encoding an antibody L chain in an expressible state, and transforming a host cell with the modified Fab' antibody expression gene to obtain A fourth step of obtaining an isoelectric point-uniformized Fab ′ antibody by culturing the obtained transformant, and a method for producing the isoelectric point-uniformized Fab ′ antibody. 2. A first step of providing an Fd chain gene encoding a VH region and a CH1 region of a Fab ′ antibody, and an L chain gene encoding an L chain of the Fab ′ antibody, and the Fd chain gene And a step of ligating the L chain gene in an expressible state to obtain a Fab ′ antibody expression gene, and a codon encoding an amide group-containing amino acid residue of the CH1 region in the Fab ′ antibody expression gene A site-specific mutation of at least one of the codons encoding an amide group-free amino acid residue except cysteine to obtain a modified Fab ′ antibody expression gene, and the modified Fab ′ antibody expression gene A fourth step of obtaining an isoelectric point-uniformized Fab ′ antibody by transforming a host cell and culturing the obtained transformant, and a method for producing the isoelectric point-uniformized Fab ′ antibody. 3. A first step of providing a CH1 gene encoding a region containing a CH1 region of a first Fab ′ antibody, wherein the CH1 gene comprises a codon encoding an amino acid residue containing an amide group in the CH1 region. At least one
The second step of obtaining a modified CH1 gene by site-specific mutation to a codon encoding an amide group-free amino acid residue except cysteine, and a gene encoding a CH1 region by cleaving the modified CH1 gene with a restriction enzyme. A fourth step of providing a third step of obtaining a gene fragment containing the same, a VH gene encoding the VH region of the second Fab ′ antibody, and an L chain gene encoding the L chain of the second Fab ′ antibody A step of ligating the gene fragment, the VH gene and the L chain gene in an expressible state to obtain a modified Fab ′ antibody expression gene; and transforming a host cell with the modified Fab ′ antibody expression gene. And a sixth step of obtaining an isoelectric point-uniformized Fab ′ antibody by culturing the resulting transformant and culturing the obtained transformant. 4. The amide group-free amino acid residue is an aspartic acid residue, a glutamic acid residue, a glycine residue, or a serine residue, according to any one of claims 1 to 3. The method for producing an isoelectric point-uniformized Fab ′ antibody according to 1. 5. The Fab ′ antibody, wherein the Fab ′ antibody has an asparagine residue at H-chain number 162 according to Kabat numbering.
A b'antibody, wherein the amide group-containing amino acid residue is the asparagine residue, and the production of an isoelectric point-uniformized Fab 'antibody according to any one of claims 1 to 3. Method. 6. A polymerase chain wherein the site-specific mutation uses a CH1 region amplification primer that substitutes at least one amide group-containing amino acid residue in the CH1 region with an amide group-free amino acid residue excluding cysteine. The method for producing an isoelectric point-uniformized Fab ′ antibody according to any one of claims 1 to 3, which is carried out by a reaction. 7. An antibody using a primer for introducing an amino acid sequence containing a cysteine residue not involved in binding to an L chain into a portion adjacent to the C-terminus of the CH1 region before the first step. CD complementary to mRNA from producer cells
The method further comprising a step of performing a polymerase chain reaction using NA as a template, wherein the isoelectric point-uniformized Fab ′ antibody has a cysteine residue that is not involved in binding to an L chain. Item 4. A method for producing an isoelectric point-uniformized Fab 'antibody according to any one of items 3. 8. L in the isoelectric point-uniformized Fab ′ antibody
The method for producing an isoelectric point-uniformized Fab ′ antibody according to claim 7, further comprising the step of binding a fluorophore dye to a cysteine residue that is not involved in chain binding. 9. The method according to claim 8, wherein the bond between the cysteine residue and the chromophore dye is at least one bond selected from the group consisting of a thioester bond, a dithioester bond and a thioether bond. Electric point uniformization Fa
Method for producing b'antibody. 10. The fluorophore dye is at least selected from the group consisting of rhodamine, fluorescein, cyanine, indocyanine, indocarbocyanine, pyronin, lucifer yellow, quinacrine, squaric acid, coumarin, fluoroanthenylmaleimide, anthracene. The method for producing an isoelectric point-uniformized Fab ′ antibody according to claim 8, which is one fluorophore dye. 11. A fluorescence-labeled isoelectric point-uniformized Fab ′ antibody produced by the method according to claim 8.