JP7317353B2 - Production of heterodimeric proteins by Brevibacillus - Google Patents

Description

The present invention relates to a method for producing recombinant heterodimeric proteins using Brevibacillus.

Brevibacillus is a host capable of extracellularly secreting and producing proteins, and because it is a Gram-positive bacterium, it does not have endotoxin, has low extracellular protease activity, is easy to culture, and is safe. It is advantageous for the production of recombinant proteins because it is high.

The production of heterodimeric proteins by Brevibacillus has been reported. For example, for antibody production, inserting both the heavy chain-encoding DNA and the light chain-encoding DNA into one plasmid, transforming the brevibacillus with the plasmid, culturing the brevibacillus, A method of forming heterodimeric proteins in the medium during culture has been reported (see Non-Patent Document 1). In addition, brevibacillus having a DNA encoding a heavy chain variable region (VH) and brevibacillus having a DNA encoding a light chain variable region (VL) are mixed and cultured, and VH and VL are mixed in the medium during culture. A method of forming a heterodimeric protein as Fv by weak interaction has been reported (see Non-Patent Document 2).

Mizukami M. et al., Protein Expr Purif 2018 Oct, 150, pp.109-118 Horita et al., Scientific Reports 6, Article number: 35297(2016)

An object of the present invention is to provide a method for producing a heterodimeric protein such as a bispecific antibody using Brevibacillus.

As described above, in producing a heterodimeric protein using Brevibacillus, conventionally, both the DNA encoding the heavy chain and the DNA encoding the light chain are inserted into one plasmid, and the plasmid and culturing the brevibacillus to form a heterodimeric protein in the culture medium, or brevi with DNA encoding the heavy chain variable region (VH) Bacillus and Brevibacillus having a DNA encoding a light chain variable region (VL) are co-cultured, and a heterodimeric protein is formed as Fv by weak interaction between VH and VL in the medium during culture. had been reported.

The present inventors inserted DNAs encoding two monomeric proteins forming a heterodimeric protein into separate expression vectors, and transformed separate Brevibacillus strains with the resulting two types of expression vectors. , co-cultivating two transformed Brevibacillus bacteria simultaneously to secrete two monomeric proteins into the same culture medium, and covalently linking the two monomeric proteins to form a heterodimeric protein. let me

By this method, it is possible to produce heterodimeric proteins such as bispecific antibodies more efficiently than before, and an appropriate combination of monomeric proteins that form a heterodimeric protein The inventors have found that efficient screening can be performed, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for producing a heterodimeric protein using Brevibacillus spp., wherein Brevibacillus is produced using an expression vector into which DNA encoding one of the monomeric proteins constituting the heterodimeric protein is inserted. Transforming a bacterium, transforming Brevibacillus using an expression vector into which DNA encoding the other monomer protein is inserted, and simultaneously mixed-cultivating the two types of transformed Brevibacillus obtained, Each of the brevibacillus bacterium is caused to secrete and produce each monomer protein in the culture solution, and the two types of monomer proteins are covalently bound in the culture solution to form a heterodimeric protein, A method of making a heterodimeric protein.
[2] The method of producing the heterodimeric protein of [1], wherein the covalent bond that binds two types of monomeric proteins is a disulfide bond.
[3] Producing the heterodimeric protein of [1] or [2], wherein the monomeric protein has an antibody variable region and a constant region, and forms a heterodimeric protein through disulfide bonds between the constant regions. how to.
[4] The method of producing the heterodimeric protein of [3], wherein the heterodimeric protein is a bispecific antibody or Fab.
[5] In a heterodimeric protein formed from monomer protein 1 and monomer protein 2, an appropriate combination from among combinations of n types of monomer protein 1 and m types of monomer protein 2 A method for screening a heterodimeric protein of n types of monomer protein 1, which is used to transform Brevibacillus spp. to produce n types of transformed Brevibacillus spp. Each of the monomeric proteins 2 is used to transform brevibacillus to produce m types of transformed brevibacillus, one of n types of brevibacillus producing monomeric protein 1 and One of the m types of brevibacillus that produces the monomeric protein 2 is simultaneously mixed-cultured, each brevibacillus is made to secrete and produce each monomeric protein in the culture solution, and heterogeneous in the culture solution. By forming a dimer protein, n × m types of heterodimeric proteins are produced, properties of the n × m types of heterodimeric proteins are tested, and heterodimeric proteins having appropriate properties are obtained. A method for screening an appropriate combination of heterodimeric proteins from combinations of n kinds of monomer proteins 1 and m kinds of monomer proteins 2, comprising selecting
[6] The covalent bond that connects two types of monomeric proteins is a disulfide bond. A method for screening the heterodimeric protein of [5].
[7] The heterodimeric protein of [5] or [6], in which the monomer has an antibody variable region and a constant region and forms a heterodimeric protein through disulfide bonds between the constant regions, is screened. Method.
[8] The method of screening a heterodimeric protein of [7], wherein the heterodimeric protein is a bispecific antibody or Fab.

DNAs encoding two monomeric proteins forming a heterodimeric protein are inserted into separate expression vectors, and the resulting two types of expression vectors are transformed into separate Brevibacillus strains, resulting in two transformations. By simultaneously co-culturing Brevibacillus spp., secreting two monomeric proteins into the same culture medium, and covalently linking the two monomeric proteins to form a heterodimeric protein, efficiency is increased. It enables the production of heterodimeric proteins such as bispecific antibodies, and enables the rapid and efficient screening of suitable combinations of monomeric proteins that form heterodimeric proteins. Became.

FIG. 2 shows the structure of the expression vector pROXb3-Fab. FIG. 2 shows the structures of expression vector pROXb3-VLCL and expression vector pROXb3-VHCH1. FIG. 2 shows the structures of expression vector pROXb3-scFv(EGFR)-CH1-His and expression vector pROXb3-scFv(CD3)-CL-DDDDK. Figure 2 shows the structures of Fab (A) and bispecific antibody (B). FIG. 1 shows the structure of a bispecific antibody.

The present invention will be described in detail below.
The present invention is a method for producing a recombinant heterodimeric protein using Brevibacillus.

Brevibacillus spp. is a bacterium that can secrete and produce protein extracellularly, including Brevibacillus choshinensis, Brevibacillus brevis, and Brevibacillus choshinensis HPD31 (FERM BP- 1087, Takagi H et al., Int J Syst Bacteriol. 1993 Apr;43(2):221-31).

A heterodimeric protein produced in the present invention is a protein in which two different monomeric proteins are covalently dimerized. The covalent bond is preferably a disulfide bond, so the two monomeric proteins have at least one cysteine residue. Examples include a dimer protein of an antibody molecule and an antibody molecule, a dimer protein of an antibody molecule and a ligand molecule, a dimer protein of an antibody molecule and a toxin protein molecule, and the like. Preferably, the dimeric protein is composed of monomeric proteins consisting of the antibody or fragment thereof. Suitable heterodimeric proteins include bispecific antibodies, Fabs, and the like. A bispecific antibody refers to an antibody in which each of the two antigen-binding sites binds to a different antigen, and in IgG antibodies, the two variable regions are different and bind separate molecules. The format (type) of the bispecific antibody is not limited, but each of the two monomeric proteins comprises an antibody constant region and a variable region, and these two monomeric proteins are separated by disulfide bonds between the constant regions. combine to form a dimeric protein.

A typical antibody is composed of a heavy chain variable region and a heavy chain constant region and a light chain variable region and a light chain constant region. The heavy chain constant region is composed of three domains CH1, CH2 and CH3. Also, the light chain constant region is composed of one domain CL. Light chain constant regions are either kappa or lambda constant regions.

The monomeric protein that constitutes the heterodimeric protein of the present invention preferably contains all or part of the constant region of an antibody, and a covalent bond such as a disulfide bond between the constant regions of the monomeric protein allows the heterodimeric protein to form a heterodimeric protein. Forms a dimeric protein. The binding between constant regions may be binding between heavy chain constant regions or binding between a heavy chain constant region such as CH1 and a light chain constant region (CL).

A bispecific antibody is composed of a heavy chain variable region, a heavy chain constant region and a light chain variable region and a light chain constant region, and has a structure in which the two variable regions bind to different antigens, like a normal antibody. You may have The constant region need not be composed of multiple domains and may contain only one domain. The variable regions also comprise a heavy chain variable region and a light chain variable region in each of the two monomeric proteins. One monomer protein may contain the heavy chain variable region and the light chain variable region as Fab, or may contain the heavy chain variable region and the light chain variable region as Fv bound by weak interaction. , may contain a heavy chain variable region and a light chain variable region linked by a single chain peptide linker as scFv (single chain Fv), or a heavy chain antibody VHH ( variable domain of heavy chain of heavy chain antibody). In addition, in monomer proteins containing the constant regions and variable regions of two antibodies, the portions containing the variable regions do not need to have the same structure, for example, one is Fab and the other is scFv good too.

Figures 4-1 and 4-2 show the structure of the Fab (Figure 4-1A) and the structure of the bispecific antibody (Figures 4-1B and 4-2A, B). The bispecific antibody shown in FIG. 4-1B is a molecule in which one of the two monomeric proteins is a heavy chain constant region (CH1) and a scFv that binds to a specific antigen (scFv1), and the other is a light chain constant region. It is a molecule bound by scFv (scFv2) that binds to a different antigen than the scFv bound to the constant region (CL) and CH1, and these two molecules are bound by disulfide bonds between CH1 and CL to form a dimer. forming proteins. The bispecific antibody shown in Figure 4-1B is sometimes called a bispecific miniantibody. The bispecific antibody shown in FIG. 4-2A has the structure of a normal IgG antibody, with different antigens bound by the variable regions of each monomeric protein of the dimeric protein. The bispecific antibody shown in FIG. 4-2B is a bispecific antibody in which the variable region of one monomer protein is Fab in the bispecific antibody shown in FIG. 4-1B. The bispecific antibodies shown in Figures 4-1 and 4-2 are exemplary, and the bispecific antibodies of the invention are not limited to these bispecific antibodies.

The heterodimeric protein of the present invention is an expression vector into which DNA encoding one monomer protein constituting the heterodimeric protein is inserted and an expression vector into which DNA encoding the other monomer protein is inserted. as separate expression vectors to prepare two types of expression vectors. These two expression vectors are then used to transform Brevibacillus strains to obtain two strains of Brevibacillus strains that produce different monomeric proteins. By co-cultivating these two types of Brevibacillus at the same time, each Brevibacillus produces a different monomeric protein. Two types of monomeric proteins secreted into the medium are bound by covalent bonds such as disulfide bonds to form a heterodimeric protein in the medium. Brevibacillus bacteria capable of producing each monomer protein are separately cultured, the resulting two types of culture solutions are mixed, and the two types of monomer proteins are bound to form a heterodimeric protein. Although it is possible to form a dimeric protein, it is possible to form a dimeric protein when two types of Brevibacillus are mixed and cultured at the same time, and the production and secretion of a monomeric protein and the formation of a dimeric protein are performed at the same time. Formation efficiency is high. If the two transformed brevibacillus strains have different monomer protein production efficiencies, the number of brevibacillus strains to be mixed may be adjusted. Preferably, the two kinds of monomeric proteins are adjusted so that similar molar numbers are produced.

The DNA to be inserted into the vector may contain DNA encoding a signal peptide that facilitates secretion of the monomeric protein from the host cell, in which case the DNA encoding the signal peptide and the DNA encoding the monomeric protein be concatenated in-frame. After the monomeric protein is produced, the signal peptide is removed and the mature protein can be obtained.

At this time, the DNA encoding one monomer protein and the DNA encoding the other monomer protein may be functionally linked to an element such as a promoter. Here, "functionally linked" means linked so that the elements perform their functions.

The promoter may be any promoter that functions in Brevibacillus spp., more preferably the promoter of the gene encoding the Brevibacillus choshinensis HPD31 cell wall protein HWP (Ebisu S et al., J. Bacteriol. 1990 172:1312-1320).

A vector for inserting the gene of the present invention is not particularly limited as long as it is replicable in Brevibacillus. Examples include pROXb3 (Protein Express Co., Ltd.), pBIC, pNY326, pNCMO2 (Takara Bio Inc.) and the like.

An expression vector can be introduced into a host cell by a known method to transform the host cell. For example, there are an electroporation method, a PEG method, and the like.

Cultivation of the transformed Brevibacillus spp. may be carried out according to a common culture method for bacteria. The medium used is not limited, but TM medium, TMN medium, 2SY medium, 2SYN medium, 2SLN medium, LB medium and the like can be used. Culturing is performed at 25 to 40° C., preferably by shaking culture under aeration.

Purification of the produced heterodimeric protein may be performed using separation and purification methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed Harlow, David Lane , Cold Spring Harbor Laboratory, 1988). Alternatively, after producing a heterodimeric protein with an affinity tag, it can be purified by affinity chromatography using the affinity tag. Affinity tag sequences include, for example, polyhistidine sequences consisting of 2 to 12 histidines, preferably 4 or more, more preferably 4 to 7, and even more preferably 5 or 6 histidines. When polyhistidine sequences are used, the synthetic protein can be purified by using nickel chelate column chromatography with nickel as a ligand. It can also be purified by affinity chromatography using a column on which an antibody against polyhistidine is immobilized as a ligand. In addition, HAT tag, HN tag, V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag09, CruzTag22, CruzTag41, Glu-Glu tag, Ha.11 tag, KT3 tag, etc. can also be used. In the case of purification using a tag, by attaching different tags to each of the two monomeric proteins that form the dimer protein, a two-step affinity purification is performed using two types of tags. It is possible to purify only the dimerized protein.

Further, the invention includes methods of screening for appropriate combinations of multiple monomeric proteins in heterodimeric proteins. The screening method is useful when screening for bispecific antibodies with the desired reactivity, particularly when making bispecific antibodies.

Bispecific antibodies bind antigens in which the variable regions of each monomeric protein of the heterodimeric protein differ. Which epitope of each antigen each variable region recognizes is important for the reactivity of the bispecific antibody, and is determined by the positional relationship between the antigen recognition sites. Therefore, in order to generate a useful bispecific antibody, it is preferable to select the appropriate combination of epitopes bound by the variable regions of each of the two monomeric proteins from among a large number of combinations. For example, if the bispecific antibody is a heterodimeric protein of monomeric proteins that bind to antigen A and antigen B, respectively, and there are five candidate epitopes each for antigen A and antigen B, then 5×5 = 25 combinations of bispecific antibody reactivities need to be examined.

DNA encoding one monomer protein of the heterodimeric protein and DNA encoding the other monomer protein are inserted into one expression vector, and the expression vector is used to transform Brevibacillus. However, when Brevibacillus is cultured, two monomeric proteins are produced and secreted, and the reactivity of the heterodimeric protein formed after secretion is examined, one of the five monomeric proteins and 5 An expression vector with a combination of monomeric proteins of the other type would need to be generated, requiring a total of 5×5=25 expression vectors. It is necessary to prepare and culture 25 types of transformed Brevibacillus using 25 types of expression vectors. On the other hand, in the present invention, DNA encoding one monomer protein of the heterodimeric protein and DNA encoding the other monomer protein are inserted into separate expression vectors, and using the expression vectors Since separate brevibacillus strains are transformed and two types of brevibacillus strains are cultured together, 5+5=10 types of expression vectors are required. Five strains of transformed brevibacillus are prepared using expression vectors containing DNAs encoding respective monomer proteins, and five strains of brevibacillus are combined and cultured together.

Both cases are the same in that 25 cultures are required, but the number of vectors to be produced differs greatly, and DNA encoding one monomer protein of the heterodimeric protein and the other monomer DNAs encoding somatic proteins are inserted into separate expression vectors, separate brevibacillus strains are transformed with the expression vectors, and two types of brevibacillus strains are mixed-cultured to produce two monomeric proteins. Screening can be performed more efficiently by producing, secreting, and examining the reactivity of the heterodimeric protein formed after secretion.

There are n types of one monomer protein 1 and m types of the other monomer protein 2, and an appropriate combination from a combination of n types of monomer protein 1 and m types of monomer protein 2 When screening for heterodimeric proteins, each of the n types of monomeric protein 1 is used to transform Brevibacillus spp. to produce n types of transformed Brevibacillus spp., and m types of monomeric protein 2 each of which is used to transform brevibacillus to produce m types of transformed brevibacillus, one of n types of brevibacillus that produces monomer protein 1 and a monomer protein One of the m types of Brevibacillus that produces 2 is mixed at the same time, and each Brevibacillus is made to produce each monomer protein and secrete it into the culture medium, and the heterodimer in the culture medium. to form body proteins; As a result, n×m types of heterodimeric proteins are produced. Properties of the obtained n×m kinds of heterodimeric proteins are tested, and heterodimeric proteins having appropriate properties are selected. Here, when the dimer protein is Fab or a bispecific antibody, n types of monomer protein 1 are all monomer proteins that bind to the same antigen, but n types of monomer protein 1 have different reactivities or different binding epitopes. Similarly, all of the m types of monomeric proteins 2 are monomeric proteins that bind to the same antigen, but the m types of monomeric proteins 2 have different reactivities or bind epitopes is different. Here, n and m are 100 or less, preferably 50 or less, more preferably 20 or less, still more preferably 10 or less. Appropriate properties in this case will depend on the bispecific antibody to be produced, but include, for example, the properties of cross-linking and contacting antigen A and antigen B to which their respective monomeric proteins bind. For example, a bispecific antibody that recognizes a cancer cell surface antigen and an effector cell surface antigen such as a lymphocyte can exert an antitumor effect by bringing these cells into contact with lymphocytes brought into close proximity. Alternatively, a bispecific antibody that recognizes active factor XI and factor X can cross-link these factors to replace the cofactor function of factor VIII, thereby exerting therapeutic effects on hemophilia. .

The present invention will be specifically described by the following examples, but the invention is not limited by these examples.

(Example 1) Construction of expression vector VLCL converted from the amino acid sequence of the Fab region of adalimumab (trade name: Humira), an anti-TNF-α antibody drug, to codons for Brevibacillus as a heterodimeric Fab protein. A gene (SEQ ID NO: 1) and a VHCH1 gene (SEQ ID NO: 2) were artificially synthesized and introduced into the BspHI/HindIII recognition sequence of the expression vector pROXb3 for Brevibacillus as shown in FIG. 1 to construct the expression vector pROXb3-Fab.

Separately, expression vectors pROXb3-VLCL and pROXb3-VHCH1 were constructed by independently introducing the VLCL gene and VHCH1 gene into the BspHI/HindIII recognition sequence of pROXb3, respectively, as shown in FIG.

As a model of bispecific miniantibody, another form of heterodimeric protein, we developed a small molecule bispecific antibody that can recognize both EGFR, a cancer cell surface protein, and CD3, a lymphocyte surface protein. Using the bispecific antibody Ex3 (MAbs. 2018 Jul 9: 1-10.) as a model, the scFv of the anti-EGFR antibody was linked to CH1 and His tag (SEQ ID NO: 3), and the scFv of the anti-CD3 antibody was cloned with CL. By artificially synthesizing the DDDDK tag-ligated gene (SEQ ID NO: 4) and introducing it into the BspHI/HindIII recognition sequence of pROXb3, expression vectors pROXb3-scFv(EGFR)-CH1-His, pROXb3-scFv(CD3) -CL- DDDDK was constructed (Fig. 3).

By introducing a Shine-Dalgarno sequence (SD sequence) and a signal peptide (SP) that functions in Brevibacillus into the upstream of each gene, secretory production outside the Brevibacillus is possible. .
Figures 4-1 and 4-2 show schematic diagrams of dimer formation of Fab and bispecific antibody.

(Example 2) Comparison of Fab Production Amount Brevibacillus choshinensis HPD31 was transformed with each of the expression vectors pROXb3-Fab, pROXb3-VLCL and pROXb3-VHCH1. Each transformant was cultured in 2 mL of TMN medium (1% polypeptone, 0.5% meat extract, 0.2% yeast extract, 0.001% iron(II) sulfate heptahydrate, 0.001% manganese(II) sulfate pentahydrate, 0.0001% Zinc sulfate heptahydrate, 1% glucose, 50 µg/mL neomycin) was cultured at 30°C for 18 hours (preculture). To 100 mL of newly prepared TMN medium, (1) 1 mL of pROXb3-Fab transformant culture medium was added (independent culture), (2) 0.5 mL of pROXb3-VLCL transformant culture medium, and pROXb3-VHCH1 transformant culture. Add 0.5 mL of the solution (mixed culture), (3) add 1 mL of pROXb3-VLCL transformant culture medium, and (4) add 1 mL of pROXb3-VHCH1 transformant culture medium. was carried out.

The supernatant was recovered from the culture medium after the culture was completed by centrifugation. The extracellularly secreted Fab was subjected to SDS-PAGE of the centrifugation supernatant, detected by Western blotting using an anti-Fab antibody, and subjected to relative quantification from the signal intensity. Table 1 shows the results. By not adding a reducing agent during sample preparation for SDS-PAGE, a signal is detected at a position of approximately 50 kDa where VLCL and VHCH1 are heterodimeric proteins via disulfide bonds.

The amount of Fab produced in the mixed culture of (2) was 1.4 times the amount of extracellular Fab produced in (1), confirming an improvement in productivity. After culturing, the culture supernatants of (3) and (4) were mixed (30°C, 24 hours) to try to form Fab as a heterodimeric protein. No bands were detected.

When two types of genes are carried in one expression vector like pROXb3-Fab, the size of the expression vector increases, making construction difficult. Furthermore, it is known that the combination of the signal peptide and the substance to be secreted and produced greatly affects its production ability. Examining signal peptides using an expression vector carrying two types of genes, for example, when examining three types of signal peptides for each gene, it is necessary to construct expression vectors with nine types of combinations. On the other hand, if mixed culture is performed as in (2), it is sufficient to construct three types of expression vectors and change the combination during culture. In other words, this mixed culture can be said to be a culture method that not only improves the productivity of heterodimeric proteins, but also allows efficient investigation of their combinations.

(Example 3) Comparison of bispecific antibody production
Mixed cultures were examined for their ability to form bispecific antibodies, as well as for Fab production. Brevibacillus choshinensis HPD31 was transformed with each of the expression vectors pROXb3-scFv(EGFR)-CH1-His and pROXb3-scFv(CD3)-CL-DDDDK. Each transformant was cultured in 2 mL of TMN medium at 30°C for 18 hours (preculture). (1) Add 0.5 mL of pROXb3-scFv(EGFR)-CH1-His transformant culture and 0.5 mL of pROXb3-scFv(CD3)-CL-DDDDK transformant culture to 100 mL of freshly prepared TMN medium. bottom. In parallel, (2) 1 mL of pROXb3-scFv(EGFR)-CH1-His transformant culture medium was added, and (3) 1 mL of pROXb3-scFv(CD3)-CL-DDDDK transformant culture medium was added. Culturing was carried out at 30° C. for 48 hours.

In the same manner as in Example 2, the extracellular culture solution after the mixed culture of (1) and the culture supernatant of (2) and (3) were mixed (30 ° C., 24 hours) and subjected to SDS-PAGE under non-reducing conditions. The amount of heterodimeric protein formation was quantified by Western blotting using an anti-His antibody. Table 2 shows the results.

As in the case of Fab in Example 2, the amount of dimer formation in the case of mixed culture is greater than when mixed after culturing each of them. It can be said that this culture method is useful not only for producing heterodimeric proteins.

INDUSTRIAL APPLICABILITY According to the method of the present invention, active ingredients of pharmaceuticals such as bispecific antibodies can be produced efficiently.

Claims (6)

A method for producing a heterodimeric protein using Brevibacillus , wherein an expression vector into which DNA encoding one of the monomer proteins constituting the heterodimeric protein is inserted is used to transform Brevibacillus. Brevibacillus was transformed with an expression vector into which DNA encoding the other monomer protein was inserted, and the resulting two types of transformed Brevibacillus were mixed and cultured at the same time. Bacillus bacterium to secrete and produce each monomer protein in the culture solution, and covalently bind the two types of monomer proteins in the culture solution to form a heterodimeric protein, A method for producing a heterodimeric protein, wherein the protein has an antibody variable region and a constant region, and disulfide bonds between the constant regions form the heterodimeric protein. 2. The method of making a heterodimeric protein of claim 1, wherein the heterodimeric protein is a bispecific antibody or Fab. In a heterodimeric protein formed from a monomer protein 1 and a monomer protein 2, an appropriate combination of heterodimer proteins 1 and m types of monomer proteins 2 is selected. A method for screening a monomeric protein, wherein the monomer has an antibody variable region and a constant region, n and m are 10 or less, and DNA encoding n kinds of monomeric protein 1 is inserted. Using each of the expression vectors , brevibacillus was transformed to prepare n types of transformed brevibacillus, and each of the expression vectors into which DNA encoding m types of monomeric protein 2 was inserted was used to transform brevibacillus. Transform the bacterium to create m types of transformed Brevibacillus strains, one of n types of Brevibacillus strains that produce monomeric protein 1 and m strains of brevibacillus that produce monomeric protein 2 Simultaneously mixed culturing one type of Bacillus, causing each brevibacillus to secrete and produce its respective monomeric protein in the culture medium, and forming a heterodimeric protein in the culture medium, n xm types of heterodimeric proteins, testing properties of nxm types of heterodimeric proteins, and selecting heterodimeric proteins with appropriate properties. A method for screening a suitable combination of heterodimeric proteins from combinations of a monomeric protein 1 and m types of monomeric proteins 2. 4. The method of screening for heterodimeric proteins according to claim 3, wherein the covalent bond that binds the two types of monomeric proteins is a disulfide bond. 5. The method of screening for heterodimeric proteins according to claim 3 or 4, wherein the monomers form heterodimeric proteins through disulfide bonds between the constant regions. 6. The method of screening for heterodimeric proteins according to claim 5, wherein the heterodimeric proteins are bispecific antibodies or Fabs.

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