Method for producing heavy chain variable domain of heavy chain antibody

Abstract

Provided is a method for producing a heavy chain variable domain (VHH) of a heavy-chain antibody at high yield. The present invention pertains to a VHH production method that includes culturing bacteria of genus Bacillus into which the gene coding the VHH has been introduced and which is deficient in extracellular proteases.

Description

[Technical field]

[0001] The present invention relates to a method for producing a heavy chain variable domain of a heavy chain antibody. [Background technology]

[0002] Small molecular weight antibodies have advantages such as production efficiency, ease of drug design, and tissue permeability, and therefore have been attracting attention as materials for antibody drugs and reagents. Representative small molecular weight antibodies include Fab, single chain antibodies (scFv), diabody, and variable domain of heavy chain of heavy chain antibody (VHH). Serum of camelids (Bactrian camels, dromedaries, llamas, etc.) and cartilaginous fish such as sharks contains antibodies consisting of only heavy chains without light chains (heavy-chain antibodies). The variable domain of the heavy-chain antibody is VHH. VHH is a molecule that can recognize an antigen with a single domain, and is the smallest unit of antibody molecules discovered to date. Pharmaceutical VHH containing multiple domains or humanized domains are also commercially available.

[0003] Conventionally, Escherichia coli has been mainly used for the microbiological production of antibodies. However, heterologous protein production using E. coli has problems such as low productivity of high molecular weight molecules, contamination of the product with endotoxin, and reduced product yield due to endotoxin removal treatment. Non-Patent Document 1 describes that Brevibacillus can overcome the above-mentioned shortcomings of E. coli, and that scFv, Fab, and VHH have been produced using a Brevibacillus expression system. Patent Document 1 describes a report that VHH has been produced using filamentous fungi such as Aspergillus.

[0004] Bacillus bacteria such as Bacillus subtilis secrete extracellular proteases such as AprE, Epr, WprA, Mpr, NprB, Bpr, NprE, Vpr, and AprX. In heterologous protein production using Bacillus bacteria, these extracellular proteases are known to be factors that reduce the productivity of the target protein. Patent Document 2 describes that the productivity of heterologous proteins is improved in Bacillus strains in which extracellular proteases such as AprX are deleted. Non-Patent Document 2 describes that a Bacillus subtilis strain that co-expresses intracellular and extracellular molecular chaperones and in which the cell wall-bound protease WprA is inactivated secretes and produces scFv derived from a fibrin-specific monoclonal antibody. On the other hand, Non-Patent Document 2 also describes that the scFv could not be produced in a Bacillus subtilis strain in which seven types of extracellular proteases are deleted.

[0005] (Patent Document 1) JP 2012-157315 A (Patent Document 2) JP 2006-174707 A (Non-patent document 1) Biotechnology, 2017, 95(11):649-651 (Non-patent document 2) Appl Environ Microbiol, 2002, p.3261-3269 Summary of the Invention

[0006] The present invention provides a method for producing a heavy chain variable domain of a heavy chain antibody, comprising culturing a Bacillus bacterium into which a gene encoding the heavy chain variable domain of a heavy chain antibody has been introduced and which is deficient in extracellular proteases. The present invention also provides a Bacillus bacterium into which a gene encoding a heavy-chain variable domain of a heavy-chain antibody has been introduced and which is deficient in extracellular proteases. [Brief description of the drawings]

[0007] [Figure 1] SDS-PAGE image of the culture supernatant from a recombinant Bacillus subtilis strain lacking a single extracellular protease and incorporating a VHH gene. The arrow indicates the position of the VHH protein band. [Diagram 2]Expression of pharmaceutical VHH molecules using the protease multiple deletion strain Dpr9ΔsigF. [Diagram 3] Western blotting images of ozoralizumab and caplacizumab expressed in Dpr9ΔsigF. * indicates full-length and < indicates degradation products. [Figure 4] Antigen (HEWL) binding activity of VHH (1ZVH) expressed in Bacillus subtilis. [Diagram 5] Western blotting images of cultures of E. coli strains into which VHH genes have been introduced. Detailed Description of the Invention

[0008] As used herein, the term "antibody" refers to a molecule capable of specifically binding to a target such as a protein via an antigen recognition domain derived from immunoglobulin.

[0009] As used herein, "VHH" refers to the variable domain of a heavy chain of a heavy chain antibody. Heavy chain antibodies have been found in camelids, cartilaginous fish such as sharks, and the like. The VHH in this specification is derived from, for example, camelids or sharks, and is preferably derived from camelids. VHH is a molecule capable of recognizing an antigen with a single domain, and is the smallest unit of antibody molecules found to date. The VHH produced in the present invention may contain one or more heavy chain variable domains derived from a heavy chain antibody, and the number of heavy chain variable domains contained in the VHH is not limited. The heavy chain variable domain contained in the VHH produced in the present invention may be a heavy chain variable domain derived from a natural heavy chain antibody or a modified version thereof. The VHH produced in the present invention may also contain a fragment other than the heavy chain variable domain derived from the immunoglobulin heavy chain (for example, a fragment of the constant region of a heavy chain antibody, or a human-derived fragment, a linker sequence, etc.), or an amino acid mutation for stabilization. The VHH produced by the present invention does not have a variable domain derived from an immunoglobulin light chain as an antigen recognition domain. For example, single-chain antibodies (e.g., scFv) containing an immunoglobulin light chain variable domain are not included in the VHH produced by the present invention.

[0010] In this specification, the camelid family includes Bactrian camels, dromedaries, llamas, alpacas, vicunas, guanacos, etc., and preferably includes alpacas.

[0011] In the present specification, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using the search homology program of the genetic information processing software Genetyx-Win with the unit size to compare (ktup) set to 2.

[0012] As used herein, "at least 85% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 85% or more, preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more.

[0013] As used herein, the term "operably linked" between a regulatory region and a gene refers to a state in which the gene and the regulatory region are linked such that the gene can be expressed under the control of the regulatory region. The procedure for "operably linked" between a gene and a regulatory region is well known to those skilled in the art.

[0014] In this specification, "upstream" and "downstream" of a gene refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter in the DNA sense strand, and "upstream" of a gene means the 5' region of the gene in the DNA sense strand.

[0015] As used herein, the term "deficiency" of a protease in a cell means that the activity of the protease by the cell is reduced compared to the activity of the cell itself (a non-deficient strain, for example, a wild-type strain). The "deficiency" of a protease includes not only a reduction or loss of expression of a protease protein, but also a reduction or loss of function of the protease protein. A cell lacking a protease (a protease-deficient strain) can be prepared by partial or complete deletion or inactivation (so-called knockdown or knockout, etc.) of the gene for the protease in the cell. Preferably, the protease-deficient strain in the present specification has a protease activity reduced to 50% or less, more preferably 25% or less, compared to that of a wild-type strain.

[0016] As used herein, the term "native" when referring to a function, property, or trait of a cell is used to indicate that the function, property, or trait is inherently present in the cell. In contrast, the term "exogenous" is used to indicate a function, property, or trait that is not inherently present in the cell, but is introduced from the outside. For example, an "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from the outside. An exogenous gene or polynucleotide may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).

[0017] In this specification, the term "bacteria of the genus Bacillus" refers to bacteria of the genus Bacillus in the family Bacillaceae. Examples of bacteria of the genus Bacillus include Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus liqueniformis. Note that bacteria of the genera Paenibacillus and Brevibacillus, which belong to the family Paenibacillaceae, are not included in the genus Bacillus in this specification.

[0018] The names of the genes of Bacillus subtilis described herein are based on the Bacillus subtilis genome data published on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) ([bacillus.genome.ad.jp / ], updated January 18, 2006). The gene numbers of Bacillus subtilis described herein refer to the gene numbers registered in BSORF DB.

[0019] The present invention provides a method for producing a heavy chain variable domain (VHH) of a heavy chain antibody.

[0020] The present inventors have found that VHH can be produced in high yields using Bacillus bacteria that have been deficient in extracellular proteases.

[0021] According to the present invention, VHH can be produced in high yield. The present invention overcomes the problems of low yield and endotoxin contamination in conventional antibody production using E. coli. Furthermore, since Bacillus bacteria have a long history of use in the production of useful substances such as enzymes and are not harmful, the method of the present invention is easy to operate and safe.

[0022] The present invention provides a method for producing a VHH, which comprises culturing a Bacillus bacterium into which a gene encoding a VHH has been introduced and which is deficient in extracellular proteases.

[0023] The VHH produced by the present invention is as defined above. More specific examples of the VHH produced by the present invention include polypeptides consisting of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38. Detailed information on these VHHs is shown in Table 1.

[0024] [Table 1]

[0025] Other examples of VHHs produced by the present invention include polypeptides consisting of an amino acid sequence that is at least 85% identical to any of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.

[0026] Further examples of VHHs produced by the present invention include polypeptides consisting of the amino acid sequences of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138. Further examples of VHHs produced by the present invention include polypeptides consisting of an amino acid sequence that is at least 85% identical to any of the amino acid sequences set forth in SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138.

[0027] The gene (polynucleotide) encoding the VHH described above can be prepared according to a conventional method. Examples of the gene encoding the VHH include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, and 137, and a polynucleotide having at least 85% sequence identity to any of these sequences. For example, the polynucleotide of the gene encoding the VHH can be chemically synthesized according to the sequence information. Alternatively, the polynucleotide of the gene encoding the VHH can be isolated from the genomic DNA of a camelid. The obtained gene may be further subjected to mutagenesis.

[0028] The gene encoding the VHH may be operably linked to a control region. In the present specification, the term "control region" refers to a region that has the function of controlling the expression of a gene located downstream thereof in a cell, and preferably has the function of constitutively expressing or highly expressing the gene located downstream. More specifically, the control region may be defined as a region that exists upstream of the coding region of a gene and has the function of controlling the transcription of the coding region by interacting with RNA polymerase. Preferably, the control region in the present specification refers to a region of about 200 to 600 nucleotides upstream of the coding region of a gene. The control region includes a transcription initiation control region and / or a translation initiation control region, or a region from the transcription initiation control region to the translation initiation control region. The transcription initiation control region is a region that includes a promoter and a transcription initiation site, and the translation initiation control region is a site corresponding to the Shine-Dalgarno (SD) sequence that forms a ribosome binding site together with an initiation codon (Proc Natl Acad Sci USA, 1974, 71:1342-1346).

[0029] Preferred examples of the control region include, but are not limited to, control regions that function in bacteria of the genus Bacillus, for example, the control region of an α-amylase gene, a protease gene, an aprE gene, or a spoVG gene derived from a bacillus bacterium, the control region of the cellulase gene of Bacillus sp. KSM-S237 strain (JP 2000-210081 A), the control region of the cellulase gene of Bacillus sp. KSM-64 strain (JP 2011-10387 A), and the control region of a kanamycin resistance gene or a chloramphenicol resistance gene derived from Staphylococcus aureus (for both, see JP 2009-089708 A). More preferred examples of the control region include the promoter of the cellulase gene of Bacillus sp. KSM-S237 strain (SEQ ID NO: 39), the promoter of the cellulase gene of Bacillus sp. KSM-64 strain (SEQ ID NO: 40), and the promoter of the spoVG gene of Bacillus subtilis (SEQ ID NO: 41). In addition, preferred control regions include polynucleotides that have at least 85% identity with any of SEQ ID NOs: 39 to 41 and have the function of controlling the transcription and translation of genes.

[0030] The gene encoding the VHH may be operably linked to a sequence encoding a secretory signal having the function of secreting an expressed protein outside the cell (referred to as a secretory signal sequence). Preferred examples of the secretory signal sequence include secretory signal sequences that function in Bacillus bacteria, such as secretory signal sequences derived from Bacillus bacteria. Preferred examples of secretory signal sequences derived from Bacillus bacteria include the secretory signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain (SEQ ID NO: 42), the secretory signal sequence of the cellulase gene of Bacillus sp. KSM-64 strain (SEQ ID NO: 43), and the secretory signal sequence of the amylase gene amyE of Bacillus subtilis (SEQ ID NO: 44). Further examples of secretory signal sequences derived from Bacillus bacteria include polynucleotides that have at least 85% identity with any of SEQ ID NOs: 42 to 44 and have the function of secreting an expressed protein outside the cell.

[0031] The gene encoding the VHH may contain a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF). For example, the gene may contain the above-mentioned promoter, secretory signal sequence, and terminator.

[0032] The gene can be introduced into a Bacillus bacterium according to a standard method. For example, the gene or a vector containing the gene can be introduced into a host Bacillus cell to integrate the gene into the genome of the host cell. Alternatively, an expression vector containing the gene can be introduced into a host Bacillus cell.

[0033] To introduce a gene or vector into a host Bacillus cell, known transformation techniques such as the competent cell method, electroporation, protoplast method, particle gun method, and PEG method can be applied.

[0034] Examples of host Bacillus bacteria into which the gene encoding the VHH is introduced include the aforementioned bacteria of the genus Bacillus in the family Bacillaceae, among which Bacillus subtilis or a mutant strain thereof is preferred.

[0035] A vector containing the gene encoding the VHH can be constructed by inserting and ligating the gene and, if necessary, a polynucleotide containing a regulatory region or secretory signal sequence into any vector by a conventional method. The type of the vector is not particularly limited, and may be any vector, such as a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC vector, or a shuttle vector. Furthermore, the vector is preferably a vector that can be amplified in a host cell, and more preferably an expression vector. Examples of preferred vectors include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmids usable for transformation of bacteria of the genus Bacillus, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmids derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0036] The Bacillus bacterium used for producing VHH in the present invention has a gene encoding the VHH introduced therein and is also deficient in extracellular protease. The extracellular protease deficient in the Bacillus bacterium is preferably at least one selected from the group consisting of epr, wprA, bpr, vpr, aprE, aprX, and extracellular proteases encoded by genes corresponding thereto, more preferably at least one selected from the group consisting of proteases encoded by epr, wprA, bpr, vpr, aprE, and apr, even more preferably a protease encoded by epr, wprA, bpr, vpr, aprE, or apr, and even more preferably a protease encoded by wprA or bpr.

[0037] All of epr, wprA, bpr, vpr, aprE, and aprX are Bacillus subtilis genes. The gene numbers of these genes and the functions of the proteins they encode are shown in Table 2. Genes equivalent to epr, wprA, bpr, vpr, aprE, and aprX include genes derived from the genus Bacillus that have at least 85% identity in nucleotide sequence to epr, wprA, bpr, vpr, aprE, and aprX, respectively, and encode proteins with the same functions (listed in Table 2). These genes can be searched for in the BSORF DB mentioned above.

[0038] [Table 2]

[0039] The Bacillus bacteria used in the present invention may be deficient in extracellular proteases other than the above-mentioned extracellular proteases encoded by epr, wprA, bpr, vpr, aprE, and aprX. For example, the Bacillus bacteria used in the present invention may be deficient in all of the extracellular proteases encoded by epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX. Furthermore, the Bacillus bacteria used in the present invention may be modified in a manner other than the deficiency of extracellular proteases, for example, deletion of a sigma factor such as sigF.

[0040] Bacillus bacteria lacking extracellular proteases can be produced according to standard methods. For example, a Bacillus bacterium lacking extracellular proteases can be produced by deleting or inactivating a gene encoding an extracellular protease in the genomic DNA of the bacterium. Thus, in one embodiment of the present invention, the Bacillus bacteria used for producing VHHs have at least one gene selected from the group consisting of epr or a gene equivalent thereto, wprA or a gene equivalent thereto, bpr or a gene equivalent thereto, vpr or a gene equivalent thereto, aprE or a gene equivalent thereto, and aprX or a gene equivalent thereto deleted or inactivated. Preferably, the Bacillus bacteria have at least one gene selected from the group consisting of epr, wprA, bpr, vpr, aprE, and aprX deleted or inactivated. More preferably, the Bacillus bacteria have epr, wprA, bpr, vpr, aprE, or aprX deleted or inactivated. More preferably, the Bacillus bacterium has wprA or bpr deleted or inactivated.

[0041] Means for deleting or inactivating a gene include, for example, mutagenesis of the gene or its structural region, such as substitution or insertion of one or more nucleotides in the polynucleotide of the gene or its structural region, or deletion of a part or the whole of the sequence; inactivation of the promoter by substitution or insertion of another sequence into the promoter region of the gene; introduction of an antisense nucleic acid for the gene, etc. Specific methods for mutagenesis include ultraviolet irradiation, site-specific mutagenesis, SOE-PCR (splicing by overlap extension PCR: Gene, 1989, 77: 61-68), and homologous recombination. The position on the genomic DNA and the nucleotide sequence of the gene to be deleted or inactivated can be confirmed in the BSORF DB mentioned above.

[0042] In the present invention, a recombinant Bacillus bacterium is used that has been introduced with the polynucleotide encoding VHH obtained by the above-mentioned procedure and that is deficient in extracellular protease. In the procedure for preparing the recombinant Bacillus bacterium, the order of introduction of the polynucleotide encoding VHH and the protease deletion treatment is not particularly limited, as long as the desired recombinant Bacillus bacterium is obtained.

[0043] In the present invention, a recombinant Bacillus bacterium into which a polynucleotide encoding the above-mentioned VHH has been introduced and which is deficient in extracellular protease is cultured to produce a desired VHH. The recombinant Bacillus bacterium may be cultured according to a general method for culturing Bacillus bacteria. For example, a medium for culturing Bacillus bacteria contains a carbon source and a nitrogen source necessary for the growth of the bacteria. Examples of carbon sources include glucose, dextran, soluble starch, sucrose, and methanol. Examples of nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. If necessary, the medium may contain other nutrients, such as inorganic salts (e.g., sodium chloride, calcium chloride, sodium dihydrogen phosphate, and magnesium chloride), vitamins, and antibiotics (e.g., tetracycline, neomycin, kanamycin, spectinomycin, and erythromycin). Culture conditions, such as temperature, aeration and agitation conditions, medium pH, and culture time, can be appropriately selected depending on the bacterial species, characteristics, culture scale, etc.

[0044] By culturing the recombinant Bacillus bacteria, the VHH is expressed intracellularly. Furthermore, when the gene for the VHH is linked to a secretion signal, the expressed VHH is secreted outside the cells. The VHH produced by the method of the present invention can be recovered from the culture medium by a general method used for protein purification, such as cell disruption, centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination.

[0045] The present invention also includes the following substances, manufacturing methods, uses, methods, etc. as exemplary embodiments, but the present invention is not limited to these embodiments.

[0046] [1] A method for producing a heavy chain variable domain of a heavy chain antibody, comprising culturing a Bacillus bacterium into which a gene encoding the heavy chain variable domain of a heavy chain antibody has been introduced and which is deficient in extracellular proteases. [2] The extracellular protease, Preferably, it is at least one selected from the group consisting of epr, wprA, bpr, vpr, aprE, aprX, and extracellular proteases encoded by genes corresponding thereto; More preferably, it is an extracellular protease encoded by epr, wprA, bpr, vpr, aprE or aprX. More preferably, it is an extracellular protease encoded by wprA or bpr. The method described in [1]. [3] The method of [1] or [2], wherein the heavy chain variable domain of the heavy chain antibody preferably consists of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138, or an amino acid sequence that is at least 85% identical to said sequence. [4] The method according to any one of [1] to [3], wherein the Bacillus bacterium is Bacillus subtilis or a mutant strain thereof. [5] The method according to any one of [1] to [4], wherein the Bacillus bacterium lacking an extracellular protease is a Bacillus bacterium in which a gene encoding the extracellular protease has been deleted or inactivated.

[0047] [6] A Bacillus bacterium having a gene encoding a heavy-chain variable domain of a heavy-chain antibody introduced therein and deficient in extracellular proteases. [7] The extracellular protease, Preferably, it is at least one selected from the group consisting of epr, wprA, bpr, vpr, aprE, aprX, and extracellular proteases encoded by genes corresponding thereto; More preferably, it is an extracellular protease encoded by epr, wprA, bpr, vpr, aprE or aprX. More preferably, it is an extracellular protease encoded by wprA or bpr. The Bacillus bacterium described in [6]. [8] The Bacillus bacterium according to [6] or [7], wherein the heavy chain variable domain of the front heavy chain antibody preferably consists of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138, or an amino acid sequence that is at least 85% identical to said sequence. [9] The Bacillus bacterium according to any one of [6] to [8], which is preferably Bacillus subtilis or a mutant strain thereof.

[10] The Bacillus bacterium according to any one of [6] to [9], preferably wherein a gene encoding the extracellular protease is deleted or inactivated. EXAMPLES

[0048] The present invention will be described in more detail below with reference to examples, although the technical scope of the present invention is not limited to these examples.

[0049] material (1) Strains From Bacillus subtilis 168 strain (hereinafter referred to as 168 strain), strains deficient in extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE and aprX) were prepared according to the method described in Patent Document 2. Hereinafter, the obtained extracellular protease single-deficient strains are respectively referred to as Δepr, ΔwprA, Δmpr, ΔnprB, Δbpr, ΔnprE, Δvpr, ΔaprE and ΔaprX. In addition, according to the method described in Patent No. 4336082, the sigF gene involved in sporulation was deleted from the Bacillus subtilis strain Dpr9, which is deficient in all of the above nine extracellular protease genes. The obtained extracellular protease multiple-deficient strain is referred to as Dpr9ΔsigF. The Escherichia coli used was the Rosetta-gami 2(DE3)pLysS strain (Novagen).

[0050] (2) Culture medium LB medium: 1% Bacto TM Tryptone, 0.5% Bacto TM Yeast Extract, 1% sodium chloride. 1.5% agar was added to the plate medium. Tetracycline (50 ppm) was added as necessary. DM3 medium: 1% CMC (Kanto Chemical), 0.5% Bacto TM Casamino Acids, 0.5% Bacto TM Yeast Extract, 8.1% disodium succinate 6H2O, 0.35% dipotassium hydrogen phosphate, 0.15% potassium dihydrogen phosphate, 0.5% glucose, 20 mM magnesium chloride, 0.01% BSA, 50 ppm tetracycline. 1% agar was added to the plate medium. 2×L-mal medium: 2% Bacto TM Tryptone, 1% Bacto TM Yeast Extract, 1% Sodium Chloride, 7.5% Maltose Monohydrate, 7.5 ppm Manganese Sulfate, 15 ppm Tetracycline.

[0051] (3) Reagents Unless otherwise specified, reagents used were manufactured by Wako.

[0052] Example 1. VHH production by protease-deficient recombinant Bacillus subtilis (1) Artificial synthesis of genes The VHH genes (polynucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, encoding the VHHs of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, respectively) were artificially synthesized by Thermo Fisher Scientific. GCAGCTCTTGCAGCA (SEQ ID NO: 45) and TCTATTAAACTAGTT (SEQ ID NO: 46) were added to the 5' end and 3' end, respectively, of the artificially synthesized genes as sequences for inserting the genes into a plasmid vector.

[0053] (2) Construction of plasmid for expression of VHH The plasmid sequence was amplified by PCR using the recombinant plasmid pHY-S237 (JP Patent Publication 2014-158430) as a template and a primer set of SEQ ID NO: 47 (5'-TGCTGCAAGAGCTGCCGGAAATAAA-3') and SEQ ID NO: 48 (5'-TCTATTAAACTAGTTATAGGG-3') with PrimeSTAR Max DNA polymerase (TaKaRa). DNA containing the artificial synthetic gene of (1) was inserted into the obtained PCR fragment using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid containing each of the artificial synthetic VHH genes shown in (1). The constructed plasmid was introduced into Dpr9ΔsigF according to the procedure shown in the following (3).

[0054] For the gene encoding the VHH of SEQ ID NO: 26, the promoter region was changed to that derived from the Bacillus subtilis spoVG gene by the following procedure. Using a VHH expression plasmid containing the gene of SEQ ID NO: 25 as a template, the plasmid sequence was amplified by PCR using a primer set of SEQ ID NO: 49 (5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3') and SEQ ID NO: 50 (5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3') and PrimeSTAR Max DNA polymerase (TaKaRa). Using the genome of strain 168 as a template, the promoter DNA derived from the spoVG gene was amplified by PCR using a primer set of SEQ ID NO: 51 (5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3') and SEQ ID NO: 52 (5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3'). The obtained promoter DNA was incorporated into a plasmid sequence using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid containing the VHH gene of SEQ ID NO: 25 linked to the spoVG promoter. The constructed plasmid was introduced into each of the protease single-deficient strains Δepr, ΔwprA, Δmpr, ΔnprB, Δbpr, ΔnprE, Δvpr, ΔaprE, and ΔaprX according to the procedure shown in (3) below.

[0055] (3) Construction of recombinant Bacillus subtilis Plasmids were introduced into Bacillus subtilis strains by the protoplast method described below. Various Bacillus subtilis strains stocked in glycerol were inoculated into 1 mL of LB liquid medium and cultured overnight at 30°C and 210 rpm with shaking. The next day, 10 μL of this culture was inoculated into a new 1 mL of LB liquid medium and cultured at 37°C and 210 rpm with shaking for about 2 hours. This culture was collected in a 1.5 mL tube, centrifuged at 1,2000 rpm for 5 minutes, and the pellet from which the supernatant was removed was suspended in 500 μL of SMMP containing 4 mg / mL of Lysozyme (SIGMA) and incubated at 37°C for 1 hour. Next, the mixture was centrifuged at 3,500 rpm for 10 minutes, and the pellet from which the supernatant was removed was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with various plasmids, and 100 μL of 40% PEG was added and vortexed. To this solution, 350 μL of SMMP was added and mixed by inversion, and after shaking at 30° C. and 210 rpm for 1 hour, the entire amount was spread onto a DM3 agar medium plate and incubated at 30° C. for 2 to 3 days.

[0056] (4) VHH production The recombinant Bacillus subtilis prepared in (3) was inoculated into 1 mL of LB medium containing 50 ppm tetracycline and shaken overnight at 32°C to prepare a preculture solution. For the protease single deletion strain, the preculture solution was inoculated at 1% into 5 mL of 2×L-mal medium placed in a test tube and cultured at 30°C for 72 hours with shaking. For Dpr9ΔsigF, the preculture solution was inoculated at 1% into 20 mL of 2×L-mal medium placed in a pleated Erlenmeyer flask and cultured at 30°C for 72 hours with shaking. At the end of the culture, 1 mL of the culture solution was centrifuged in a microtube at 4°C, 15,000 rpm, and 5 minutes, and the supernatant was collected.

[0057] (5) SDS-PAGE The culture supernatant obtained in (4) was mixed with an equal volume of Laemmli Sample Buffer (BIO-RAD) containing 50 mM DTT, and then heat-treated at 99°C for 5 minutes to prepare a sample. Mini-PROTEIN TGX Stain-Free (BIO-RAD) was used for the gel. 5 μL of sample was applied to each well, and electrophoresis was performed at 210 V for 25 minutes. Precision Plus protein Unstained standard (BIO-RAD) was used as the molecular weight marker. Protein bands were detected using a ChemiDoc MP Imaging System. The detected proteins were quantified using a calibration curve created based on a lysozyme preparation (Sigma-Aldrich). Protein quantification was performed using Image Lab, a software manufactured by BIO-RAD.

[0058] (6) Western blotting Proteins were transferred from SDS-PAGE gel to a PVDF membrane using Trans-Blot Turbo Mini PVDF Transfer Packs (BIO-RAD) and Trans-Blot Turbo System (BIO-RAD). 6x-His Tag Monoclonal Antibody (3D5), HRP (Invitrogen) were used as antibodies, and iBind Western System (Invitrogen) was used for antibody reaction. The target protein was detected using 1-Step Ultra TMB-Blotting Solution (Thermo Scientific). Proteins were quantified using a calibration curve created based on His-N15 purified protein (SEQ ID NO: 26) with a known concentration. Protein quantification was performed using Image Lab, a software manufactured by BIO-RAD.

[0059] (7) Intermolecular interaction measurement The antigen binding activity of 1ZVH (SEQ ID NO: 38) produced by Dpr9ΔsigF was analyzed by molecular interaction measurement using Biacore T200 (GE Healthcare). The sensor chip was Series S Sensor Chip CM5, and the running buffer was HBS-EP+. HEWL (lysozyme, egg white-derived; Wako) was adjusted to 0.7 g / L with 25 mM Na phosphate, pH 6.5 buffer to prepare an antigen solution. The antigen solution was diluted 100-fold with an aqueous acetic acid solution (pH 4.0, 4.5, 5.0, or 5.5) to adjust to 7 μg / mL and used as a substrate. 1ZVH diluted to 8.6 nM, 26 nM, 78 nM, 230 nM, or 700 nM with the running buffer was used as a sample. The following items were set or executed on the control software attached to the Biacore T200 using the substrate and sample, and the binding between HEWL and 1ZVH was monitored. Immobilisation pH scouting was performed to determine the optimum pH for immobilisation on the sensor chip. The optimum pH condition was determined to be pH 5.5. Lysozyme was immobilised in flow path 2 by the amine coupling method, with the target set at 150 RU under the aim for immobilised level. Blank immobilisation was performed on flow path 1. The binding between the substrate and the sample was monitored in flow path 2-1. The regeneration solution used was 10 mM glycine-HCl (pH 1.5). The contact time for binding monitoring was 120 sec, the flow rate was 30 μL / min, the dissociation time was 120 sec, the contact time for regeneration was 30 sec, and the stabilisation period was 120 sec. Kinetics analysis was performed using the obtained sensorgram. 1:1 Binding was selected as the reaction model formula.

[0060] (8) Results Figure 1 shows SDS-PAGE images of culture supernatants from recombinant B. subtilis strains deficient in a single extracellular protease, Δepr, ΔwprA, Δmpr, ΔnprB, Δbpr, ΔnprE, Δvpr, ΔaprE, and ΔaprX. "+" in the figure indicates the intensity of the antibody band (indicated by the arrow in the figure). VHHs were highly expressed in Δepr, ΔwprA, Δbpr, Δvpr, ΔaprE, and ΔaprX. In particular, VHH expression in ΔwprA and Δbpr was significantly higher. These results indicated that the deletion of epr, wprA, bpr, vpr, aprE, and aprX affects the expression of VHHs in B. subtilis.

[0061] Table 3 shows the expression levels of VHHs of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 in the extracellular protease multiple-deficient recombinant Bacillus subtilis strain Dpr9ΔsigF. The expression of SEQ ID NO: 4 and SEQ ID NO: 28 was quantified by Western blotting, and the remaining expression was quantified by SDS-PAGE. Dpr9ΔsigF was able to produce various VHHs. Figure 2 also shows the expression levels of pharmaceutical VHH molecules (Ozoralizumab and Caplacizumab) of SEQ ID NOs: 30 and 32, and pharmaceutical humanized VHH molecules (NbHul6_VGLW and h-NbBcII10_FGLA) of SEQ ID NOs: 34 and 36 in Dpr9ΔsigF. Figure 3 shows the Western blotting images of Ozoralizumab and Caplacizumab expressed in Dpr9ΔsigF. Dpr9ΔsigF was able to express all four types of pharmaceutical VHH molecules in full length (see Figures 2 and 3). These results demonstrated that the protease-deficient Bacillus subtilis strain is capable of expressing VHHs, humanized VHHs, and molecules in which multiple VHHs are linked together.

[0062] [Table 3]

[0063] FIG. 4 shows electrophoretic images of the culture supernatant of Dpr9ΔsigF and fractions obtained during purification (left), and the antigen (HEWL) binding activity of the purified VHH (1ZVH, SEQ ID NO: 38) measured in (7) (right). In the electrophoresis, the Dpr9ΔsigF culture supernatant (S) was applied to a column packed with Ni-NTA Agarose (Wako), and fraction FT that passed through the column was obtained. Next, washing buffer (30 mM imidazole, 0.25 M NaCl, 10 mM Tris pH 8.0) was applied to the column, and fraction W that passed through the column was obtained. Then, elution buffer (500 mM imidazole, 0.5 M NaCl, 20 mM Tris pH 7.5) was applied to the column, and fraction E that passed through the column was obtained. The obtained S, and fractions FT, W, and E were treated with Laemmli Sample Buffer (BIO-RAD) without DTT. Fraction E was also treated with Laemmli Sample Buffer (BIO-RAD) containing 50 mM DTT. The treated sample was subjected to SDS-PAGE. Compared with fraction E treated with a buffer without DTT (non-reduced E), the band shifted upward in fraction E treated with a buffer containing DTT (reduced E). This suggested that disulfide bonds were formed in the VHH expressed in Bacillus subtilis. Furthermore, as shown in the right of Figure 4, the expressed VHH retained antigen-binding activity, as a concentration-dependent increase in the response value to the antigen immobilized on the sensor was observed by intermolecular interaction measurement. This result demonstrated that it is possible to produce active VHH using Bacillus subtilis.

[0064] Comparative Example 1: Production of VHH by recombinant E. coli (1) Construction of recombinant E. coli DNA containing the artificially synthesized genes shown in Example 1(1) (polynucleotides of SEQ ID NOs: 1, 3, 5, 7, and 9, encoding VHHs of SEQ ID NOs: 2, 4, 6, 8, and 10, respectively) was amplified by PCR using the primers shown in Table 4 and PrimeSTAR Max DNA polymerase (TaKaRa). pET22b was digested with Nco I and Hind III, and the resulting PCR fragment was inserted using the In-Fusion HD Cloning Kit to construct a plasmid. The constructed plasmid was mixed with competent cells of the Rosetta-gami 2(DE3)pLysS strain, left on ice for 10 minutes, and then treated at 42°C for 1 minute to introduce it into the E. coli strain.

[0065] [Table 4]

[0066] (2) VHH production The recombinant E. coli prepared in (1) was inoculated into 2 mL of LB medium containing 50 ppm sodium ampicillin, and the mixture was shaken overnight at 30°C to prepare a preculture solution. TM The bacteria was inoculated into Instant TB Medium and cultured at 30°C for 24 hours.

[0067] (3) SDS-PAGE and Western blotting The culture obtained in (2) was centrifuged to collect the cells. The obtained cells were resuspended in an equal volume of PBS buffer to the culture medium subjected to centrifugation, and Laemmli Sample Buffer (BIO-RAD) containing 50 mM DTT was added to prepare a sample. The obtained sample was subjected to SDS-PAGE in the same manner as in Example 1 (5). Western blotting was performed in the same manner as in Example 1 (6).

[0068] (4) Results SDS-PAGE images of the E. coli strain cultures are shown in Figure 5. Protein quantification showed that all strains had productivity of 15 mg / L or less. When compared with the VHH expression levels in the Dpr9ΔsigF strain (Table 3), it was shown that the VHH productivity of B. subtilis was more than 20 times that of E. coli.

[0069] Example 2 VHH production by protease-deficient recombinant Bacillus subtilis Polynucleotides of SEQ ID NOs: 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, and 137 (SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, A plasmid for expressing VHH was constructed in the same manner as in Example 1 (1) and (2), except that VHHs encoding VHHs 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138 (referred to as VHH1 to 38, respectively) were used. The constructed plasmid was introduced into Dpr9ΔsigF according to the procedure shown in Example 1 (3). The prepared recombinant Bacillus subtilis was cultured in the same manner as in Example 1 (4) to express VHH.

[0070] The VHH expressed from recombinant Bacillus subtilis was quantified by ELISA. ELISA was performed according to the manual for His Tag Antibody Plate (GenScript). The supernatant of the culture of the recombinant Bacillus subtilis obtained above was diluted 20,000 times with PBS, and 100 μL of the diluted solution was added to each well of the His Tag Antibody Plate and left to stand overnight at 4 ° C. The wells were washed three times with PBS containing 0.05% Tween (registered trademark) 20 (PBST), and then 100 μL of Peroxidase-conjugated AffiniPure Goat Anti-Alpaca IgG, VHH domain (Jackson ImmunoResearch 128-035-232) diluted 5000 times with PBST was added and left to stand at room temperature for 30 minutes. Thereafter, the wells were washed three times with PBST, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB [for ELISA]; Tokyo Chemical Industry) was added and left to stand at room temperature for 20 minutes. After adding 50 μL of TMB stop solution (CeraCare Life Sciences) to stop the reaction, the absorbance of each well was measured at 450 nm. Protein concentration was calculated using the absorbance of VHH1 of known concentration in ELISA according to the Ni-NTA agarose (Fujifilm Wako Pure Chemical) manual.

[0071] The expression levels of VHH1 to 38 in the recombinant Bacillus subtilis strain Dpr9ΔsigF are shown in Table 5. All of VHH1 to 38 were produced from the recombinant Bacillus subtilis strain.

[0072] [Table 5]

[0073] Although the embodiments of the present invention have been described above, it should be understood that they are not intended to limit the present invention to the specific embodiments described. Various other changes and modifications within the scope of the present invention will be apparent to those skilled in the art. The literature and patent applications cited in this specification are incorporated by reference as if fully set forth herein.

Claims

1. The method includes culturing Bacillus subtilis or a mutant thereof into which a gene encoding a heavy-chain variable domain of a heavy-chain antibody has been introduced and which is deficient in extracellular protease, the Bacillus subtilis or a mutant thereof is deficient in at least one extracellular protease selected from the extracellular proteases encoded by wprA and bpr, or is deficient in all extracellular proteases encoded by epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE and aprX, The heavy chain variable domain of the heavy chain antibody is a molecule capable of recognizing an antigen in a single domain. A method for producing a heavy chain variable domain of a heavy chain antibody.

2. The method according to claim 1, wherein the Bacillus subtilis or a mutant thereof has been introduced with a plasmid vector in which a gene encoding a heavy-chain variable domain of the heavy-chain antibody is located downstream of a promoter of a cellulase gene of Bacillus sp. KSM-S237 strain shown in SEQ ID NO: 39, a promoter of a cellulase gene of Bacillus sp. KSM-64 strain shown in SEQ ID NO: 40, a promoter of a Bacillus subtilis spoVG gene shown in SEQ ID NO: 41, or a polynucleotide having at least 85% sequence identity thereto and having a function of controlling gene transcription and translation.

3. The method of claim 1 or 2, wherein the heavy chain variable domain of the heavy chain antibody is a molecule consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

4. The method according to any one of claims 1 to 3, wherein the Bacillus subtilis or a mutant thereof lacking an extracellular protease is a Bacillus subtilis or a mutant thereof in which a gene encoding the extracellular protease has been deleted or inactivated.

5. A gene encoding a heavy chain variable domain of a heavy chain antibody is introduced, and The strain is deficient in at least one extracellular protease selected from the extracellular proteases encoded by wprA and bpr, or is deficient in all extracellular proteases encoded by epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX, The heavy chain variable domain of the heavy chain antibody is a molecule capable of recognizing an antigen in a single domain. Bacillus subtilis or a mutant thereof.

6. The Bacillus subtilis or a mutant thereof according to claim 5, wherein a plasmid vector in which a gene encoding a heavy-chain variable domain of the heavy-chain antibody is located downstream of a promoter of a cellulase gene of Bacillus sp. KSM-S237 strain shown in SEQ ID NO: 39, a promoter of a cellulase gene of Bacillus sp. KSM-64 strain shown in SEQ ID NO: 40, a promoter of a Bacillus subtilis spoVG gene shown in SEQ ID NO: 41, or a polynucleotide having at least 85% sequence identity thereto and having a function of controlling gene transcription and translation, is introduced.

7. The Bacillus subtilis or a mutant thereof according to claim 5 or 6, wherein the heavy chain variable domain of the heavy chain antibody is a molecule consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

8. The Bacillus subtilis or a mutant thereof according to any one of claims 5 to 7, wherein the gene encoding the extracellular protease is deleted or inactivated.

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