JP6781854B1 - Method for producing and screening monoclonal antibody with yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a monoclonal antibody more quickly and efficiently without using an individual animal. A DNA fragment containing a gene encoding a secretory signal, a gene encoding a nanobody, and a gene encoding a peptide bar code, or a yeast into which a vector containing the DNA fragment has been introduced, wherein the peptide bar code is used. Discloses a yeast represented by an amino acid sequence consisting of 5 to 30 amino acids, wherein the amino acids are independently selected from the group consisting of A, F, G, K, L, P, R, V and W. .. Monoclonal antibodies consisting of Nanobodies and polypeptides containing the peptide barcodes, as well as the barcode peptides, are also disclosed. [Selection diagram] Fig. 1

Description

The present invention relates to a method for producing a monoclonal antibody with yeast and a method for screening.

An antibody is a large protein molecule of 150 kDa consisting of a heavy chain (H chain) and a light chain (L chain). So far, antibodies have been produced over time, for example, by administering an antigen to an individual vertebrate. The antigenic determination site of an antibody is encoded as a gene of various sites on the genome, and the diversity is combined against the invasion of various foreign substances from the outside to create a so-called polyclonal antibody protein molecule. Furthermore, monoclonal antibodies with improved specificity are produced by preparing hybridoma cells (fusion cells) with myeloma cells (myeloma cells) in vitro and culturing them.

Thus, polyclonal antibodies have been produced by administration to vertebrates, and monoclonal antibodies by cell culture in vitro. However, since a full-bodied antibody consisting of a heavy chain (H chain) and a light chain (L chain) is a large protein molecule, both methods for producing polyclonal antibodies and monoclonal antibodies are costly and time-consuming methods. It was.

On the other hand, for the production of antibodies, display techniques (eg, phage display or yeast display) have been used to screen for antibodies produced in animals or by cultured cells. In display technology, antibodies are immobilized on the surface of cells and phages. Antibodies immobilized in this way are known to have enhanced structural stability. Therefore, when the antibody obtained by screening is used as a free antibody, which is the actual usage situation, the structural stability may be lost and the antibody may not be used. In addition, since a plurality of antibodies are presented to one carrier (for example, phage or yeast cells), a proximity inhibitory effect may occur and an antibody having a low binding ability may be obtained.

Monoclonal antibodies are attracting attention as antibody drugs. Monoclonal antibodies bind to a single antigen and have high specificity, so they are expected to be applied to cancer cells, autoimmune diseases, infectious diseases, and the like. For example, a monoclonal antibody can specifically bind to a target antigen and attack and destroy cells containing that antigen (eg, cancer cells, cells infected with an infectious agent) by an immune system. Therefore, there is a need for a simpler method for producing a monoclonal antibody. In addition, there is also a need for a method for more easily identifying monoclonal antibodies that specifically bind to the target antigen.

Non-Patent Documents 1 and 2 describe that NestLink, a method for selecting and identifying a binding substance capable of characterizing thousands of library members at a time, has been developed as a method for examining the binding ability without immobilizing an antibody. Has been done. This NestLink is based on the gene-encoded peptide barcode "flycode", which was designed to enhance the detection power of mass spectrometry and to function as a unique identifier during sequence analysis. It is stated that.

However, in order to facilitate the identification of antibodies capable of binding, a method with increased sensitivity and specificity and less discrimination bias is still required for the detection of peptides by mass spectrometry.

Egloff P. et al., BioRxiv: 287813 (December 21, 2018) Egloff P. et al., NATURE METHOD VOL 16 MAY 2019 421-428

An object of the present invention is to provide a method for producing a monoclonal antibody more quickly and efficiently.

The present invention provides a method of producing a monoclonal antibody, which method is:
A step of introducing a DNA fragment containing a gene encoding a secretory signal, a gene encoding a nanobody and a gene encoding a peptide bar code, or a vector containing the DNA fragment into a yeast cell, and expressing in the cell and the cell. Including the step of recovering a polypeptide containing the nanobody and the peptide bar code secreted outside the cell.

In one embodiment, the yeast is a yeast belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces, Zygosaccharomyces, Candida, Trulopsis, Yarrowia or Hansenula.

In one embodiment, the secretory signal is an α-factor secretory signal, a glucoamylase secretory signal or a PHO1 secretory signal.

In one embodiment, the DNA fragment further comprises a promoter that is the AOX1 promoter, GAP promoter, FLD1 promoter, PEX8 promoter or YPT1 promoter.

In one embodiment, the DNA fragment is selected from the group consisting of FLAG tag, His tag, calmodulin protein (CBP) tag, Strep tag, StrepII tag, GST tag, Myc tag, Maltose binding protein (MBP) tag. It further comprises a gene encoding at least one tag.

In one embodiment, the peptide barcode is represented by an amino acid sequence consisting of 6 to 16 amino acids, the amino acids independently A, F, G, K, L, P, R, V and W. Selected from the group consisting of.

In one embodiment, the production method comprises combining the recovered polypeptide with an antigen to obtain a polypeptide containing Nanobodies that specifically bind to the antigen.
The peptide barcode is cut out from the obtained polypeptide, and the cut out peptide barcode is identified by mass spectrometry, and the peptide barcode is identified based on the base sequence of the nucleic acid encoding the identified peptide barcode. The identified peptide barcode further comprises the step of identifying the nanobody contained in the excised polypeptide.

In one embodiment, the DNA fragment further comprises a site that is cleaved by a specific protease.

In one embodiment, the step of identifying the peptide barcode is performed by detecting peaks using a tandem mass spectrometer (MS / MS) connected to a high performance liquid chromatograph (LC).

In one embodiment, the high performance liquid chromatograph comprises a monolith long column.

In one embodiment, the production method further comprises the step of determining the base sequence of the DNA fragment.

In one embodiment, the step of introducing into the yeast cell is the step of introducing at least two of the DNA fragments or vectors into the yeast cell, and the gene encoding the peptide bar code in each DNA fragment is each. It encodes a peptide bar code represented by a different amino acid sequence.

In one embodiment, the DNA fragment comprises a gene encoding two or more peptide barcodes, with cleavage sites located between the two or more peptide barcodes.

The present invention further provides a vector for producing a monoclonal antibody in yeast, which contains a DNA fragment containing a gene encoding a secretory signal, a gene encoding a nanobody and a gene encoding a peptide bar code. And, it is introduced into the cells of the yeast and expressed so as to secrete the polypeptide containing the nanobody and the peptide bar code to the outside of the cells of the yeast.

In one embodiment, the secretory signal is an α-factor secretory signal, a glucoamylase secretory signal or a PHO1 secretory signal.

In one embodiment, the DNA fragment further comprises a promoter that is the AOX1 promoter, GAP promoter, FLD1 promoter, PEX8 promoter or YPT1 promoter.

In one embodiment, the DNA fragment is selected from the group consisting of FLAG tag, His tag, calmodulin protein (CBP) tag, Strep tag, StrepII tag, GST tag, Myc tag, Maltose binding protein (MBP) tag. It further comprises a gene encoding at least one tag.

In one embodiment, the peptide barcode is represented by an amino acid sequence consisting of 6 to 16 amino acids, the amino acids independently A, F, G, K, L, P, R, V and W. Selected from the group consisting of.

In one embodiment, the DNA fragment further comprises a site that is cleaved by a specific protease.

The present invention further provides a method of screening for monoclonal antibodies, which methods are:
(I) A step of expressing an antibody library from a gene library.
The gene library contains at least two gene members, each gene member containing a DNA fragment containing a gene encoding a Nanobody and a gene encoding at least one peptide barcode.
The DNA fragment of each gene member of the gene library encodes the polypeptide of each antibody member of the antibody library.
A DNA fragment in which the polypeptide of each antibody member of the antibody library comprises a Nanobody and at least one peptide barcode, and the Nanobody and the at least one peptide barcode are contained in each gene member of the gene library. Coded by
Each peptide barcode of the antibody member is represented by a different amino acid sequence.
Process;
(Ii) A step of combining the antibody library with an antigen and selecting an antibody member of the antibody library containing a Nanobody bound to the antigen from the antibody library;
(Iii) A step of cutting out a peptide bar code contained in an antibody member of the selected antibody library and identifying the cut out peptide bar code by mass analysis; and (iv) encoding the identified peptide bar code. A step of determining the base sequence of a gene based on the base sequence of the gene library and identifying the nanobody of the antibody member from which the identified peptide barcode was excised is included.
The expression step is carried out by introducing the above vector into yeast cells.

In one embodiment, the screening method further comprises the step of determining the base sequence of the DNA fragment.

According to the present invention, a monoclonal antibody that specifically binds to an arbitrary antigen can be rapidly obtained. Further, in the present invention, since the binding ability is measured using a free antibody, a monoclonal antibody that specifically binds to an antigen can be obtained based on the binding ability of the antibody according to the actual usage situation.

It is a schematic diagram which shows an example of the analysis of the binding based on the antigen-antibody interaction of the peptide barcode-added Nanobody produced by the secretion expression by yeast in this invention. It is a schematic diagram which shows 4 kinds of Nanobodies designed in Example 1. FIG. 3 is an electrophoretic photograph showing the results of SDS-PAGE of anti-CD4-FLAG, anti-CD4-FLAG-barcode 1, anti-GFP-FLAG and anti-GFP-FLAG-barcode 2 produced from Pichia pastris transformants. .. It is a graph which shows the production amount of anti-CD4-FLAG, anti-CD4-FLAG-barcode 1, anti-GFP-FLAG and anti-GFP-FLAG-barcode 2 produced from the Pichia-pastris transformant. 3 is a fluorescence micrograph showing the results of immunofluorescence staining of CD4 using Nanobodies to which peptide barcodes have been added. It is a schematic diagram which shows the scheme for quantifying the binding ability of a Nanobody using mass spectrometry of a peptide barcode. 6 is a graph showing analysis results of various amounts of LC-MS / MS for barcode 1 (A) and barcode 2 (B). It is a graph which shows the result of having quantified the peptide bar code cut out from 250 fmol anti-CD4-FLAG-bar code 1 and 250 fmol anti-GFP-FLAG-bar code 2 by LC-MS / MS, respectively. 500 μL of Nanobody mixture (including 0.1 μM anti-CD4-FLAG-barcode 1 and 0.1 μM anti-GFP-FLAG-barcode 2) was subjected to a simultaneous binding assay using CD4 fixed magnetic beads and then excised from the beads. It is a graph which shows the result of quantifying the obtained peptide barcode by LC-MS / MS. It is a graph which shows the peak capacity when each monolith column of 100 μm inner diameter and 75 μm inner diameter is used in LC-MS / MS. It is a graph which shows the peak capacity when each monolith column of 500mm length and 1000mm length is used in LC-MS / MS.

(Definition)
The terms herein are used essentially in terms commonly used in biology and immunology, but the following terms will be described.

The term "nucleic acid" refers to a polymeric form of any length of nucleotide, deoxyribonucleotide or ribonucleotide or an analog thereof. The term includes, for example, DNA, RNA and their modified forms. Nucleic acids may be linear or cyclic. The term "gene" refers to a region of nucleic acid that contains the genetic information encoded by its base sequence. A gene encodes a "peptide," "polypeptide," or "protein," and can express a "peptide," "polypeptide," or "protein" based on its sequence information.

The term "peptide", "polypeptide" or "protein" refers to a polymeric form of an amino acid. A "peptide", "polypeptide" or "protein" may include, for example, an amino acid encoded by a nucleic acid as a constituent molecule, and a chemically or biochemically modified or derivatized amino acid may also be included as a constituent molecule. .. As used herein, "polypeptide" or "protein" is used interchangeably.

As used herein, the term "antibody" is a polypeptide or protein commonly used in biology and immunology and is also referred to as "immunoglobulin". In full body, the antibody consists of two heavy chains (H chain) and two light chains (L chain), and the chains are linked by a disulfide bond. The term "antibody" herein includes an antibody or fragment of an antibody of any isotype that retain specific binding to an antigen, for example, Fab, Fv, scFv, Fd , V H H ( "nanobodies"), Also included are chimeric antibodies, humanized antibodies, single chain antibodies and fusion proteins containing the antigen binding portion of the antibody and non-antibody proteins. Antibodies can be detectably labeled using, for example, radioactive isotopes, enzymes that produce detectable products, fluorescent proteins, peptide barcodes, and the like.

A "Nanobody", also referred to as a single domain antibody, is a type of antibody fragment that consists of a single monomeric variable antibody domain and lacks the CH domain of the light chain and the heavy chain of the conventional Fab region. Nanobodies have a molecular weight of 12 to 15 kDa, which is about one-tenth that of a normal fullbody antibody with a molecular weight of 150 kDa. Despite these small molecules, Nanobodies have properties similar to fullbody antibodies. For example, an antibody consisting of V _{H H} domain is the variable region of a single domain antibody with the camelid. Nanobodies are usually polypeptides with approximately 120 amino acid residues. Nanobodies are basically similar in composition to the sequence of the variable regions of a typical immunoglobulin and are hypervariable regions of complementarity with four framework regions of FR1, FR2, FR3 and FR4 in between. Three CDR1, CDR2 and CDR3 are seen, called determining regions (CDRs).

A "gene library" is a group containing at least two genes, and each gene constituting the group is also referred to as a "gene member". In the present invention, in a population of gene libraries, each gene of a gene member can be integrated into a separate vector (eg, a plasmid) and each can exist independently. Furthermore, each of these genes or vectors may be introduced into an individual host cell (eg, yeast) and exist separately. An "antibody library" is a group containing at least two antibodies, and each antibody constituting the group is also referred to as an "antibody member". In the present invention, each antibody member of the antibody library is obtained by expression of each gene member of the gene library in a host cell. In a population of antibody libraries, each antibody member may be present as a free antibody, for example, in the supernatant of the host cell culture used for expression from a gene member of the gene library.

The term "base sequence" is the sequence of consecutive nucleotide bases in a gene and is responsible for genetic information. The term "amino acid sequence" is the sequence of consecutive amino acids in a peptide or protein.

A gene that "encodes" a peptide or protein is, for example, transcribed (in the case of DNA) and translated into a peptide or protein (mRNA) when the gene is placed, for example, under the control of an appropriate regulatory or regulatory element. in the case of).

"Peptide barcode" refers to a peptide having a sequence that can identify the molecule (eg, antibody) to which the peptide barcode is fused and / or distinguish it from one or more different molecules. The peptide barcode is fused to the antibody so as not to affect the binding of the antibody to which the peptide barcode is fused to the antigen, and has a size and an amino acid sequence that does not affect such influence. Is preferable. As used herein, when a peptide (eg, peptide bar code) "fuse" with a molecule (eg, antibody or tag), the molecule is linked to the C-terminus or N-terminus of the peptide to form a larger molecule. However, another molecule or region (for example, a cleavage site) may be arranged between the peptide and the molecule, and / or one or more (for example, 2 to 10 or more). Amino acids may be present. The "peptide barcode" is encoded by a gene, and the base sequence of the encoding gene can function as a gene barcode.

As used herein, "specific binding" means that binding partners (eg, antigens and antibodies) bind to each other, but under given conditions (eg, physiological conditions), the environment (eg, biological sample, eg). A bond based on an interaction between binding partners that does not adequately or substantially bind to other molecules that may be present in the tissue.

(1. Manufacturing method of monoclonal antibody)
The present invention provides a method for producing a monoclonal antibody. This production method is a step of introducing a DNA fragment containing a gene encoding a secretory signal, a gene encoding a nanobody and a gene encoding a peptide bar code, or a vector containing the DNA fragment into yeast cells (step (A)). ); And a step (step (B)) of recovering a polypeptide containing the nanobody and the peptide bar code expressed in the cell and secreted outside the cell.

(1-1. Step (A): Expression of secretion by yeast)
In the present invention, a monoclonal antibody is produced by secretory expression by yeast. Yeast is endotoxin-free and can simplify the process of purifying the antibody obtained by expression. Examples of yeast include Saccharomyces, Pichia, Schizosaccharomyces, Zygosaccharomyces, Candida, Torulopsis, and Yarrowia. Alternatively, yeast belonging to the genus Hansenula can be mentioned. For example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like. Pichia pastoris is preferred.

As the secretory signal, any secretory signal can be used as long as the host yeast expressing the monoclonal antibody can secrete the monoclonal yeast extracellularly. In terms of efficient yeast secretion expression, for example, α-factor secretory signal (eg, α-factor prepro sequence derived from Saccharomyces cerevisiae), glucoamylase secretory signal (eg, Rhizopus oryzae-derived glucoamylase) Secretory signal) or PHO1 secretory signal (eg, secretory signal of phosphatase (PHO1) derived from Pitia pastris).

In the present invention, monoclonal antibodies can be produced by expressing genes encoding Nanobodies. Nanobodies consist of a single variable domain (VHH) of the antibody heavy chain, which can bind to a single antigen. The gene encoding the Nanobody may be either a known base sequence or an unknown base sequence. As the Nanobody, a chimeric one may be used.

When the base sequence information is known, a gene encoding a Nanobody can be obtained by PCR using available genomic DNA, cDNA, etc. as a template using a primer pair designed based on the sequence information. An artificial gene may be produced by chemically synthesizing a DNA having the base sequence. Genes encoding Nanobodies can also be obtained, for example, by amplifying variable regions from DNA extracted from leukocytes such as thymus, bone marrow, and peripheral blood of vertebrates including camelids by PCR or the like.

For the gene encoding Nanobodies, for example, various mutations may be introduced by a comprehensive mutation method using NNK codons, or error prone PCR may be used in combination. According to these methods, genes encoding various antibodies can be designed without using individual animals.

The DNA fragment can further include a promoter. A promoter can be placed upstream of the secretory signal. Any promoter capable of inducing gene expression in yeast can be used. The DNA fragment can further include a promoter. The promoter may be either an inducible promoter or a constitutive expression promoter. One of the inducible promoters is the AOX1 promoter (promoter of the gene encoding alcohol oxidase (AOX)), the FLD1 promoter (promoter of the gene encoding formaldehyde dehydrogenase (FLD)), and the PEX8 promoter (peroxin (PEX)). (Promoters of genes encoding), and constitutive expression promoters include, for example, the GAP promoter (promoter of the gene encoding glyceraldehyde-3-phosphate dehydrogenase) and the YPT1 promoter (these promoters). Can all be derived from Pichia pastris). When the yeast is Pichia pastris, methanol-induced promoters (eg, AOX1 promoter, FLD1 promoter and PEX8 promoter (above)) can be used and may be constitutive expression promoters. The methanol-inducible promoter is preferably used in methanol-utilizing yeast.

The DNA fragment may further contain a gene encoding a protein tag. Examples of protein tags include FLAG tags, His tags, CBP (calmodulin protein) tags, Strep tags, StrepII tags, GST tags, Myc tags and MBP (maltose-binding protein) tags, and combinations thereof. The addition of such a tag makes it easier to recover and purify the extracellularly secreted monoclonal antibody of yeast, and further enables more accurate mass spectrometry (MS) of peptide barcodes. ..

The base sequence of the gene encoding the peptide barcode can be prepared based on the amino acid sequence of the peptide barcode described below. The amino acid sequence of the peptide barcode is, for example, 5 to 30, preferably 6 to 20, more preferably 6 to 16, still more preferably 6, 7, 8, 9 or 10. Consists of amino acids. When the amino acid sequence of the peptide barcode is within such a length range, the ionization efficiency is high, the detection and quantification can be performed better by mass spectrometry, and the peptide barcode can be easily identified. .. Further, if the length is within the above range, it can be verified in advance that the ionization efficiency of the designed peptide barcode is high, and the identification by mass spectrometry (MS) is unlikely to cause a bias. Therefore, the binding ability of all Candidate Nanobodies can be measured without omission. For example, a peptide barcode consisting of 6, 7, 8, 9 or 10 amino acids provides high detection sensitivity by mass spectrometry and the ability of the barcode-added nanobody to bind to an antigen. A wide variety of barcodes can be produced without substantial degradation, or such peptide barcodes do not substantially affect the binding capacity of the nanobody and the detection sensitivity of mass spectrometry. Further protein tags can be added to the peptide barcode.

In one embodiment, the amino acids that make up the peptide barcode are independently alanine (A), phenylalanine (F), glycine (G), lysine (K), leucine (L), proline (P), arginine. It is selected from the group consisting of (R), valine (V) and tryptophan (W). These amino acids are amino acids without ion suppression in terms of ionization efficiency. For example, residues having an amino group such as lysine (K) and arginine (R) are likely to be positively charged, which enhances ionization efficiency. Glycine (G) and proline (P) residues also enhance ionization efficiency. On the other hand, histidine (H) reduces ionization efficiency, cysteine (C) and methionine (M) are easily oxidized, and asparagine (N), serine (S), and threonine (T) can be glycosylated. , These amino acids can be excluded from the amino acids that make up the peptide bar code. Further, hydrophobic amino acids are also included in order to appropriately disperse the elution time (retention time) by the liquid chromatograph (LC). In one embodiment, the peptide barcodes used are those in which A, F, G, K, L, P, R, V and W are randomly arranged. By constructing a peptide barcode with these types of amino acids, a peptide barcode having high ionization efficiency can be obtained with the above number of amino acids (particularly 6, 7, 8, 9 or 10 amino acids). Can be made. Therefore, the identification by mass spectrometry (MS) is less likely to be biased, and the binding ability of all candidate Nanobodies can be measured without omission. In addition, the retention time of separation by mass spectrometry (MS) prior liquid chromatography (LC) used for identification of peptide barcodes can be appropriately dispersed to improve the specificity of detection by mass spectrometry. Bar code can be created.

For example, the case of producing a peptide bar code consisting of six amino acids by the above types of amino acids can be designed 9 6 ⁼ 531,441 different unique bar code.

The DNA fragment can contain a gene encoding a cleavage site, which is a site that can be degraded by enzymatic or chemical means. In order to facilitate excision of the peptide barcode as described below, the cleavage site is preferably a site that can be cleaved by a protease having cleavage specificity. It is preferable to have an amino acid sequence recognized by a protease having cleavage specificity (also referred to as "specific protease" in the present specification). Specific proteases include, but are not limited to, enterokinase, for example. For example, if the specific protease is enterokinase, it cleaves behind the lysine of the amino acid sequence DDDDK (4 aspartic acid-lysines) (ie, the C-terminal side of the 5 amino acid peptide above).

The base sequence of the gene may adopt codons optimized according to the host.

The DNA fragment may further contain a terminator. As the terminator, any terminator can be used as long as it functions in yeast. One example is the AOX1 terminator.

The synthesis and binding of DNA containing various components (ie, various genes, promoters and terminators) can be performed by techniques commonly used by those skilled in the art. For example, binding of a gene encoding a secretory signal peptide to a gene encoding a Nanobody can be performed using a site-specific mutation method. By using this method, more accurate cleavage of the secretory signal peptide and expression of Nanobodies are possible.

The DNA fragment constructed as described above is introduced into yeast cells. The DNA fragment may be incorporated into the vector and introduced. As the above-mentioned components of the DNA fragment, for example, promoters and terminators, and / or genes encoding secretory signals, those previously provided in a commercially available yeast expression vector may be used. "Introduction" means not only introducing a gene in a DNA fragment into a cell, but also expressing it. Examples of such introduction methods include transformation, transduction, transfection, cotransfection, electroporation and the like. In the case of introduction into yeast cells, specific examples include a method using lithium acetate and a protoplast method. The introduction of the DNA fragment may be inserted into the host gene or homologous recombination with the host gene to be incorporated into the chromosome. Commercially available kits can also be used for introduction, for example Frozen EZ Yeast Transformation II Kit (manufactured by Zymo Research) when the host cell is yeast.

(1-2. Step (B): Recovery of secreted and expressed polypeptide)
With the above introduction, the polypeptide containing the Nanobody and the peptide barcode is expressed in yeast cells and secreted out of the cells. For example, when Pichia pastris, which is methanol-utilizing, is used as a host cell, for example, a secretory signal peptide (eg, α-factor secretory signal peptide) and a peptide bar code are placed downstream of a methanol-inducible promoter (eg, AOX1 promoter). Design a plasmid vector containing a DNA fragment containing a gene encoding a fusion protein with the added nanobody. When such a plasmid vector is introduced into a host cell to express the fusion protein, the α factor secretory signal is cleaved during the secretion process, and finally, the nanobody to which the peptide bar code is added is secreted into the medium supernatant. .. By recovering the culture supernatant, the polypeptide containing the Nanobody and the peptide barcode can be recovered. Polypeptides containing Nanobodies and the peptide barcodes can be recovered without the need to disrupt cells to obtain the expressed antibody. The polypeptide (monoclonal antibody to which a peptide barcode is added) may be purified from the culture supernatant, if necessary.

The production method of the present invention can further include the following steps: combining the recovered polypeptide with an antigen to obtain a polypeptide containing a nanobody that specifically binds to the antigen (step (C)). The step (step (D)) of cutting out the peptide bar code from the obtained polypeptide and identifying the cut out peptide bar code by mass analysis; and the nucleic acid encoding the identified peptide bar code. A step of identifying the nanobody contained in the polypeptide from which the identified peptide bar code was cut out based on the base sequence (step (E)).

(1-3. Step (C): Binding based on antibody-antigen interaction)
In order to more reliably obtain a monoclonal antibody that specifically binds to the target antigen, the polypeptide recovered in step (B) is combined with the antigen to obtain a polypeptide containing a nanobody that specifically binds to the antigen. May be done (step (C)). For example, when the culture medium supernatant is collected by the above step (B), Nanobodies to which the peptide barcode is added are contained in the culture medium supernatant. be able to. The medium supernatant can be used as it is for measuring the antibody binding ability without the need to disrupt cells to obtain the expressed antibody, and the antigen binding ability can be measured quickly and at low cost without the need for pre-purification. It is possible.

The antigen is not particularly limited as long as it can produce an antibody by an immune reaction, and examples thereof include cells collected from a living body such as proteins, peptides, polysaccharides, lipids, and low molecular weight compounds, and preferably. It is a protein.

The antigen can be immobilized on a carrier. As such a carrier, magnetic beads (for example, NHS activated magnetic beads (manufactured by Thermo Scientific)) can be used). For example, cells that present the antigen on the surface (for example, yeast) can also be used. It is also possible to use a cell sorter (FCS). The methods and conditions of the binding assay for such antigen-antibody binding can be performed by methods commonly used by those skilled in the art. For example, when magnetic beads on which an antigen is immobilized are used, the polypeptide containing the antibody that is not bound to the antigen is washed away by washing the beads with an appropriate solvent after the binding reaction between the antigen and the antibody. Polypeptides containing antibodies bound to the antigen above can be left behind.

Assays based on surface plasmon resonance can also be used, for example, to determine the ability to bind antigen. Such an assay can be performed, for example, by measuring kinetic parameters using a Biacore T-200 (manufactured by GE Healthcare).

(1-4. Step (D): Identification of peptide barcode)
In order to identify and / or obtain an antibody whose binding to an antigen has been confirmed, a peptide barcode is excised from a polypeptide in which Nanobodies are bound to the antigen in step (C), and the excised peptide barcode is subjected to mass spectrometry. Can be identified (step (D)).

From a polypeptide in which Nanobodies are bound to an antigen, a peptide barcode contained in the polypeptide together with Nanobodies is cut out. Cutting out such a peptide barcode is performed, for example, by adding an enzyme that recognizes the cleavage site and cleaving at the cleavage site of the polypeptide. Preferably, a specific protease (eg, enterokinase) is used. This leaves the antibody bound to the antigen and cleaves the peptide barcode. This detached peptide barcode can be recovered.

The excised peptide barcode is then identified by mass spectrometry. For example, a peptide barcode can be identified by comparing the molecular weight determined based on the peak detected by mass spectrometry with the molecular weight estimated by the composition of the peptide barcode (amino acid sequence).

For such mass spectrometry, for example, a mass spectrometer (MS) or tandem mass spectrometer (MS / MS) connected to a high speed liquid chromatograph (LC) can be used (ie, LC-MS method or LC-MS / MS method). The LC-MS / MS method is preferable in that the specificity of detection is increased. As the tandem mass spectrometer, a triple quadrupole mass spectrometer (for example, LCMS-8060: manufactured by Shimadzu Corporation) is preferable in that it has high sensitivity, is specific, and is quantitative. As the method for measuring MS / MS, selective reaction monitoring (SRM: a method for detecting a specific m / z product ion generated by dissociating a specific m / z precursor ion) is preferably used.

As the column of the liquid chromatograph (LC), for example, a monolith column, preferably a long type monolith column is used. The monolith column is a rod-shaped integrated column for liquid chromatography having pores (mesopores and macropores) of two sizes, and monolith silica gel is used as a separating agent. The long type refers to a monolith column having a column length of, for example, 100 mm to 10000 mm. The inner diameter of the monolith column is, for example, 1 μm to 200 μm, preferably 30 μm to 75 μm. The long type, more preferably the inner diameter within the above range, enhances the column separation ability, enables more efficient detection of peptide barcodes, and facilitates identification. Further, by using a long monolith column, absolute quantification of peptides can be performed by SRM.

(1-5. Step (E): Identification of monoclonal antibody)
Based on the base sequence of the gene encoding the peptide barcode identified as described above, the antibody contained in the polypeptide containing the identified peptide barcode can be identified (step (E)).

When the peptide barcode is identified by mass spectrometry, the nanobody to which the peptide barcode is added can be estimated from the base sequence of the gene encoding the peptide barcode. The base sequence information of the Nanobody estimated in this way can be acquired, and the Nanobody can be identified. For example, when an unknown base sequence is used as a gene encoding a Nanobody, the production method of the present invention further includes a step of determining the base sequence of the DNA fragment in order to obtain the base sequence of the gene encoding the Nanobody. You may. A method usually used by those skilled in the art can be used for determining such a base sequence.

In this way, a monoclonal antibody that specifically binds to the antigen of interest can be identified.

Furthermore, the antibody can also be obtained by expressing a gene encoding the identified monoclonal antibody. For example, a primer pair is designed from the base sequence information of the nucleic acid encoding the identified antibody, and the nucleic acid encoding the antibody is obtained by PCR using the primer pair. An expression vector can be constructed using such a nucleic acid as described above, and the expression vector can be introduced into a host cell to obtain an expressed antibody. For the expression of such a monoclonal antibody, preferably secretory expression by yeast as described above is used.

In the production method of the present invention, two or more DNA fragments can also be used. In this case, the step of introducing into the yeast cell (step (A)) is the step of introducing at least two of the DNA fragments or vectors into yeast cells, and the gene encoding the peptide barcode in each DNA fragment is , Each encode a peptide bar code represented by a different amino acid sequence. When two or more DNA fragments are used, the peptide barcode detected by mass spectrometry in step (D) can be identified by differentiating the amino acid sequences of the peptide barcodes in each DNA fragment. ..

Also, one DNA fragment may contain genes encoding two or more peptide barcodes. In this case, the two or more peptide barcodes can be arranged in tandem for Nanobodies encoded by one DNA fragment. A cleavage site can be placed between the two or more peptide barcodes. For example, the diversity of peptide barcodes can be increased by arranging two or more peptide barcodes in tandem and placing a specific protease cleavage sequence between them. For example, when a library is constructed with a polypeptide sequence of NH _2- [nanobody] -cleaved site- [peptide bar code A] -cleaved site- [peptide bar code B] -COOH, the peptide near the COOH terminal is used. When naming a peptide bar code A that is close to the bar code B or NH ₂ terminal, the end of the peptide bar code (which may be either the C-terminal or the N-terminal) is used so that the position of the peptide bar code can be identified. One amino acid as an identifier is added so as to be different for each peptide bar code. Any of A, F, G, K, L, P, R, V and W can be used as the amino acid of the identifier. For example, if K (lysine) is added to the C-terminal of peptide barcode A and R (arginine) is added to the C-terminal of peptide barcode B, each peptide barcode is derived from peptide barcode A or peptide barcode. It becomes possible to distinguish whether it is derived from B by mass analysis. In this case, when arranging two peptides bar code in ^tandem, it becomes possible to evaluate ⁽⁹ 6) 2 = 531 441 ² types of diversity. If we lined three peptides bar code in ^tandem, it is possible to assess ⁽⁹ 6) 3 = 531441 ^three diversity. Since it is possible to tandemize four or more types of peptide barcodes, the size of the quantifiable nanobody library is increased.

(2. Vector for producing monoclonal antibody in yeast)
The present invention further provides a vector for producing a monoclonal antibody in yeast. The vector of the present invention contains the above DNA fragment. Components of the DNA fragment (as described above, genes encoding secretory signals, genes encoding nanobodies and peptides barcodes, and promoters, genes encoding protein tags, genes encoding cleavage sites, and genes. The terminator) is as described above.

The vector may optionally include factors such as selectable markers, origins of replication, enhancers and the like. The vector is, for example, in the form of a plasmid. For example, a plasmid having an origin of replication of yeast ColE1 is preferably used. The plasmid preferably has a selectable marker in that it facilitates plasmid preparation and detection of transformants. Selectable markers include, for example, drug resistance genes and auxotrophic genes. Examples of the drug resistance gene include, but are not limited to, an ampicillin resistance gene (Ampr) and a kanamycin resistance gene (Kanr). Examples of the nutritionally demanding gene include N- (5'-phosphoribosyl) anthranilate isomerase (TRP1) gene, tryptophan synthase (TRP5) gene, malate β-isopropyl dehydrogenase (LEU2) gene, and imidazole glycerol phosphate dehydration. Examples thereof include, but are not limited to, the elementary enzyme (HIS3) gene, histidineol dehydrogenase (HIS4) gene, dihydroorotoic acid dehydrogenase (URA1) gene, and orotidine-5-phosphate decarboxylase (URA3) gene. The replication gene for yeast can be selected as needed.

(3. Method for screening monoclonal antibodies)
The present invention also provides a method of screening for monoclonal antibodies. This method is (i) a step of expressing an antibody library from a gene library.
The gene library contains at least two gene members, each gene member containing a DNA fragment containing a gene encoding a Nanobody and a gene encoding at least one peptide barcode.
The DNA fragment of each gene member of the gene library encodes the polypeptide of each antibody member of the antibody library.
The polypeptide of each antibody member of the antibody library comprises a Nanobody and at least one peptide barcode, and the Nanobody and the at least one peptide barcode are encoded by a DNA fragment contained in each gene member of the gene library. Being done
Each peptide barcode of the antibody member is represented by a different amino acid sequence.
Process;
(Ii) A step of combining the antibody library with an antigen and selecting an antibody member of the antibody library containing a Nanobody bound to the antigen from the antibody library;
(Iii) A step of cutting out a peptide bar code contained in an antibody member of the selected antibody library and identifying the cut out peptide bar code by mass analysis; and (iv) encoding the identified peptide bar code. The base sequence of the gene is determined based on the base sequence of the gene library, and the step of identifying the nanobody of the antibody member from which the identified peptide barcode has been cut out is included. The expression step (step (i)) is carried out by introducing a vector as described in "2. Vector for producing a monoclonal antibody in yeast" into yeast cells.

(3-1. Step (i): Expression of gene library)
According to the present invention, a monoclonal antibody that specifically binds to a target antigen can be selected and identified by expressing a plurality of types of Nanobodies at one time. For this, a gene library can be created. The gene library contains at least two gene members, each gene member containing a DNA fragment containing a gene encoding a Nanobody and a gene encoding at least one peptide barcode. This DNA fragment can be constructed as described above and incorporated into the vector if desired.

The DNA fragment of each gene member of the above gene library encodes the polypeptide of each antibody member of the antibody library. Thus, the polypeptide of each antibody member of the antibody library contains Nanobodies and at least one peptide barcode. The Nanobodies of each antibody member and at least one peptide barcode are encoded by the DNA fragments contained in each gene member of the gene library.

The peptide barcode of each antibody member of the antibody library is represented by a different amino acid sequence. The antibody members of the antibody library have a unique (unique) peptide bar code added to the randomized antibody, and the gene members of the gene library are the genes of the randomized antibody (nucleic acid encoding the antibody). On the other hand, a gene encoding a unique peptide bar code (nucleic acid (for example, DNA)) (hence, a unique DNA bar code) is added. As a result, there is a 1: 1 correspondence between the randomized antibody gene and the DNA barcode in the gene library, and the randomized antibody and the peptide barcode. The peptide barcodes of each antibody member of the antibody library can be efficiently detected by mass spectrometry. Since the amino acid sequence of the peptide barcode is different for each antibody member, the base sequence of the nucleic acid (DNA barcode) encoding the peptide barcode is different. The amino acid sequence of the peptide barcode of each antibody member can be used for identification by mass spectrometry so that different retention times can be obtained by separation by LC in liquid chromatography (LC) -tandem mass spectrometry (MS / MS). The base sequence of the gene can be designed to encode the designed amino acid sequence.

Therefore, if the base sequence information of the DNA barcode can be obtained, the base sequence of the antibody gene (nucleic acid encoding the antibody) to which the DNA barcode is added can be determined. In the nucleic acid library, the nucleic acids encoding the peptide barcodes of each nucleic acid member are all different, but the antibodies may be duplicated (in other words, one or more kinds (for example, one kind) for one kind of antibody. 2 to 10 types of peptide barcodes) may be added). This makes it possible to prepare for cases where the expression of a certain nucleic acid member is not successful, or where the antibody to which the peptide barcode is added is lost before mass spectrometry for some reason.

In one embodiment, the antibody member of the antibody library comprises two or more peptide barcodes and the cleavage site is located between the two or more peptide barcodes. The peptide barcode and cleavage site are as described above. By arranging two or more peptide barcodes in tandem, the combination of barcodes can be increased, for example, the degree of diversity found in mammals or more diversity can be manifested. ..

The number of gene members contained in the gene library may be at least 2 or more, and is, for example, 2 to ⁸ pieces, preferably 10 ² to 10 ⁷ pieces, or 10 ³ to ⁶ pieces. With the number of gene members within such a range, acquisition of the base sequence of the gene library and / or simultaneous binding assay based on antigen-specific binding can be performed more efficiently.

As the gene library, a new sequence may be designed and constructed when screening a monoclonal antibody that specifically binds to an antigen, or a gene library prepared in advance using existing sequence information may be used.

The expression of the antibody library from the gene library is as described above for the method for producing the monoclonal antibody and the vector. That is, DNA fragments of each gene member of the gene library are introduced into yeast cells, and nanobody and peptide barcodes are secreted and expressed from yeast. The above-mentioned vector is used for the expression of secretion by this yeast.

(3-2. Step (ii): Selection of antigen-binding antibody members)
In the screening method of the present invention, an antibody library is combined with an antigen, and an antibody member of the antibody library containing an antibody bound to the antigen is selected from the antibody library (step (ii)). In this step, the antibody library is screened for binding to the antigen of interest. After introduction into yeast cells in step (i) above, each antibody member of the antibody library (Nanobody to which a peptide barcode is added) is contained in the culture medium supernatant for the expression of secretion by yeast. Therefore, in order to combine the antibody library with the antigen, the culture medium supernatant may be added to the antigen as it is. If necessary, the polypeptide may be purified from the culture medium supernatant and then added to the antigen. This step (ii) can be basically performed in the same manner as the above-mentioned "step (C): binding based on antibody-antigen interaction" (step (C)). Preferably, a simultaneous binding assay is used in which antibody members of the antibody library are added all at once to the antigen.

(3-3. Step (iii): Identification of peptide barcode)
In the screening method of the present invention, a peptide barcode contained in an antibody member of the selected antibody library is cut out, and the cut out peptide barcode is identified by mass spectrometry (step (iii)). This step (iii) can be basically performed in the same manner as the above-mentioned "Step (D): Identification of peptide barcode".

(3-4. Step (iv): Identification of monoclonal antibody)
In the screening method of the present invention, the nucleotide sequence of the gene encoding the identified peptide barcode is determined based on the nucleotide sequence of the gene library, and the identified peptide barcode is excised from the antibody member Nanobody. (Step (iv)). This step (iv) can be basically performed in the same manner as the above-mentioned "Step (E): Identification of monoclonal antibody".

When the gene library contains a nucleic acid whose base sequence is unknown or the combination of the peptide barcode and the antibody is unknown, the step of obtaining the base sequence of the gene library may be further included.

The base sequence of the gene library can be obtained by large-scale sequence analysis of the gene library. Large-scale sequence analysis can be performed according to a method usually used by those skilled in the art, for example, using a DNA sequencer capable of processing a large amount of base sequence information. For large-scale sequence analysis, for example, a next-generation sequencer (NGS) such as MiSeq (manufactured by Illumina) can be used.

A database may be created using the information of the base sequence. After identification of the peptide bar code by mass analysis, such a database contains the nucleotide sequence information of the nucleic acid encoding the member of the antibody library containing the identified peptide bar code (gene encoding the peptide bar code and the gene encoding the antibody). Can be used to identify the antibody from the sequence information of the amino acid sequence (including the peptide barcode and the antibody) deduced from the base sequence information, if necessary.

Further, the present invention will be described with reference to FIG.

FIG. 1 is a schematic diagram showing an example of analysis of binding based on an antigen-antibody interaction of a peptide barcode-added Nanobody produced by secretory expression by yeast in the present invention. With respect to FIG. 1, (a) at least two gene members to which a unique DNA bar code (gene encoding peptide bar code) is added to each of the randomized nanobody genes (genes encoding nanobodies). A gene library containing the above is provided, and each of these gene members is introduced into yeast cells; (b) Expression in yeast cells produces antibody members containing antibodies (nanobodies) to which the respective peptide barcodes have been added, and antibodies are produced. Constituting the library, each antibody member of this antibody library is secreted from yeast cells into the supernatant of the medium; (c) each antibody member of this antibody library is combined with the antigen and bound to the antigen; ( d) Wash, remove antibody members containing unbound antibody, leave and select antibody members containing antibody bound to the antigen; (e) Cut out peptide barcodes from the selected antibody members by enterokinase; Then, (f) the excised peptide bar code is identified by mass analysis.

Based on the sequence information from the gene library of FIG. 1 (a), the 1: 1 correspondence between the base sequence of the randomized antibody gene and the base sequence of the DNA bar code (gene encoding the peptide bar code) is determined. The correspondence between the randomized amino acid sequence of the antibody (encoded by the base sequence of the antibody gene) and the amino acid sequence of the peptide bar code can be abbreviated. According to the above (b) to (f), an antibody member containing an antibody bound to an antigen is selected from the antibody library, a peptide bar code is cut out from the selected antibody member, and the cut out peptide bar code is identified by mass analysis. Then, together with the known sequence information of the nucleic acid library or the sequence information obtained by the sequence analysis, the base sequence of the gene encoding the antibody member containing the identified peptide bar code was obtained, and which antibody was specific to the antigen. It can be determined whether it binds to the antigen, and thus a nanobody that is monoclonal to the antigen can be obtained. Further, the antibody thus identified can be produced by obtaining a gene encoding the antibody based on the sequence information of the nucleic acid library and expressing the antibody from the gene.

As described above, according to the present invention, a monoclonal antibody can be produced in vitro without using an animal, and the free antibody can be obtained through mass spectrometry of a unique peptide barcode added to the free antibody. The binding ability can be identified. Further, according to the present invention, instead of costly and time-consuming antibody production using vertebrates and cultured cells, antibodies having various properties can be produced in vitro and in a laboratory. Furthermore, according to the present invention, when a large number of candidate Nanobodies for monoclonal antibodies are used, binding of these Nanobodies to the antigen of interest through mass spectrometric quantification of the unique peptide barcodes added to each candidate Nanobody. Capabilities can be comprehensively identified in a single experiment. Therefore, it is possible to produce a monoclonal Nanobody that specifically binds to the target antigen at a lower cost, faster, and more efficiently. INDUSTRIAL APPLICABILITY The present invention is useful for the production of experimental reagents for basic imaging research and sensing research, diagnostic agents using molecular target antibodies, and pharmaceuticals.

(Example 1: Preparation of Nanobody fused with peptide barcode)
Four Nanobodies were designed based on anti-CD4 Nanobodies (US Patent Application Publication 2011/0318347) and anti-GFP (Green Fluorescent Protein) Nanobodies (Mol. Cell. Proteomics. 2008: 7: 282-289). FIG. 2 is a schematic diagram of four types of Nanobodies designed in this embodiment. Two of the Nanobodies were added with a unique peptide barcode consisting of 6 amino acid residues, and the other two were without peptide barcodes. Barcode 1 was added as a peptide barcode to the anti-CD4 Nanobody, and barcode 2 was added as a peptide barcode to the anti-GFP Nanobody (Fig. 2).

The peptide barcode was designed as follows. The ionization efficiency of a peptide varies greatly depending on the length of the peptide and the constituent amino acid residues. Peptides with 6 to 16 residues show high ionization efficiency. Ionization efficiency also depends on the types of amino acid residues that make up the peptide. For example, residues having an amino group, such as lysine (K) and arginine (R), are positively charged and thus increase the ionization efficiency. Glycine (G) and proline (P) residues increase the ionization efficiency, and histidine (H) decreases the ionization efficiency. Since cysteine (C) and methionine (M) are easily oxidized and asparagine (N), serine (S), and threonine (T) can be glycosylated, these residues are SRM analyzes that analyze a given target. Not suitable for. In addition, hydrophobic amino acids were included in order to appropriately disperse the elution time (retention time) of the liquid chromatograph.

Considering these factors, nine amino acids (A, F, G, K, L, P, R, V, and W) were selected as constituent amino acid residues, and two peptide barcodes having different molecular weights were selected. Designed:
Barcode 1: WLFPVG (SEQ ID NO: 1)
Barcode 2: FVGARL (SEQ ID NO: 2)

For both barcodes 1 and 2, a FLAG tag peptide (DYKDDDDK: SEQ ID NO: 3) was fused to the N-terminal thereof. In addition, both barcodes 1 and 2 were fused with a FLAG tag peptide at the C-terminal thereof (Fig. 2).

"FLAG Tag Peptide-Barcode 1-FLAG Tag Peptide" (the amino acid sequence is shown in SEQ ID NO: 4) is fused to the C-terminus of the anti-CD4 nanobody, and "FLAG Tag Peptide-Barcode 2-FLAG Tag Peptide" (Amino Acid). The sequence shown in SEQ ID NO: 5) was fused to the C-terminus of the anti-GFP nanobody (these were referred to as "anti-CD4-FLAG-barcode 1" and "anti-GFP-FLAG-barcode 2": FIG. 2). ..

In the case of anti-CD4 Nanobodies and anti-GFP Nanobodies without barcodes, FLAG tag peptides (DYKDDDDK: SEQ ID NO: 3) were fused to the C-terminus of these Nanobodies (these were "anti-CD4-FLAG" and "anti-CD4-FLAG". It was called "GFP-FLAG": FIG. 2).

A plasmid encoding these Nanobodies was constructed. The plasmid was designed to place genes encoding α-factor secretory signals and Nanobodies downstream of the AOX1 promoter, respectively. A gene encoding the nanobody designed in this example (anti-CD4-FLAG, anti-CD4-FLAG-barcode 1, anti-GFP-FLAG or anti-GFP-FLAG-barcode 2) was synthesized to obtain a DNA fragment, which was obtained. The DNA fragment was pre-cut with EcoRI and NotI (both manufactured by Toyo Boseki Co., Ltd.) into a pPIC9K vector (manufactured by Invitrogrn: this vector contains the AOX1 promoter and α-factor secretion signal) and the In Fusion HD Cloning Kit (Takara Bio). Incorporated using (manufactured by Co., Ltd.).

The entire sequence of each plasmid is shown in SEQ ID NOs: 6-17 (anti-CD4-FLAG plasmid: total sequence (SEQ ID NO: 6) and expressed peptide sequence (SEQ ID NOs: 7-8); anti-CD4-FLAG-barcode 1 plasmid: total). Sequence (SEQ ID NO: 9) and expressed peptide sequence (SEQ ID NOs: 10-11); anti-GFP-FLAG plasmid: full sequence (SEQ ID NO: 12) and expressed peptide sequence (SEQ ID NOs: 13-14); and anti-GFP-FLAG-bar Code 2 plasmid: full sequence (SEQ ID NO: 15) and expressed peptide sequence (SEQ ID NOs: 16-17).

The constructed plasmid was digested with SacI (manufactured by Toyobo Co., Ltd.) and purified with the MinElute PCR purification kit (manufactured by QIAGEN). This plasmid was transformed into Pichia pastris GS115 strain using Frozen EZ Yeast Transformation II Kit (manufactured by Zymo Research). Transformed cells were seeded on MD solid medium (1.34% w / v yeast nitrogen base (without amino acids), 2% w / v D-glucose, and 2% w / v agar) to obtain colonies. .. The obtained colonies (transformants) were cultivated in 20 mL of BMGY medium (1% w / v yeast extract, 2% w / v peptone, 1% w / v glycerol, 0.1 M potassium phosphate buffer (pH 6.0)). 2.68% w / v yeast nitrogen base (without amino acids), 400 μg / mL biotin) was inoculated and cultured at 30 ° C. at 250 rpm for 48 hours. The grown bacteria are collected by centrifugation and collected in 10 mL of BMMY medium (1% w / v yeast extract, 2% w / v peptone, 0.1 M potassium phosphate buffer (pH 6.0), 2.68% w /. It was suspended in v yeast nitrogen base (without amino acids) 400 μg / mL biotin and 0.5% v / v methanol) and cultured at 30 ° C. and 250 rpm for 24 hours. After culturing, the BMMY medium was centrifuged and the supernatant was filtered through a 0.22 μm filter. This filtered supernatant was analyzed by SDS-PAGE to confirm Nanobody production.

Centrifuge 10 mL of the filtered supernatant into Amicon Ultra-15 Centrifugal Filters Ultracel-3K (Merck Millipore, Burlington, MA, USA) for 60 minutes at 8000 g, and add 10 mL of Phosphate Buffered Saline (PBS) to Amicon Ultra- It was added to a 3k unit and centrifuged at 8000g for 60 minutes. This buffer exchange procedure was repeated twice. Concentration was performed while performing buffer substitution in this manner, and this concentrated solution was used as a monoclonal antibody solution.

FIG. 3 shows the results of SDS-PAGE of anti-CD4-FLAG, anti-CD4-FLAG-barcode 1, anti-GFP-FLAG and anti-GFP-FLAG-barcode 2 produced from Pichia pastris transformants. It is an electrophoretic photograph. In FIG. 3, bands were observed at positions of corresponding molecular weights in all of anti-CD4-FLAG, anti-CD4-FLAG-barcode 1, anti-GFP-FLAG and anti-GFP-FLAG-barcode 2. As can be seen from the results of FIG. 3, as a result of culturing the transformants in a methanol-containing medium, it was confirmed that the production of all the designed Nanobodies was successful. FIG. 4 is a graph showing the production of anti-CD4-FLAG, anti-CD4-FLAG-barcode 1, anti-GFP-FLAG and anti-GFP-FLAG-barcode 2 produced from the Pitia-pastris transformant ( Each value represents the mean ± standard deviation from at least 5 samples. Statistical analysis by t-test was performed. Stars represent significant differences (p <0.05)). There was a slight difference in productivity between anti-CD4-FLAG and anti-CD4-FLAG-barcode 1 (Fig. 4), which is believed to be due to copy number variation of the Pichia-Pastris transformant. Be done.

(Example 2: Evaluation of binding characteristics of Barcode-added Nanobody)
We investigated whether the addition of peptide barcodes affected the properties of Nanobodies.

First, using a sensor chip with CD4 or GFP fixed, surface plasmon resonance was performed according to the following procedure, and the kinetic parameters of Nanobodies were measured.

Kinetic parameters were measured using a Biacore T-200 (manufactured by GE Healthcare). Recombinant human sCD4 CF (manufactured by R & D Systems) and recombinant GFP (ProSpec, Rehovot, Israel) were immobilized on Series S Sensor Chip M5 (manufactured by GE Healthcare) and found to be 2486.9 RU and 1189.8 RU, respectively. It was. Anti-CD4 Nanobodies (anti-CD4-FLAG, anti-CD4-FLAG-barcode 1) in HBS-EP buffer (0.01M HEPE (pH 7.4), 0.15M NaCl, 3 mM EDTA, and 0.005% v / v Surfactant) to 0.2, 0.4, 0.6, 0.8, and 1.0 μM to anti-GFP Nanobodies (anti-GFP-FLAG, anti-GFP-FLAG-barcode 2) It was set to 0.5, 1, 5, 10, and 50 nM. The flow rate, contact time, and dissociation time were 30 μL / min, 120 seconds, and 120 seconds, respectively. The CD4 fixation chip was regenerated with 10 mM NaOH (flow rate 30 μL / min and contact time 30 seconds), and the GFP-fixed chip was regenerated with 50 mM NaOH (flow rate 30 μL / min and contact time 30 seconds).

The results are shown in Table 1 below.

All Nanobodies showed specific binding to each antigen, and there was no significant difference in kinetic parameters depending on the presence or absence of peptide barcodes (Table 1).

Next, immunofluorescence staining of CD4 using Nanobodies was performed. pCAGGS-CD4-myc was donated by Jacob Yount (Addgene plasmid # 58537; http://n2t.net/addgene: 58537; RRID: Addgene_58537). HEK293 cells were transfected with the pCAGGS-CD4-myc plasmid by using Xfect Transfection Reagent (manufactured by Takara Bio Inc.) and cultured in DMEM low glucose medium (manufactured by Nacalai Tesque, Inc.) for 1 day. Transfected cells were transferred onto a cover glass coated with poly-L-lysine hydrobromide (manufactured by Sigma-Aldrich) and cultured in DMEM low glucose medium for 1 to 2 hours. After removing the medium, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes. Fixed cells were rinsed 3 times with PBS and blocked with PBS containing 10% FBS (GE Healthcare) for 30 minutes at room temperature. Incubate anti-CD4 Nanobodies (anti-CD4-FLAG-barcode 1) (2.7 μg / ml) or anti-GFP Nanobodies (anti-GFP-FLAG-barcode 2) (3.3 μg / ml) with fixed cells for 90 minutes. After that, the cells were rinsed 3 times with PBS and washed 3 times with PBS for 5 minutes. Anti-DDDDDK tag mAb Alexa Fluor 488 (anti-FLAG-AF488: showing green under fluorescence microscopy) (manufactured by Medical Biology Laboratory Co., Ltd.) as a secondary antibody to 1 μg / mL in PBS containing 10% FBS. After diluting to and incubating with the cells for 1 hour at room temperature, the cells were rinsed 3 times with PBS and washed 3 times with PBS for 5 minutes. The nuclei were stained with 1 μg / mL of 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI: manufactured by Nacalai Tesque, Inc.) for 1 minute (stained in blue). Stained cells were visualized using a confocal laser scanning fluorescence microscope LSM700 (manufactured by Carl Zeiss). As a control, wild-type HEK293 cells were used.

FIG. 5 is a fluorescence micrograph showing the results of immunofluorescence staining of CD4 using Nanobodies to which peptide barcodes have been added. In pCAGGS-CD4-mycfr-transfected HEK293 cells (CD4 surface-expressing cells) and control wild-type HEK293 cells, anti-CD4 Nanobodies (anti-CD4-FLAG-barcode 1), anti-GFP nanobodies (anti-GFP-FLAG-barcodes) Nuclear staining was observed in both 2) and without Nanobody treatment, whereas the green color of the secondary antibody was when anti-CD4 Nanobodies (anti-CD4-FLAG-barcode 1) were incubated with CD4 surface-expressing cells. Only observed. Therefore, the results shown in FIG. 5 showed that anti-CD4-FLAG-barcode 1 recognized CD4 on the surface of HEK293 cells, but anti-GFP-FLAG-barcode 2 did not. These results indicate that the addition of peptide barcodes did not affect the properties of Nanobodies.

(Example 3: Characterizing the binding ability of Nanobodies by mass spectrometry)
A principle proof experiment was designed to show that the peptide barcode addition technique can be a useful technique for measuring the binding ability of multiple binding substances in a single experiment (Fig. 6). FIG. 6 is a schematic diagram showing a scheme for quantifying the binding ability of Nanobodies by LC-MS. In the designed experiment, first, a mixture of anti-CD4-FLAG-barcode 1 and anti-GFP-FLAG-barcode prepared in equimolarity was reacted with CD4 fixed magnetic beads (simultaneous binding assay), and the mixture was bound in this reaction. The missing antibody is washed away, then enterokinase is added to cut out peptide barcodes from the beads, and the cut out peptide barcodes are quantified by LC-MS or LC-MS / MS selective reaction monitoring (SRM) method. Is. Specifically, it was carried out as follows.

(Simultaneous binding assay: enrichment of CD4 Nanobodies by antigen-antibody interaction)
Recombinant CD4 (0.1 mg / mL) was mixed with 30 μL of NHS activated magnetic beads (manufactured by Thermo Scientific) prewashed with 1 mM ice-cold HCl. After the reaction, the beads were washed twice with 0.1 M glycine-HCl (pH 2.0) and once with distilled water and blocked with 3 M ethanolamine (pH 9.0) at room temperature for 2 hours. After blocking, the magnetic beads were suspended in 30 μL of 50 mM borate buffer containing 0.05% sodium azide to give CD4 fixed magnetic beads.

CD4 fixed magnetic beads were mixed with 500 μL of Nanobody mixture (including 0.1 μM anti-CD4-FLAG-barcode 1 and 0.1 μM anti-GFP-FLAG-barcode 2) and incubated at room temperature for 2 hours. After removing the supernatant, the beads were washed twice with 1 mL of TBS containing 0.05% Tween 20 and once with 1 mL of distilled water.

Beads were incubated with 20 μL of 50 ng / mL enterokinase (manufactured by New England Bio-Labs) at 25 ° C. for 16 hours to cleave the peptide barcode. The supernatant was desalted using MonoSpin C18 (manufactured by GL Sciences, Inc.) and lyophilized. Dried pellets are lysed with 20 μL of 50 mM TEAB, filtered through Ultrafree-MC-HV Centrifugal Filters Durapore PVDF 0.45 μm (Merck Millipore) and at −20 ° C. until subjected to LC-MS / MS analysis. saved.

(Quantification of peptide barcode by LC-MS / MS)
High Performance Liquid Chromatography (LC) (Nexera UHPLC / HPLC System: Shimadzu Corporation) -Triple Tandem Mass Spectrometry (MS / MS) (LCMS-8060: Co., Ltd.) from peptide barcodes cut out by enteroxinase. Analyzed by Shimadzu Corporation).

A 5 μL solution containing a peptide barcode is injected into a 50 mm Inert SustainSwift ^TM C18 column (PN 5020 to 88228, inner diameter 2.1 mm, particle size 1.9 μm; manufactured by GL Sciences Co., Ltd.) and the barcode. To separate. The column is kept at 40 ° C., and the solution is subsequently injected into the MS through a 6-port injection / switching valve (Valco Instruments). Peptide barcode analysis was performed at a flow rate of 600 μL / min for 3.5 minutes. Gradients were produced by varying the mixing ratio of the two eluents: A, 0.1% (v / v) formic acid and B, 0.1% (v / v) formic acid containing acetonitrile. The gradient starts with a 5% B 0.5 minute hold, then raises this to 50% B in 2 minutes, then holds for 0.5 minutes and raises to 95% B, and finally raises the B concentration to 5% immediately. And held for 0.5 minutes to rebalance the column. The autosampler was kept at 4 ° C and equipped with a black door. The interface temperature, heat block temperature and desolvation part (DL) temperature were set to 300 ° C, 400 ° C and 250 ° C, respectively. The flow rates of the nebulizer gas (N ₂ ), the drying gas (N ₂ ), and the heating gas (dry air) for producing the droplets were set to ₂ L / min, 10 L / min, and 10 L / min, respectively.

For the optimization of the SRM method, each synthetic peptide prepared by Skyline software (Proteomics 2012: 12: 1134-1141) (a peptide in which a FLAG tag peptide is fused to the C-terminal of bar code 1 and a C-terminal of bar code 2) All transitions of the peptide (peptide fused with FLAG tag peptide) were analyzed. Two sensitive transitions were selected for each peptide and the collision energy with the highest peak intensity was adopted (Table 2).

In addition, the time was determined for these transitions based on the retention time obtained. Ionized peptide barcodes were analyzed by this method with a residence time of 5 minutes.

(result)
In order to verify the sensitivity and quantification of SRM, a synthetic peptide (a peptide in which a FLAG tag peptide is fused to the C-terminal of bar code 1 (SEQ ID NO: 18) and a peptide in which a FLAG tag peptide is fused to the C-terminal of bar code 2) (SEQ ID NO: 19)) were continuously diluted and quantified.

FIG. 7 is a graph showing the analysis results of various amounts of LC-MS / MS for barcode 1 (A) and barcode 2 (B). Each value in FIG. 7 represents the mean ± standard deviation of the three samples. From the results of FIGS. 7A and 7B, it was found that both the barcode 1 and the barcode 2 were detected and quantified with high sensitivity by the LC-MS / MS analysis used in this example.

As a positive control experiment, from 250 fmol anti-CD4-FLAG-bar code 1 or 250 fmol anti-GFP-FLAG-bar code 2 to peptide bar code (ie, bar code 1-FLAG (SEQ ID NO: 18) and bar code 2-FLAG, respectively). (SEQ ID NO: 19)) were cut out and quantified by LC-MS / MS.

FIG. 8 is a graph showing the results of quantifying peptide barcodes cut out from 250 fmol anti-CD4-FLAG-bar code 1 and 250 fmol anti-GFP-FLAG-bar code 2 by LC-MS / MS, respectively. Each value in FIG. 8 represents the mean ± standard deviation of the three samples. From the results of FIG. 8, both barcode 1 and barcode 2 were detected with high sensitivity by the LC-MS / MS used in this example, and were successfully quantified with minimal loss during the sample preparation procedure. It turned out that.

Next, a 500 μL Nanobody mixture (containing 0.1 μM anti-CD4-FLAG-barcode 1 and 0.1 μM anti-GFP-FLAG-barcode 2) was added to the CD4 immobilized magnetic beads for a simultaneous binding assay. Peptide barcodes cut out from the beads were quantified.

FIG. 9 is a graph showing the results of quantifying peptide barcodes cut out from beads by LC-MS / MS after the simultaneous binding assay. Each value in FIG. 9 represents the mean ± standard deviation of the three samples. As seen in FIG. 9, the peptide of barcode 1 derived from anti-CD4-FLAG-barcode 1 was detected, but the peptide of barcode 2 derived from anti-GFP-FLAG-barcode 2 was not detected. It was. Therefore, it was found that the peptide barcode derived from anti-CD4-FLAG-barcode 1 bound to the CD4 antigen carried on the beads after being subjected to the simultaneous binding assay was specifically detected by LC-MS / MS. It was.

(Example 4: Examination of the effect of various peptide barcodes on the binding assay)
The following eight peptide barcodes were designed. These peptide barcodes were designed with varying degrees of hydrophobicity. Thereby, the elution time of the liquid chromatograph can be appropriately shifted:
Barcode 4-1: DIVVLGVEK (SEQ ID NO: 20)
Barcode 4-2: LIHVLDAGR (SEQ ID NO: 21)
Barcode 4-3: LHAILFGLPR (SEQ ID NO: 22)
Barcode 4-4: LEDLLLDR (SEQ ID NO: 23)
Barcode 4-5: LAEIHGVPR (SEQ ID NO: 24)
Barcode 4-6: FQFLWGPR (SEQ ID NO: 25)
Barcode 4-7: VELQQEVERK (SEQ ID NO: 26)
Barcode 4-8: NIFEQLHR (SEQ ID NO: 27)

A plasmid for secreting and expressing anti-CD4 Nanobodies or anti-GFP Nanobodies to which the above various peptide barcodes were added was constructed in yeast. The plasmid was constructed according to Example 1 except that the barcode portions of anti-CD4-FLAG-barcode 1 and anti-GFP-FLAG-barcode 1 were changed to genes expressing the above-mentioned various peptide barcodes. I went in the same way. In this example, the peptide barcode has a FLAG-tag peptide (DYKDDDDK: SEQ ID NO: 3) fused to its N-terminus, but not to its C-terminus. Nucleotide sequence and expression of DNA fragment encoding each barcoded nanobody fusion protein are shown in SEQ ID NOs: 28 to 75: Nucleotide sequence of barcode 4-1 addition anti-CD4: Nucleotide sequence of DNA fragment (SEQ ID NO: 28) and expression. Peptide sequence (SEQ ID NOs: 28-30); Barcode 4-2 addition anti-CD4: Nucleotide sequence of DNA fragment (SEQ ID NO: 31) and expression peptide sequence (SEQ ID NOs: 31-33); Barcode 4-3 addition anti-CD4: Nucleotide sequence of DNA fragment (SEQ ID NO: 34) and expressed peptide sequence (SEQ ID NOs: 34-36); Barcode 4-4 Additive Anti-CD4: Nucleotide sequence of DNA fragment (SEQ ID NO: 37) and expressed peptide sequence (SEQ ID NOs: 37-36) 39); Barcode 4-5 Additive Anti-CD4: Nucleotide Sequence of DNA Fragment (SEQ ID NO: 40) and Expressed Peptide Sequence (SEQ ID NOs: 40-42); Barcode 4-6 Additive Anti-CD4: Nucleotide Sequence of DNA Fragment (Sequence) No. 43) and expressed peptide sequence (SEQ ID NOs: 43-45); Barcode 4-7 Addition Anti-CD4: Nucleotide sequence of DNA fragment (SEQ ID NO: 46) and expressed peptide sequence (SEQ ID NOs: 46-48); Barcode 4- 8 Additive anti-CD4: DNA fragment base sequence (SEQ ID NO: 49) and expressed peptide sequence (SEQ ID NO: 49-51); Barcode 4-1 Additive anti-GFP: DNA fragment base sequence (SEQ ID NO: 52) and expressed peptide sequence (SEQ ID NOs: 52 to 54); Nucleotide sequence of DNA fragment (SEQ ID NO: 55) and expression peptide sequence (SEQ ID NO: 55-57); Barcode 4-3 added anti-GFP: DNA fragment Nucleotide sequence (SEQ ID NO: 58) and expressed peptide sequence (SEQ ID NO: 58-60); Barcode 4-4 addition anti-GFP: Nucleotide sequence of DNA fragment (SEQ ID NO: 61) and expressed peptide sequence (SEQ ID NOs: 61-63) Barcode 4-5 Additive Anti-GFP: Nucleotide Sequence of DNA Fragment (SEQ ID NO: 64) and Expressed Peptide Sequence (SEQ ID NOs: 64-66); Barcode 4-6 Additive Anti-GFP: Nucleotide Sequence of DNA Fragment (SEQ ID NO: 67) ) And the expressed peptide sequence (SEQ ID NOs: 67-69); barcode 4-7 added anti-GFP: base sequence of DNA fragment (SEQ ID NO: 70) and expressed peptide sequence (SEQ ID NOs: 70-72); and barcode 4-8. Nucleotide sequence of DNA fragment (SEQ ID NO: 73) and expression peptide sequence (SEQ ID NOs: 73-75).

Monoclonal antibodies to which peptide barcodes were added were obtained from various plasmids in the same manner as in Example 1. The obtained monoclonal antibody was examined for its binding ability to each antigen using kinetic parameters in the same manner as in Example 2. As a control, Nanobodies to which no peptide barcode was added (“Anti-CD4-FLAG” and “Anti-GFP-FLAG” of Example 1) were used. The results are shown in Table 3 (CD4) and Table 4 (GFP) below. In each case, it was found that the addition of peptide barcodes did not affect the ability of Nanobodies to bind to antigens.

(Example 5: Improvement of mass spectrometer detection power using a long monolith column)
The long monolith column was further improved in order to improve the detection ability of peptides by LC-MS / MS. In order to evaluate the detection power, the peak capacity was measured using the same LC-MS / MS apparatus as in Example 3. The higher the peak capacity, the higher the separation performance.

For example, FIG. 10 is a graph showing the peak capacity when each monolith column having an inner diameter of 100 μm and an inner diameter of 75 μm is used in LC-MS / MS measurement of peptide barcode 1 (SEQ ID NO: 18) (both are 500 mm). Long). By changing the inner diameter from 100 μm inner diameter to 75 μm inner diameter, the peak capacity was increased (FIG. 10).

In addition, the longer columns also increased the peak capacity. For example, FIG. 11 is a graph showing the peak capacity when each monolith column having a length of 500 mm and a length of 1000 mm is used in the LC-MS / MS measurement of peptide barcode 1 (SEQ ID NO: 18) (both inner diameters). 75 μm). It was confirmed that the peak capacity was improved by lengthening the column having a length of 500 mm to 1000 mm (FIG. 11). Further improvement in performance is expected by making the monolith column longer.

By using a 1000 mm monolith column having an inner diameter of 75 μm, the peptide separation performance by liquid chromatography was improved, and the detection sensitivity of peptides by mass spectrometry could be greatly improved.

Using a 1000 mm monolith column with a 75 μm inner diameter, for example, a tryptic digest of bovine serum albumin (BSA) was analyzed by LC-MS / MS measurement, and it was confirmed that the sensitivity was improved more than twice.

The present invention is useful, for example, in the manufacture of experimental reagents, diagnostic agents, and pharmaceuticals.

Claims (6)

A composition comprising a DNA fragment containing a gene encoding a secretory signal, a gene encoding a Nanobody and a gene encoding a peptide barcode, or a yeast into which a vector containing the DNA fragment has been introduced.
The peptide barcode is represented by an amino acid sequence consisting of 5 to 30 amino acids, and the amino acids are independently selected from the group consisting of A, F, G, K, L, P, R, V and W. ,
A polypeptide containing the Nanobody and the peptide barcode is expressed intracellularly in the yeast and secreted extracellularly, and through identification of the peptide barcode excised from the polypeptide, specifically with the antigen. A composition used to identify the Nanobodies of a monoclonal antibody that binds . The composition according to claim 1, wherein the yeast belongs to the genus Saccharomyces, Pichia, Schizosaccharomyces, Zygosaccharomyces, Candida, Trulopsis, Yarrowia or Hansenula. The composition according to claim 1 or 2, wherein the secretory signal is an α-factor secretory signal, a glucoamylase secretory signal, or a PHO1 secretory signal. The composition according to any one of claims 1 to 3, wherein the DNA fragment further comprises a promoter which is an AOX1 promoter, a GAP promoter, an FLD1 promoter, a PEX8 promoter or a YPT1 promoter. A composition comprising a polypeptide containing Nanobodies and peptide barcodes.
The peptide barcode is represented by an amino acid sequence consisting of 5 to 30 amino acids, and the amino acids are independently selected from the group consisting of A, F, G, K, L, P, R, V and W. ,
A composition used to identify Nanobodies of monoclonal antibodies that specifically bind to an antigen through the identification of the peptide barcode excised from the polypeptide . Represented by the amino acid sequence consisting of 5-30 amino acids, and the amino acid is independently A, F, G, K, L, P, R, is selected from the group consisting of V and W, the peptide barcode A composition comprising, wherein the peptide barcode is fused to the nanobody and used to identify the fused nanobody through identification of the peptide barcode .