IMMUNOLOGICAL EVALUATION METHOD FOR mRNA EXPRESSION LEVELS

Abstract

To provide a simple and accurate method of quantitatively making immunological analysis of every protein in order to compare and evaluate functionality and effects of two or more mRNAs with different sequences in terms of expression levels of proteins encoded thereby.SOLUTION: A method disclosed herein involves preparing a labeled mRNA linked with a labeling nucleotide encoding an identical labeled peptide at the end of each mRNA, and quantifying an expression level of a labeled polypeptide expressed from each labeled mRNA using an identical antibody against the labeled peptide.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for immunologically evaluating mRNA expression levels. [Background technology]

[0002] mRNA vaccines are introduced into the body by injection or other means, and function by expressing the proteins encoded by the mRNA. Their effectiveness is evaluated based on the proteins expressed in the body.

[0003] However, the inherent function and efficacy of mRNA vaccines depend on the base sequence and length of the vaccine. For example, two mRNA vaccines encoding the same protein using degenerate codons express proteins with the same amino acid sequence. However, because the base sequences differ, they do not necessarily behave identically after in vivo administration. Specifically, the amount of protein expressed by each mRNA vaccine may differ. Therefore, the function and efficacy of mRNA vaccines are evaluated relative to the expression level of the target protein expressed by the mRNA vaccine. Protein expression levels have traditionally been measured using immunoassays using antibodies that specifically bind to each protein, as shown in Figure 1. However, this measurement method has several problems.

[0004] First, measurement itself is impossible unless highly specific antibodies are available. Although there are commercially available antibodies for some target proteins, many of them lack specificity and binding ability, and selecting an antibody suitable for measurement requires a great deal of time and cost. Furthermore, if it is difficult to obtain antibodies because they are not commercially available, it is necessary to produce the antibodies yourself, which requires an even greater amount of time and effort.

[0005] Second, detection accuracy is reduced when the target protein is endogenously present in cells. Even when a highly specific antibody is used, if the target protein derived from the introduced mRNA and the endogenous target protein are identical, the antibody cannot distinguish between the two proteins. This poses the problem of being unable to accurately measure the expression level of the target protein derived from the introduced mRNA.

[0006] The third problem is the low versatility of the measurement method. When measuring and comparing the expression levels of multiple target proteins, highly specific antibodies must be prepared for each target protein. However, the first problem makes measurement difficult except for target proteins for which highly specific antibodies are readily available. Another problem is that the binding ability between antibodies and proteins is not necessarily the same for each antibody, making it impossible to simply compare the expression levels of each target protein.

[0007] Patent Document 1 discloses multiple compositions containing a protein-binding reagent conjugated to an oligonucleotide containing a unique identifier for quantitative analysis of multiple proteins. The protein-binding reagent can specifically bind to a protein target, thereby enabling quantitative analysis of multiple protein targets in a sample. Patent Document 1 also discloses a method and kit for simultaneous quantitative analysis of protein and nucleic acid targets in a sample, as well as a system for preparing labeled biomolecular reagents. This method can solve the first and second problems. However, the third problem remains: measured expression levels cannot be simply compared because the binding affinity between each protein-binding reagent and the protein to which it specifically binds is not identical. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to develop and provide a highly versatile method for easily and accurately quantitatively analyzing target proteins immunologically, in order to evaluate the functions and effects of multiple target mRNAs with different base sequences as the expression level of target proteins expressed from each target mRNA. [Means for solving the problem]

[0009] To solve the above problems, the present inventors conducted extensive research and developed a method using a fusion mRNA in which a labeling nucleotide encoding the same common labeling peptide is linked to the terminus of multiple target mRNAs with different base sequences, as shown in Figure 2. This method makes it possible to express the fusion mRNA and then easily and accurately quantitatively analyze all target proteins using a single common antibody that binds to the labeling peptide, and is highly versatile. The present invention is a method based on the results of this development and provides the following (1) and (2).

[0010] (1) A method for immunologically evaluating the amount of mRNA expression, comprising: an expression step of expressing labeled mRNAs in which labeled nucleotides encoding the same labeled peptide are linked to the ends of two or more target mRNAs having different base sequences; and a quantification step of quantifying the labeled polypeptides expressed from each labeled mRNA in the expression step using the same antibody against the labeled peptide; (2) A method for immunologically evaluating the expression level of mRNA, comprising: an expression step of expressing labeled mRNA in which a labeled nucleotide encoding a labeled peptide is linked to the end of the target mRNA; a quantification step of quantifying the labeled polypeptide expressed from the labeled mRNA in the expression step using an antibody against the labeled peptide; and an analysis step of comparing the quantified value of the labeled polypeptide obtained in the quantification step with a reference value and analyzing the expression level of the target mRNA from the result, wherein the reference value is a value obtained by quantifying, using the antibody, a labeled reference polypeptide expressed from a labeled reference mRNA in which the same labeled nucleotide is linked to a reference mRNA having a base sequence different from that of the target mRNA. [Effects of the Invention]

[0011] According to the immunological evaluation method of the present invention, the expression levels of proteins expressed by multiple mRNAs with different base sequences can be quantified and compared using a single common antibody, thereby enabling a simple and accurate immunological evaluation of the function and effect of each mRNA. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a conceptual diagram of a conventional general method for immunologically measuring mRNA expression levels. [Figure 2] FIG. 1 is a conceptual diagram of the method of the present invention for immunologically measuring mRNA expression levels. [Figure 3] FIG. 1 is a flow diagram of the immunological evaluation method of the present invention. [Figure 4] Figures showing the results of an example of the present invention. After EGFP mRNA or EGFP-Flag mRNA was introduced into HEK293 cells, the expression level of EGFP in each cell was measured by EGFP fluorescence intensity (A) and measured by immunostaining using an anti-Flag antibody (B). HEK293 cells not introduced with either mRNA served as a control. DETAILED DESCRIPTION OF THE INVENTION

[0013] 1. Immunological evaluation method for mRNA expression level (1) Overview A first aspect of the present invention is a method for immunologically evaluating mRNA expression levels. To evaluate the expression levels of two or more target mRNAs with different nucleotide sequences, the present invention involves preparing labeled mRNAs in which the same labeled nucleotide is linked to the end of each target mRNA, and quantifying the expression levels of labeled polypeptides expressed from the labeled mRNAs using the same antibody against the labeled peptide. This method does not require the preparation of antibodies that specifically bind to each target mRNA, and can accurately evaluate the expression levels of target mRNAs using readily available antibodies without being affected by endogenous mRNAs.

[0014] 1-2.Definition of Terms Terms used in this specification are defined below. As used herein, the term "expression level of mRNA" refers to the protein expression activity of a single mRNA. This expression level is expressed as the amount of protein expressed from a specific mRNA introduced into a cell. The amount of protein used herein may be a relative value such as fluorescence intensity, or an absolute value such as mass.

[0015] As used herein, "immunological evaluation of mRNA expression level" refers to measuring and evaluating the expression level of a specific mRNA as the expression level of a protein expressed from that mRNA by an immunological assay method using an antibody.

[0016] As used herein, the term "target mRNA" refers to an mRNA that is the target of measurement in the method of the present invention. A polypeptide encoded by a target mRNA is referred to herein as a "target polypeptide."

[0017] As used herein, the term "label peptide" refers to a tag peptide consisting of a known amino acid sequence of 7 to 40 amino acids. A label peptide can be linked to the amino acid sequence terminal of another polypeptide to label the polypeptide. As used herein, the label peptide is preferably an epitope tag.

[0018] As used herein, the term "epitope tag" refers to a labeled peptide containing at least one epitope in its amino acid sequence. Specific examples of epitope tags include, but are not limited to, the Flag® tag represented by SEQ ID NO: 1 (DYKDDDDK), the HA tag represented by SEQ ID NO: 2 (YPYDVPDYA), the 6xHis tag represented by SEQ ID NO: 3 (HHHHHH), the Myc tag represented by SEQ ID NO: 4 (EQKLISEEDL), the V5 tag represented by SEQ ID NO: 5 (GKPIPNPLLGLDST), the T7 tag represented by SEQ ID NO: 6 (MASMTGGQQMG), the S tag represented by SEQ ID NO: 7 (KETAAAKFERQHMDS), the E tag represented by SEQ ID NO: 8 (GAPVPYPDPLEPR), and the Glu-Glu tag represented by SEQ ID NO: 9 (EEEEYMPME). For epitope tags, known antibodies (tag antibodies) that specifically recognize the epitopes contained therein are usually available. Therefore, even if an antibody that specifically recognizes the polypeptide of the labeled polypeptide has not been prepared, the labeled polypeptide can be immunologically detected and quantified using a tag antibody against the labeled peptide.

[0019] As used herein, the term "labeled nucleotide" refers to a nucleotide encoding the labeled peptide. The labeled nucleotide is linked in frame to the end of the base sequence of the target mRNA. The end to which the labeled nucleotide is linked may be the 5' end, the 3' end, or both. The 3' end is preferred.

[0020] As used herein, "labeled mRNA" refers to a target mRNA linked to a labeled nucleotide, which encodes a labeled polypeptide, as described below. As used herein, labeled mRNA can encompass two or more target mRNAs with different nucleotide sequences and multiple labeled mRNAs containing the same labeled nucleotide linked to the end of each target mRNA (often referred to herein as "two or more different labeled mRNAs"). Here, "two or more target mRNAs with different nucleotide sequences" includes not only mRNAs that encode different polypeptides, but also mRNAs that encode the same polypeptide but differ in nucleotide sequence due to differences in peptide-encoding codons or the presence or absence of nucleic acid base modifications. A single labeled mRNA may contain two or more different labeled nucleotides. In this case, each labeled nucleotide may be linked to a different end of the target mRNA, or may be linked in tandem to the same end. Furthermore, when two or more different labeled RNAs each contain two or more different labeled nucleotides, the type of labeled nucleotide contained in each labeled RNA is not particularly limited as long as at least one of them is common, but it is desirable that the number of labeled nucleotides contained in each labeled mRNA and the number of bases of the labeled nucleotide are the same or similar. Furthermore, the design may be such that a linker is inserted between the labeled nucleotide and the target mRNA. There are no particular limitations on the linker, but it is preferable that it does not impair the three-dimensional structure or function of the target polypeptide, and examples thereof include, but are not limited to, a GS linker.

[0021] As used herein, the term "labeled polypeptide" refers to a polypeptide encoded by a labeled mRNA and to be measured in the immunological evaluation method of the present invention. Labeled polypeptides expressed from two or more different labeled mRNAs are often referred to herein as "two or more different labeled polypeptides." However, two or more different labeled polypeptides may be composed of the same amino acid sequence.

[0022] As used herein, the term "reference mRNA" refers to an mRNA having a nucleotide sequence different from that of a target mRNA, which serves as a comparison standard when quantifying the expression level of the target mRNA in the immunological evaluation method of the present invention. The nucleotide sequence of the reference mRNA is not limited, but preferably has a sequence similar to that of the target mRNA, for example, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more nucleotide identity with the nucleotide sequence of the target mRNA. The nucleotide length of the reference mRNA is not limited, but preferably is identical to or differs from that of the target mRNA by 1 to 20 nucleotides.

[0023] As used herein, a "labeled reference mRNA" refers to a reference mRNA to which a labeled nucleotide is linked, and which encodes a labeled reference polypeptide (described below). The labeled nucleotide linked to the labeled reference mRNA is identical to the labeled nucleotide linked to the labeled mRNA.

[0024] As used herein, the term "labeled reference polypeptide" refers to a polypeptide encoded by a labeled reference mRNA.

[0025] As used herein, the term "quantitative value" refers to the amount of labeled polypeptide expressed from labeled mRNA. This quantitative value is determined using an antibody against a labeled peptide contained in the labeled polypeptide. This value may be a relative amount indicated by fluorescence intensity, luminescence intensity, turbidity, absorbance, radiation dose, ionic strength, or concentration, or may be an absolute amount such as the weight or volume of the labeled polypeptide contained in a sample.

[0026] As used herein, the term "reference value" refers to a value obtained by quantifying a labeled reference polypeptide expressed from a labeled reference mRNA. This value, like the quantitative value described above, may be a relative value or an absolute value.

[0027] 1-3. Method A flow diagram of the immunological evaluation method of this embodiment is shown in Figure 3. The immunological evaluation method of the present invention includes, as essential steps, an expression step (S0320) and a quantification step (S0340), and, as selection steps, an introduction step (S0310), a protein extraction step (S0330), and an analysis step (S0350). Each step will be explained below.

[0028] 1-3-1.Introduction process The "introduction step" (S0310) is a step of introducing two or more different labeled mRNAs into cells. This step is selected when the labeled mRNAs are to be expressed in cells.

[0029] The preparation of the labeled mRNA used in this step is not particularly limited. For example, a labeled mRNA in which a target mRNA and a labeled nucleotide are linked may be prepared using genetic recombination technology. In this case, for example, a target DNA sequence corresponding to the target mRNA may be inserted into an expression vector already containing the labeled nucleotide, and then expressed in a host cell to obtain the desired labeled mRNA. Alternatively, the entire base sequence of the labeled mRNA may be designed, and then the sequence may be converted into DNA to artificially synthesize labeled DNA in vitro. In this case, the obtained labeled DNA may be inserted into an expression vector and then expressed in a host cell in the same manner as above to obtain the desired labeled mRNA. Furthermore, labeled mRNA may be directly artificially synthesized in vitro.

[0030] The cells used in this step are not particularly limited. For example, bacteria, yeast, insects or their cultured cells, cultured animal cells, or cultured plant cells can be used. Examples of bacteria include Escherichia coli and Bacillus subtilis. Examples of yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. Examples of insects include Bombyx mori, Antheraea yamamai, and Samia cynthia. Examples of insect cultured cells include Sf9 cells, Sf9plus cells, Sf21 cells, High Five cells, and Ea4 cells. Examples of animal cultured cells include primary cultured cells derived from differentiated cells such as somatic cells, undifferentiated stem cells, and established cell lines. Examples of stem cells include embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells. Examples of established cell lines include HeLa cells, HEK293 cells, NIH3T3 cells, CHO cells, human fibroblasts, FL cells, COS-7 cells, Vero cells, L cells, and GH3 cells. Examples of cultured plant cells include BY-2 cells. These cells may be cells collected from a living organism and cultured, or genetically modified cells.

[0031] The method for introducing labeled mRNA into cells is not particularly limited. Considering the type of cell to be introduced and the type of labeled mRNA to be expressed, any transformation or transfection method known in the art can be used. For example, electroporation, heat shock, protoplast, lipofection, PEG (polyethylene glycol), calcium phosphate, DEAE-dextran, protoplast, particle gun, Agrobacterium, and viral infection can be used. Regarding transformation and transfection methods, reference can be made to gene transfer methods described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc.

[0032] 1-3-2. Expression process The "expression step" (S0320) is a step of expressing labeled polypeptides from two or more different labeled mRNAs. This step may be performed in vivo in cells into which labeled mRNAs have been introduced, or in vitro using a cell-free protein expression system, but each labeled mRNA must be expressed under the same conditions. For example, when expression is performed in vivo, all labeled mRNAs are expressed using the same type of cells cultured under the same conditions.

[0033] When expression is carried out in vivo, this step can usually be achieved by culturing cells into which labeled mRNA has been introduced. In principle, the expression of labeled mRNA in this step is transient.

[0034] The method for culturing cells involves seeding the cells in a medium and culturing them under predetermined culture conditions. The medium and predetermined culture conditions used at this time may be any culture method known in the art depending on the type of cell. In principle, it is desirable to carry out all culture under sterile conditions.

[0035] For example, when E. coli is used, E. coli into which labeled mRNA has been introduced can be inoculated into a known medium such as LB medium or ND medium, and then cultured at 37°C by osmotic culture.

[0036] When yeast is used, the yeast into which the labeled mRNA has been introduced may be inoculated into a known medium such as YEP medium, YPD medium, YNB medium, or SD medium, and then cultured at 30°C by osmotic culture.

[0037] When using insect cells, Sf900 TM After adding serum as needed to a known medium such as II, TC-100, or SFM-PB, the insect cells into which the labeled mRNA has been introduced are inoculated and cultured at 27°C.

[0038] When using cultured animal cells, such as mammalian cells, the animal cells transfected with labeled mRNA can be seeded in standard cell culture medium, optionally supplemented with serum, and then cultured at 37°C under 5% CO2. The term "standard cell culture medium" refers to a versatile basal medium primarily used for culturing various types of mammalian cells. Specific examples include Eagle Minimum Essential Medium (Eagle MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F10 (Ham's F10), Ham's Nutrient Mixture F12 (Ham's F12), M199, High Performance Medium 199, Roswell Park Memorial Institute-1640 (RPMI-1640), and Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F12 (DMEM / F12). There are no particular limitations on the mixing ratio of DMEM / F12 medium. Preferably, DMEM and F12 are mixed in a weight concentration ratio of the components ranging from 6:4 to 4:6.

[0039] The specific composition of the above-mentioned medium is known in the art, and therefore, it may be prepared based on the composition described in an appropriate document (for example, a standard cell culture medium is described in Kaech S. and Banker G., 2006, Nat. Protoc., 1(5): 2406-2415). However, commercially available media from life science manufacturers such as Thermo Fisher Scientifics and FUJIFILM Wako Pure Chemical Industries may also be used.

[0040] The culture period varies depending on the type of cells to be cultured, but is sufficient as long as it is long enough for the labeled mRNA introduced into the cells to be expressed, and is generally 12 to 72 hours, preferably 24 to 48 hours.

[0041] When expression is performed in vitro, labeled polypeptides are expressed from each labeled mRNA using a cell-free protein expression system as described above. A "cell-free protein expression system" refers to a system that utilizes the biomolecular translation machinery contained in a cell extract extracted from cells to express a protein from a gene of interest in the cell extract. Cells from which the cell extract used in the cell-free protein expression system is derived may be any cells known in the art. Suitable examples include, but are not limited to, rabbit reticulocytes, wheat germ, and Escherichia coli. Labeled mRNA expression using a cell-free protein expression system may be performed according to methods known in the art.

[0042] 1-3-3. Protein extraction process The "protein extraction step" (S0330) is a selection step for extracting protein from cells after the expression step when the introduction step (S0310) is selected. The purpose of this step is to extract the labeled polypeptide expressed from the labeled mRNA in the expression step. Although not limited thereto, it is preferable to select this step in conjunction with the introduction step.

[0043] In general, proteins are extracted from cells by lysing the cells and then extracting the proteins from the cell lysate. The cell lysis method may be selected and performed according to an optimal known method depending on the type of cell and the location of the labeled polypeptide of interest (cytoplasm, cell wall, culture supernatant, etc.).

[0044] When the cells used in the introduction step are bacteria such as Escherichia coli, insect cells, cultured animal cells, etc., the method for lysing the cells is not limited to, but suitable methods include, for example, osmotic shock, freeze-thaw, surfactant extraction, or a combination thereof. Furthermore, when cells having a cell wall such as yeast or plant cells are used, the method for lysing the cells is not limited to, but suitable methods include, for example, enzymatic digestion, ultrasonic disruption, French press, homogenizer, glass bead method, or a combination thereof.

[0045] The osmotic shock method involves suspending cells in a hypotonic solution such as sterile water and disrupting the cell membranes by the osmotic pressure difference, resulting in cell lysis. The freeze-thaw method involves rapidly freezing a cell suspension using liquid nitrogen or other fluids and then thawing it to disrupt the cell membrane and lyse the cells. The detergent extraction method is the most common and preferred protein extraction method, as the addition of a detergent promotes the solubility of hydrophobic proteins, thereby lysing cells and solubilizing proteins. The detergent used is preferably, but not limited to, a nonionic or zwitterionic detergent. The enzymatic digestion method involves treating cells with enzymes specific to each cell type (e.g., lysozyme, cellulase, pectinase), thereby disrupting the insoluble cell walls and lysing the cells. The ultrasonic disruption method involves disrupting the cell membrane and cell walls by ultrasonication, resulting in cell lysis. The French press method involves forcibly extruding a cell suspension through a small hole under high pressure, disrupting the cells by shear force. The homogenizer method is a method in which cells are mechanically homogenized and disrupted together with the tissue or in a cell suspension state, while the glass bead method is a method in which cell walls and membranes are physically disrupted by the collision and friction of glass beads.

[0046] All of the above methods are known in the art, and specific methods may be carried out in accordance with these methods.

[0047] The labeled polypeptide to be quantified in the present invention is localized in, but not limited to, the cytoplasm or the culture supernatant. Therefore, by centrifuging the cell lysate prepared above and recovering the supernatant, a protein extract containing the labeled polypeptide of interest expressed in the cells can be obtained.

[0048] 1-3-4.Quantitative process The "quantification step" (S0340) is a step in which labeled polypeptides expressed from two or more different labeled mRNAs in the expression step (S0320) are measured and quantified using an antibody against a labeled peptide common to each labeled polypeptide. This step is the most distinctive and important essential step in the immunological evaluation method of the present invention. After this step, a quantitative value of each labeled polypeptide is obtained. This quantitative value may be a relative value or an absolute value as described above, but it must be a value measured and quantified using the same quantification method and the same standard for each labeled polypeptide.

[0049] Each labeled mRNA encoding two or more different labeled polypeptides of the present invention contains a different base sequence in the target mRNA region, but shares at least one labeled nucleotide linked to its terminus. Therefore, the labeled polypeptides expressed from each labeled mRNA in the expression step contain the same labeled peptide. The quantification in this step uses an antibody that specifically recognizes and binds to this common labeled peptide, i.e., an anti-labeled peptide antibody.

[0050] As used herein, the term "antibody" refers to a polypeptide that contains a framework region (FR) and a complementarity determining region (CDR) derived from an immunoglobulin and has the function of specifically binding to an antigen. The term "antibody" as used herein includes not only full-length antibodies but also functional fragments (polypeptide fragments) of antibodies that retain antigen-binding ability. Antibodies include both natural and artificial antibodies.

[0051] As used herein, the term "natural antibody" refers to an antibody produced in vivo by a vertebrate, or an antibody that has the same amino acid sequence as that antibody and is produced by an artificially produced antibody-producing cell (e.g., a hybridoma cell). Natural antibodies include, for example, polyclonal antibodies and monoclonal antibodies. Although not limited thereto, monoclonal antibodies are preferred due to the specificity of the antibody for the labeled peptide.

[0052] The term "polyclonal antibody" refers to a group of immunoglobulins that recognize and bind to different epitopes of the same antigen. Polyclonal antibodies can be obtained from the serum of an animal immunized with a target molecule as an antigen.

[0053] A "monoclonal antibody" refers to a group of clones of a single immunoglobulin. Each immunoglobulin constituting a monoclonal antibody contains a common framework region and a common complementarity-determining region, and is capable of recognizing and binding to the same epitope of the same antigen. Monoclonal antibodies can be obtained from hybridomas derived from a single cell.

[0054] When the antibody is a polyclonal or monoclonal antibody, the immunoglobulin molecule may be of any class (e.g., IgG, IgA, IgE, IgM, IgD, and IgY), and may also include single-chain antibodies such as VHH antibodies, which lack L chains and are composed only of the VH region of an H chain, such as single-chain antibodies produced by animals of the Camelidae family.

[0055] As used herein, "artificial antibodies" refer to artificially constructed antibodies. Examples include antibodies in which appropriate mutations have been introduced into the amino acid sequence of the natural antibodies, as well as single-chain antibodies that have undergone structural modifications that do not generally occur in nature. Specific examples of the latter artificial antibodies include recombinant antibodies. "Recombinant antibodies" refer to chimeric antibodies, humanized antibodies, synthetic antibodies, and antibody fragments.

[0056] A "chimeric antibody" is an antibody created by combining the amino acid sequences of antibodies derived from different animals, in which the variable region (V region) of one antibody is replaced with the V region of another antibody. For example, an antibody in which the V region of a mouse monoclonal antibody is replaced with the V region of a human antibody results in an antibody with a variable region (V region) derived from mouse and a C region derived from human.

[0057] A "humanized antibody" is a graft antibody in which the complementarity-determining regions (CDRs; CDR1, CDR2, and CDR3) in the variable region (V region) of an antibody from a non-human mammal, such as an appropriate mouse, have been replaced with the CDRs of a human monoclonal antibody. In chimeric and humanized antibodies, the heavy and light chain V regions or the complementarity-determining regions in the heavy and light chain V regions are derived from a non-human animal antibody such as a mouse, but the C region or the framework regions (FRs; FR1, FR2, FR3, and FR4) in the V region and the heavy and light chain C regions are derived from a human antibody, thereby reducing the immune response to the antibody in the human body.

[0058] The term "synthetic antibody" refers to an antibody synthesized using chemical or recombinant DNA techniques. For example, it refers to a monomeric polypeptide molecule in which one or more VLs and one or more VHs of a specific antibody are artificially linked via a linker peptide or the like having an appropriate length and sequence, or a multimeric polypeptide thereof. Specific examples of such polypeptides include single-chain Fvs (scFv: single-chain fragment of variable region), diabodies, triabodies, and tetrabodies. These synthetic antibodies are bivalent to tetravalent antibody fragments having dimeric to tetrameric structures based on the single-chain Fv structure. Diabodies and higher may also be multispecific antibodies. The term "multispecific antibody" refers to a multivalent antibody, i.e., an antibody having multiple antigen-binding sites in a single molecule, each of which binds to a different epitope.

[0059] The term "antibody fragment" refers to, for example, Fab, F(ab')2, Fv, and the like.

[0060] The antibody used in this step may be a modified antibody (modified labeled antibody). In particular, in the case where a secondary antibody that recognizes and specifically binds to the anti-labeled peptide antibody as the primary antibody is used in the quantification of the labeled polypeptide in this step, as described below, it is preferable that the secondary antibody is a modified antibody.

[0061] Antibody modifications include functional modifications or labeling modifications. Functional modifications include, for example, glycosylation, acetylation, formylation, amidation, phosphorylation, or PEGylation. Labeling modifications include fluorescent dyes (fluorescein, FITC, rhodamine, Texas Red, Cy3, Cy5), fluorescent proteins (e.g., PE, APC, GFP), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase), radioisotopes (e.g., 3 H, 14 C. 35 Examples of labeling include labeling with biotin or (strept)avidin.

[0062] In this step, the expressed labeled polypeptide is quantified by immunological measurement from the cell-free protein expression system solution obtained after the expression step or the protein extract obtained after the protein extraction step.

[0063] An "immunological assay method" is a method in which a target molecule is used as an antigen and an antibody that specifically binds to the antigen is used to form an immune complex with the target molecule, thereby detecting and quantifying the target molecule. As described above, in the immunological evaluation method of the present invention, the labeled peptide contained in the labeled polypeptide corresponds to the target molecule. Therefore, the labeled polypeptide is detected and quantified using an anti-labeled peptide antibody.

[0064] Immunological assay methods include, for example, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), radioimmunoassay (RIA), immunoturbidimetry, latex agglutination immunoassay, latex turbidimetry, particle agglutination reaction, gold colloid method, capillary electrophoresis, Western blotting, and immunohistochemistry (immunostaining). All of these methods are well known and may, in principle, be carried out in accordance with conventional methods in the relevant field. For example, Current protocols in Protein Sciences, 1995, John Wiley & Sons Inc.; Current protocols in Immunology, 2001, John Wiley & Sons Inc.; Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; "Clinical Pathology Special Issue No. 53, Immunoassays for Clinical Testing - Technology and Applications -" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Association, 1983; "Enzyme Immunoassays" edited by Ishikawa Eiji et al., 3rd Edition, Igaku-Shoin, 1987; "Protein, Nucleic Acid, Enzyme Special Issue No. 31 Enzyme Immunoassays" edited by Kitagawa Tsunehiro et al., Kyoritsu Shuppan, 1987; "Radioimmunoassays" edited by Irie Minoru, Kodansha Scientific, 1974; "Radioimmunoassays Continued" edited by Irie Minoru, Kodansha Scientific, Reference can be made to the methods described in, for example, Nagata Kazuhiro and Handa Hiroshi (eds.), Experimental Methods for Real-Time Analysis of Interactions between Biological Materials, Springer-Verlag Tokyo, 1988; Moriizumi Toyoe and Nakamoto Takamichi, Sensor Engineering, Shokodo, 1997.

[0065] "Enzyme-linked immunosorbent assay" is a method in which a primary antibody bound to a target molecule is detected via a modified, labeled secondary antibody or the like, and the target molecule is quantified based on the color density or fluorescence intensity generated by the modified label. For example, an anti-labeled peptide antibody of a primary antibody bound to a labeled peptide is captured with a labeled secondary antibody that binds to the primary antibody, and the labeled polypeptide containing the labeled peptide is indirectly measured based on the signal intensity from the label. ELISA and sandwich ELISA fall under this category.

[0066] The "Surface Plasmon Resonance (SPR) method" is a highly sensitive method for detecting and quantifying adsorbates on a metal thin film surface, utilizing the phenomenon of surface plasmon resonance, whereby the intensity of reflected light significantly attenuates at a specific angle of incidence (resonance angle) when the angle of incidence of laser light irradiated onto the metal thin film is changed. In the present invention, for example, an anti-labeled peptide antibody is immobilized on the surface of the metal thin film, and other portions of the metal thin film surface are blocked. Then, a cell-free protein expression system solution or a protein extract is passed over the surface of the metal thin film, and the labeled polypeptide can be detected and quantified from the difference in measured values ​​before and after sample passage. Detection and quantification by surface plasmon resonance can be performed, for example, using an SPR sensor commercially available from Biacore.

[0067] The "quartz crystal microbalance (QCM) method" is a mass measurement method that utilizes the phenomenon in which the resonant frequency of a quartz crystal oscillator decreases in accordance with the mass of a substance adsorbed onto the surface of an electrode attached to the quartz crystal oscillator. This method quantitatively captures minute amounts of adsorbed substances based on the change in resonant frequency. Similar to the SPR method, detection and quantification using this method can also be performed using a commercially available QCM sensor to detect and quantify target molecules. In the present invention, for example, labeled polypeptides can be quantified by an antigen-antibody reaction between an anti-labeled peptide antibody immobilized on the electrode surface and a labeled peptide contained in the labeled polypeptide in a sample.

[0068] The anti-labeled peptide antibody used in this step is determined by the labeled peptide contained in the labeled polypeptide. For example, if the labeled peptide is a Flag tag, an anti-Flag antibody may be used as the anti-labeled peptide antibody. In general, if the labeled peptide is an epitope tag, specific antibodies against each epitope tag are commercially available from various life science manufacturers, and these can also be used.

[0069] Two or more different labeled polypeptides may each contain two or more types of partially identical label peptides. Three or more different labeled polypeptides may be used, with each labeled polypeptide having a different combination of label peptides, allowing quantification in various combinations. For example, when three or more different labeled mRNAs are expressed and all of the labeled polypeptides expressed according to the same criteria are quantified, quantification can be performed using an antibody against a label peptide common to all of the labeled polypeptides. On the other hand, when three or more different labeled mRNAs are expressed and two specific labeled polypeptides are expressed according to the same criteria, quantification can be performed using an antibody against a label peptide common to only those two labeled polypeptides.

[0070] 1-3-5.Analysis process The "analysis step" (S0350) is a step of analyzing the expression levels of target mRNAs contained in two or more different labeled mRNAs that encode each labeled polypeptide by comparing the respective quantitative values ​​of the two or more different labeled polypeptides obtained in the quantification step (S0340).

[0071] There are no particular limitations on the comparison of quantitative values ​​obtained from two or more different labeled polypeptides. As described above, the quantitative values ​​are measured and quantified using the same quantitative method and the same standard for each labeled polypeptide, so direct comparison is possible. Therefore, by comparing the quantitative values ​​of each labeled polypeptide, it is possible to determine whether they are larger or smaller.

[0072] The magnitude of the quantitative value of a labeled polypeptide reflects the expression level of the target mRNA contained in the labeled mRNA encoding that labeled polypeptide. Therefore, by comparing the quantitative values ​​between two or more different labeled polypeptides, it is possible to analyze the expression level of the target mRNA contained in each labeled mRNA and determine which target mRNA is expressed more abundantly. This makes it possible, for example, in screening mRNA vaccines, to select the target mRNA vaccine with the highest intracellular expression level from among multiple target mRNA vaccines consisting of different nucleotide sequences encoding the same amino acid sequence.

[0073] 2. Immunological evaluation method for mRNA expression level (2) 2-1. Overview The second aspect of the present invention is a method for immunologically evaluating mRNA expression levels, similar to the first aspect. However, in this aspect, the expression levels of two or more different target mRNAs are not comparatively evaluated, but rather the expression levels of the target mRNA are evaluated by comparison with a reference value. This method, similar to the first aspect, does not require the preparation of an antibody that specifically binds to the target mRNA, and allows accurate evaluation of the expression level of the target mRNA using a readily available antibody, without being affected by endogenous mRNA.

[0074] 2-2. Method The flow diagram of the immunological evaluation method of this embodiment, like the first embodiment, is shown in Figure 3. That is, the immunological evaluation method of this embodiment also includes an expression step (S0320) and a quantification step (S0340) as essential steps, and an introduction step (S0310), a protein extraction step (S0330), and an analysis step (S0350) as selection steps. These steps basically conform to the descriptions of the corresponding steps in the first embodiment. Therefore, descriptions of the same content as in the first embodiment will be omitted, and the method and configuration characteristic of this embodiment will be specifically described below.

[0075] 2-2-1.Introduction process The "introduction step" (S0310) is a step of introducing into cells labeled mRNA in which a labeled nucleotide encoding a labeled peptide is linked to the end of the target mRNA. This step may be basically the same as the introduction step (S0310) of the first embodiment, except that the labeled mRNA to be introduced may be of one type.

[0076] 2-2-2. Expression process The "expression step" (S0320) is a step of expressing labeled mRNA. This step may be basically the same as the expression step (S0320) of the first embodiment, except that the number of labeled mRNAs to be expressed may be one.

[0077] 2-2-3. Protein extraction process The "protein extraction step" (S0330) is a selection step for extracting a protein from cells after the expression step when the introduction step (S0310) is selected. This step may be the same as the protein extraction step (S0330) of the first embodiment.

[0078] 2-2-4.Quantitative process The "quantification step" (S0340) is a step of quantifying the labeled polypeptide expressed from the labeled mRNA in the expression step (S0320) using an antibody against the labeled peptide. This step may also be basically the same as the quantification step (S0340) of the first embodiment, except that the number of labeled mRNAs to be expressed may be one.

[0079] 2-2-5.Analysis process The "analysis step" (S0350) is a step of comparing the quantitative value of the labeled polypeptide obtained in the quantification step with a reference value and analyzing the expression level of the target mRNA from the results. This step is the most distinctive step that differs from the immunological evaluation method of the first embodiment. In the first embodiment, quantitative values ​​of two or more different labeled polypeptides were compared and analyzed, but in this step, the quantitative value is compared with a reference value, and the expression level of the target mRNA is analyzed based on the comparison result with the reference value, which is a major difference from the first embodiment.

[0080] The reference value is a value obtained by quantifying a labeled reference polypeptide. A labeled reference polypeptide is a polypeptide expressed from a labeled reference mRNA in which a labeled nucleotide is linked to a reference mRNA. If the reference mRNA and labeled nucleotide are not limited, a huge variety of labeled reference polypeptides can be included. Therefore, there can be many reference values. These reference values ​​can be obtained in advance and compiled into a database, and can be obtained as needed when carrying out this embodiment.

[0081] The reference value used in this step is the value obtained by quantifying a labeled reference polypeptide using "the same antibody as that used to quantify the labeled polypeptide" in the quantification step (S0340). That is, in this step, the reference value must be selected based on the labeled mRNA encoding the labeled polypeptide to be quantified in the quantification step. For example, the reference mRNA must be composed of a base sequence different from that of the target mRNA. Furthermore, the labeled nucleotide must encode the same labeled peptide as the labeled polypeptide. Furthermore, the measurement and quantification must be performed using the same standards as those for the labeled polypeptide. A reference value that satisfies these conditions may be selected and obtained, for example, from the database described above. The reference value used in this step may be 2 or more as long as the above conditions are met.

[0082] The comparison between the quantitative value and the reference value may be performed in accordance with the analysis step (S0350) of the first embodiment. As described above, the reference value is a value measured and quantified by the same method and with the same standards as the quantitative value, and therefore, the magnitude of the quantitative value relative to the reference value can be determined by direct comparison.

[0083] As in the first embodiment, the magnitude of the quantitation value of the labeled polypeptide reflects the expression level of the target mRNA contained in the labeled mRNA encoding that labeled polypeptide. The expression level of the target mRNA contained in the labeled mRNA can be analyzed from the results of comparison with the reference value.

[0084] In this step, the reference value can be used, for example, as a "cutoff value" for classifying quantitative values. For example, if the reference value is based on a reference mRNA that exhibits the minimum expression level required for functioning as an mRNA vaccine, and the quantitative value is lower than this reference value, the target mRNA can be determined to be unsuitable as an mRNA vaccine. Furthermore, for example, if overexpression of an mRNA vaccine can cause side effects in the human body, the efficacy and safety of the mRNA vaccine can be determined by using an upper reference value indicating the expression level and a lower reference value indicating the minimum expression level required for function, and confirming that the quantitative value is within the range between the lower and upper reference values.

[0085] In this step, the reference value can also be used to determine whether there is a statistically significant difference from the quantitative value. Specifically, for example, the function of the target mRNA can be evaluated based on whether the quantitative value is statistically significantly higher than the reference value.

[0086] As used herein, "statistically significant" refers to a case where the risk rate (significance level) of the obtained value is small, specifically, p<0.05 (less than 5%), p<0.01 (less than 1%), or p<0.001 (less than 0.1%). Here, "p (value)" indicates the probability that an assumption will be correct by chance in a hypothetical distribution of statistics in a statistical test. Therefore, the smaller the p value, the closer the assumption is to the truth. "Statistically significantly different" refers to a significant difference between the measured value of a subject and the measured value of a population when the difference between the two is statistically processed. The statistical processing test method is not particularly limited and may be any known test method capable of determining the presence or absence of significance. For example, the Student's t-test can be used. [Example]

[0087] (the purpose) To demonstrate that the method of the present invention captures the actual expression level of a protein, a comparative study is carried out for the same protein, EGFP, by measuring the fluorescence intensity and by immunostaining using an antibody against the labeled peptide.

[0088] (method) (1) Preparation of mRNA The two types of mRNA used in the examples were EGFP-GS-Flag mRNA synthesized using unmodified nucleic acids, and mEGFP-GS-Flag mRNA synthesized by substituting all U (uracil) in EGFP-GS-Flag mRNA with modified nucleic acid N1-methyl-psudo-U. Both consist of the nucleotide sequence shown in SEQ ID NO: 11, which is constructed by linking a Flag tag to the 3' end of EGFP shown in SEQ ID NO: 10 via a GS linker shown in SEQ ID NO: 12. Although SEQ ID NOs: 10 to 12 may be RNA sequences, they are shown as DNA sequences in the sequence listing.

[0089] A DNA template with a PolyA tail was prepared by tailed PCR using a plasmid containing the EGFP-GS-Flag mRNA sequence shown in SEQ ID NO: 11 as a template. The DNA template was subjected to agarose gel electrophoresis, and bands of the desired size were excised and purified using a Gel Extraction kit (QIAGEN). mRNA was synthesized by in vitro transcription using unmodified and modified nucleic acids. T7 RNA Polymerase Mix (NEB) was used for in vitro transcription. After DNase treatment, the fragment was purified using a Monarch RNA Cleanup Kit (NEB). After capping with Vaccinia Capping Enzyme (NEB) and mRNA Cap 2'-O-Methyltransferase (NEB), the fragment was purified again using a Monarch RNA Cleanup Kit (NEB). The mRNA concentration was measured and used for mRNA transfection.

[0090] (2) mRNA introduction HEK293 cells were seeded onto a 96-well plate at 20,000 cells per well and cultured overnight at 37°C under 5% CO. DMEM (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific) was used as the culture medium.

[0091] Each well contained 100 ng of each mRNA prepared in (1) and 0.2 μL of Lipofectamine MessengerMAX Reagent (Thermo Fisher Scientific) in 10 μL of Opti-MEM (Thermo Fisher Scientific). The mixture was added to the 96-well plate and transfected into HEK293 cells. Each mRNA was transfected into three wells (N=3). Three wells were also prepared for a negative control (no mRNA transfection). Each dish was then cultured at 37°C under 5% CO2 for 24 hours to allow expression of the transfected mRNA.

[0092] (3) Fluorescence measurement Using a portion of each of the three samples, the EGFP fluorescence intensity in the cells was measured using a cell counter NC3000 (M&S TechnoSystems).

[0093] (4) Immunostaining The expression levels of the introduced miRNA were measured using a portion of each of the three samples by immunostaining with anti-Flag antibody. After fixation with 4% PFA and blocking with 3% BSA, detection was performed with a 1 / 1000 diluted Flag antibody (Sigma). The secondary antibody used was a 1 / 2000 diluted fluorescently labeled anti-mouse IgG (H+L) antibody (Thermo Fisher Scientific).

[0094] (result) The results are shown in Figure 4. Panel A shows the measurement results of EGFP fluorescence intensity for each sample, and panel B shows the results of immunostaining using an anti-Flag antibody. A: Measurement of EGFP fluorescence intensity for each sample confirmed that when EGFP-GS-Flag mRNA synthesized using unlabeled nucleic acid was transfected into HEK293 cells (lane: EGFP-GS-Flag mRNA), EGFP protein was expressed compared to a mock control (lane: no mRNA). On the other hand, HEK293 cells transfected with mEGRP-GS-Flag mRNA synthesized using modified nucleic acid (lane: mEGFP-GS-Flag mRNA) showed significantly higher EGFP expression than EGFP-GS-Flag mRNA synthesized using unlabeled nucleic acid. B: The same sample was immunohistochemically stained using anti-Flag antibody to measure its fluorescence intensity, and the same results as in A were obtained.

[0095] These results demonstrate that the measurement of labeled polypeptides by the immunological evaluation method of the present invention reflects the expression level of the labeled polypeptide in cells. Therefore, it has been demonstrated that the method for immunological evaluation of mRNA expression level of the present invention enables accurate quantification and comparison of the expression levels of each labeled polypeptide using a single antibody that specifically recognizes a label peptide common to two or more different labeled polypeptides. [Prior art documents] [Patent documents]

[0096] [Patent Document 1] Special Publication 2020-502255

Claims

1. A method for immunologically evaluating mRNA expression level, an expression step of expressing labeled mRNAs in which labeled nucleotides encoding the same labeled peptide are linked to the ends of two or more different target mRNAs; a quantification step of quantifying the labeled polypeptides expressed from the respective labeled mRNAs in the expression step using the same antibody against the labeled peptides. The method comprising:

2. an analysis step of comparing the quantitative values ​​of each labeled polypeptide obtained in the quantification step and analyzing the expression level of each target mRNA from the results; The method of claim 1 further comprising:

3. The method of claim 1 or 2, wherein the labeled mRNA is artificially synthesized in vitro.

4. The method of claim 3 , wherein the tag peptide is an epitope tag.

5. A method for immunologically evaluating mRNA expression level, an expression step of expressing a labeled mRNA in which a labeled nucleotide encoding a labeled peptide is linked to the end of the target mRNA; a quantification step of quantifying the labeled polypeptide expressed from the labeled mRNA in the expression step using an antibody against the labeled peptide; and an analysis step of comparing the quantitative value of the labeled polypeptide obtained in the quantification step with a reference value and analyzing the expression level of the target mRNA from the results; Including, The reference value is a value obtained by quantifying, using the antibody, a labeled reference polypeptide expressed from a labeled reference mRNA in which the same labeled nucleotide is linked to a reference mRNA having a base sequence different from that of the target mRNA. The method.

6. The method of claim 5 , wherein the labeled mRNA and the labeled reference mRNA are artificially synthesized in vitro.

7. The method of claim 6 , wherein the tag peptide is an epitope tag.

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