Method for immunological evaluation of mRNA expression level

Abstract

The present invention relates to a method for immunological evaluation of mRNA expression levels. The problem addressed by the present invention is to develop a method capable of simply and accurately quantifying and analyzing a protein in immunology so as to be able to compare and evaluate the function and effect of two or more mRNAs having different sequences in the form of the expression level of a protein encoded by mRNA. Labeled mRNAs are prepared, in each labeled mRNA, a labeled nucleotide encoding the same labeled peptide is linked to the terminus of the mRNA. The respective expression levels of the labeled polypeptides expressed from the labeled mRNA are quantified using the same antibody against the labeled peptides.

Description

Technical Field

[0001] The present invention relates to methods for the immunological assessment of mRNA expression levels. Background Art

[0002] mRNA vaccines are introduced into an organism by injection and function through the expression of proteins encoded by the mRNA. The effectiveness of mRNA vaccines is evaluated based on the proteins expressed in the organism.

[0003] The main functions and effects of mRNA vaccines depend on the base sequence or the length of the base sequence that makes up the mRNA. For example, two different mRNA vaccines - their respective degenerate codons encode the same protein and express proteins with the same amino acid sequence. mRNA has different base sequences, so when the mRNA vaccine is introduced into an organism, it does not necessarily exhibit the same behavior. In specific examples, mRNA vaccines express different levels of proteins. The functions and effects of mRNA vaccines are relatively evaluated based on the expression level of the target protein expressed by the mRNA vaccine. Figure 1 As shown, immunoassays using antibodies that specifically bind to proteins are conventionally used to measure protein expression levels. Such assays have several problems.

[0004] The first problem is that measurement itself is not feasible without access to highly specific antibodies. Commercially available antibodies are available for some target proteins, but many of these antibodies lack specificity and binding capacity, leading to significant time and cost associated with selecting an antibody suitable for measurement. In cases where obtaining antibodies is difficult, for example, because they are not commercially available, in-house antibody production is unavoidable, requiring significant additional time and labor.

[0005] A second issue is that protein detection accuracy decreases when the target protein is endogenously present in cells. Even with highly specific antibodies, if the target protein derived from the introduced mRNA is identical to the endogenous target protein, the antibodies cannot distinguish between the two proteins. Consequently, there is the problem of accurately measuring the expression level of the target protein derived from the introduced mRNA.

[0006] The third problem is the low versatility of the assay. Measuring and comparing the expression levels of multiple target proteins requires providing antibodies with high specificity for each target protein. Due to the first problem, it is difficult to measure proteins other than the target protein for which antibodies with high specificity are readily available. Another problem is that since the binding ability of antibodies to proteins is not necessarily the same between antibodies, it is not feasible to simply compare the expression levels of the target proteins.

[0007] Patent Document 1 discloses a plurality of compositions for quantitatively analyzing multiple proteins, the compositions comprising a protein-binding reagent bound to an oligonucleotide comprising a unique identifier. 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, for example, the following: a method and a kit for simultaneously and quantitatively analyzing protein targets and nucleic acid targets in a sample; and a system for preparing labeled biomolecule reagents. This method can solve the first and second problems. The binding capacity between each protein-binding reagent and the protein to which the reagent specifically binds is different, and therefore, solving the third problem remains unfeasible because the measured expression levels cannot be simply compared. Summary of the Invention

[0008] The present invention aims to develop and provide a highly versatile method that can evaluate the functions and effects of multiple target mRNAs composed of different base sequences based on the expression level of each target protein expressed from the target mRNA, thereby immunologically performing simple and accurate quantitative analysis of target proteins.

[0009] In order to solve the above problems, the present inventors have actively conducted research and have therefore developed a method for Figure 2 The method for fusion mRNA described herein comprises multiple target mRNAs having different base sequences and labeled nucleotides attached to their ends, which collectively encode the same labeled peptide. This method enables simple and accurate quantitative analysis of all target proteins using a common antibody that binds to the labeled peptide after expressing the fusion mRNA. Therefore, this method has extremely high versatility. The present invention is based on the results of the development and provides the following (1) and (2).

[0010] (1) A method for immunological evaluation of mRNA expression levels, comprising: an expression step of allowing expression of labeled mRNA, wherein the labeled mRNA consists of two or more different target mRNAs; and labeled nucleotides encoding the same labeled peptide, wherein the labeled nucleotides are linked to the end of each target RNA; and a quantification step of quantifying the labeled polypeptide expressed from each labeled mRNA in the expression step using the same antibody against the labeled peptide.

[0011] (2) A method for immunological evaluation of mRNA expression levels, comprising: an expression step of allowing a labeled mRNA to be expressed, the labeled mRNA consisting of a target mRNA and a labeled nucleotide encoding a labeled peptide, the labeled nucleotide being 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 based on the comparison result, wherein the reference value is a value obtained by quantifying a labeled reference polypeptide expressed from a labeled reference mRNA using an antibody, the labeled reference mRNA comprising a reference mRNA consisting of a base sequence different from that of the target mRNA and the same labeled nucleotide linked to the reference mRNA.

[0012] The method for immunological evaluation according to the present invention enables quantitative and comparative analysis of the expression levels of proteins expressed from multiple mRNAs composed of different base sequences using a common antibody. Therefore, the function and effect of each mRNA can be immunologically evaluated simply and accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a conceptual diagram of a general method used in immunology to measure mRNA expression levels.

[0014] Figure 2 is a conceptual diagram of a method for immunologically measuring mRNA expression levels according to the present invention;

[0015] Figure 3 is a flow chart of a method for immunological evaluation according to the present invention; and

[0016] Figure 4 : is a graph showing the results of Examples in the present invention, which shows the measurement results of the fluorescence intensity of EGFP ( Figure 4 A) and the measurement results by immunostaining assay using anti-Flag antibody ( Figure 4 B) wherein two measurements of the expression level of EGFP in HEK293 cells were performed after EGFP mRNA or EGFP-Flag mRNA was introduced into HEK293 cells, and wherein HEK293 cells into which mRNA was not introduced were used as a control. DETAILED DESCRIPTION

[0017] 1. Methods for immunological evaluation of mRNA expression levels (1)

[0018] 1-1. Overview

[0019] The first aspect of the present invention is a method for the immunological evaluation of mRNA expression levels. In the present invention, in order to evaluate each expression level of two or more target mRNAs consisting of different base sequences (the same labeled nucleotide is connected to its end to prepare labeled mRNA), and the same antibody for the labeled peptide is used to quantitatively evaluate the expression level of the labeled polypeptide expressed from the labeled mRNA. This method enables the use of easily available antibodies to accurately evaluate the expression level of target mRNA without the need to prepare antibodies that specifically bind to each target mRNA, and is not affected by endogenous mRNA.

[0020] 1-2. Definition of terms

[0021] The terms used herein are defined below.

[0022] As used herein, "mRNA expression level" or "expression level of an mRNA" refers to the protein expression activity of an mRNA. The expression level is expressed as the level of protein expressed from a predetermined mRNA introduced into cells. The protein level herein can be a relative value, such as a fluorescence intensity value, or an absolute value, such as a mass value.

[0023] As used herein, "immunological evaluation of an mRNA expression level" refers to measuring and evaluating the expression level of a predetermined mRNA (in the form of the expression level of a protein expressed from the mRNA) using an immunological assay utilizing an antibody.

[0024] As used herein, "target mRNA" refers to the mRNA that is the target for measurement in the method according to the present invention. The polypeptide encoded by the target mRNA is referred to herein as the "target polypeptide."

[0025] As used herein, "labeling peptide" refers to a tag peptide consisting of a known amino acid sequence of 7 to 40. The labeling peptide can be linked to the end of the amino acid sequence of another polypeptide to label the polypeptide. The labeling peptide herein is preferably an epitope tag.

[0026] As used herein, "epitope tag" refers to a tag peptide that includes at least one epitope in its amino acid sequence. Specific examples of epitope tags include, but are not limited to, a Flag (registered trademark) tag represented by SEQ ID NO: 1 (DYKDDDDK); an HA tag represented by SEQ ID NO: 2 (YPYDVPDYA); a 6×His tag represented by SEQ ID NO: 3 (HHHHHHH); a Myc tag represented by SEQ ID NO: 4 (EQKLISEEDL); a V5 tag represented by SEQ ID NO: 5 (GKPIPNPLLGLLDST); a T7 tag represented by SEQ ID NO: 6 (MASMTGGQQMG); an S tag represented by SEQ ID NO: 7 (KETAAAKFERQHMDS); an E tag represented by SEQ ID NO: 8 (GAPVPYPDPLEPR); and a Glu-Glu tag represented by SEQ ID NO: 9 (EEEEYMPME). For epitope tags, there are generally known antibodies (tag antibodies) that specifically recognize the epitope included in the epitope tag. Even if an antibody that specifically recognizes the labeled polypeptide is not prepared, the labeled polypeptide can be immunologically detected and quantified using a tag antibody against the labeled peptide.

[0027] As used herein, "labeling nucleotide" refers to a nucleotide that encodes a labeled peptide. The labeling nucleotide is attached to the end of the base sequence of the target mRNA, and the open reading frames match each other. The end to which the labeling nucleotide is attached can be one or both of the 5' end and the 3' end. The end is preferably the 3' end.

[0028] As used herein, "labeled mRNA" is a target mRNA having a labeled nucleotide connected thereto, and encodes a labeled polypeptide described below. The mRNA labeled herein may include a plurality of labeled mRNAs (commonly referred to herein as "two or more differently labeled mRNAs"), wherein the plurality of labeled mRNAs include two or more target mRNAs, which consist of different base sequences, each end of which is connected to the same labeled nucleotide. Here, "two or more target mRNAs consisting of different base sequences" include not only mRNAs that encode different polypeptides from each other, but also target mRNAs that encode the same polypeptide but have different base sequences depending on the differences in the codons encoding the polypeptide or the presence or absence of base modifications. A labeled mRNA may include two or more different labeled nucleotides. In this case, the labeled nucleotides may be connected to different ends of the target mRNA, or may be connected in series to the same end. In the case where two or more differently labeled mRNAs each include two or more different labeled nucleotides, as long as at least one labeled nucleotide is common, the type of labeled nucleotide included in each labeled mRNA is not particularly limited. The number of labeled nucleotides included in each labeled mRNA and the number of bases of the labeled nucleotides are desirably the same or approximately the same. The labeled mRNA can be designed to insert a linker between the labeled nucleotide and the target mRNA. The linker is not particularly limited and is preferably a linker that does not impair the three-dimensional structure or function of the target polypeptide. The linker is, for example, but not limited to, a GS linker.

[0029] As used herein, "labeled polypeptide" refers to a polypeptide encoded by a labeled mRNA and is a target for measurement in the method for immunological evaluation according to the present invention. Labeled polypeptides expressed from two or more differently labeled mRNAs are generally referred to herein as "two or more differently labeled polypeptides." In some cases, the two or more differently labeled polypeptides may consist of the same amino acid sequence.

[0030] As used herein, "reference mRNA" refers to an mRNA consisting of a base sequence different from that of the target mRNA, and is used as a comparison standard, the target mRNA expression level of which is quantified in the method for immunological evaluation according to the present invention. The base sequence of the reference mRNA is preferably, but not limited to, a sequence similar to the base sequence of the target mRNA, and, for example, has 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more base identity with the base sequence of the target mRNA. The base length of the reference mRNA is preferably, but not limited to, 1 to 20 bases identical or different from the base sequence of the target mRNA.

[0031] As used herein, a "labeled reference mRNA" is a reference mRNA having a labeled nucleotide attached thereto and encoding a labeled reference polypeptide described below. The labeled nucleotide attached to the labeled reference mRNA is the same labeled nucleotide attached to the labeled mRNA.

[0032] As used herein, a "labeled reference polypeptide" refers to a polypeptide encoded by a labeled reference mRNA.

[0033] As used herein, a "quantitative value" refers to the level of a labeled polypeptide expressed from labeled mRNA. The quantitative value is a value quantified using an antibody against a labeled peptide contained in the labeled polypeptide. The 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 the sample.

[0034] As used herein, a "reference value" refers to a value obtained by quantifying a labeled reference polypeptide expressed from a labeled reference mRNA. This value may be a relative value or an absolute value in the same manner as the above-mentioned quantitative value.

[0035] 1-3. Methods

[0036] Figure 3 The flow chart of the method for immunological evaluation according to the present invention is shown in FIG. The method for immunological evaluation according to the present invention 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 optional steps. The steps are as follows.

[0037] 1-3-1. Introduction steps

[0038] The "introduction step" (S0310) is a step of introducing each of two or more different labeled mRNAs into cells. This step is selected when the labeled mRNA is to be expressed in the cell.

[0039] The preparation of the mRNA of the mark used in this step is not particularly limited. For example, using gene recombination technology, the mRNA of the mark in which the mark nucleotide is connected to the target mRNA can be prepared. In this case, for example, the target DNA sequence corresponding to the target mRNA may be inserted into an expression vector that contains the mark nucleotide in advance (preliminarily), and then expressed in a host cell to produce the mRNA of the mark interested. The whole base sequence of the mRNA of the design mark, and then artificially synthesizing the DNA of the mark composed of the base sequence of the DNA being converted into in vitro is also possible. In this case, the DNA of the mark obtained is inserted into the expression vector, and then expressed in a host cell in the same manner as above, thereby making it possible to obtain the mRNA of the mark interested. The mRNA of the mark can be directly artificially synthesized in vitro.

[0040] 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 utilized. 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 asiatica. Examples of cultured insect cells include Sf9 cells, Sf9 plus cells, Sf21 cells, High Five cells and Ea4 cells. Examples of cultured animal cells include primary culture cells derived from differentiated cells (such as somatic cells), undifferentiated stem cells and cell lines established. Examples of stem cells include embryonic stem cells, mesenchymal stem cells and induced pluripotent stem cells. Examples of cell lines established 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 cultivated plant cells include BY-2 cells. These cells can be any cells collected or cultivated from an organism, or genetically modified cells.

[0041] The method for introducing the mRNA cell of labeling is not particularly limited. The method can be based on transformation methods known in the art or transfection methods, considering the kind of cell to be introduced and the kind of the mRNA of the labeling that allows to express. The example of available such method includes electroporation method, heat shock method, protoplast method, liposome infection method, PEG (polyethylene glycol) method, calcium phosphate method, DEAE-dextran method, protoplast method, particle gun method, Agrobacterium method and virus infection method. For transformation method or transfection method, reference can be made to, for example, Green & Sambrook, Molecular Cloning: A Laboratory Manual fourth edition, 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, describing gene transfer method.

[0042] 1-3-2. Expression steps

[0043] The "expression step" (S0320) is a step of expressing a labeled polypeptide from two or more differently labeled mRNAs. This step can be performed in vivo in cells having the labeled mRNA introduced therein, or can be performed in vitro using a cell-free protein expression system. It is preferred that each labeled mRNA be expressed under the same conditions. For example, in the case of in vivo expression, all labeled mRNAs are expressed using the same type of cells and cultured under the same conditions.

[0044] In the case of in vivo expression, this step can generally be achieved by culturing cells having the labeled mRNA introduced therein. In principle, the expression of the labeled mRNA in this step is transient.

[0045] In the method for culturing cells, cells are seeded in a culture medium and then cultured under predetermined culture conditions. The culture medium used in this cultivation and the predetermined culture conditions can be according to culture methods known in the art, depending on the type of cell. In principle, it is desired to cultivate under complete (whole) aseptic conditions.

[0046] For example, in the case of using Escherichia coli, Escherichia coli having the labeled mRNA introduced therein can be inoculated in a known culture medium (such as LB medium or ND medium), and then subjected to permeation culture at 37°C.

[0047] In the case of using yeast, the yeast having the labeled mRNA introduced therein can be inoculated in a known medium (such as YEP medium, YPD medium, YNB medium, or SD medium), and then subjected to permeation culture at 30°C.

[0048] When insect cells are used, a known culture medium (such as Sf900 TM II, TC-100, or SFM-PB) is optionally supplemented with serum, and then the insect cells having the labeled mRNA introduced therein are inoculated in the culture medium, followed by culturing at 27°C.

[0049] In the case of using cultured animal cells such as those from mammals, the animal cells with the labeled mRNA introduced therein can be inoculated into a standard cell culture medium optionally supplemented with serum and then cultured at 37° C., 5% CO 2 . “Standard cell culture medium” as used herein refers to a highly versatile basic culture medium for culturing various cells mainly derived from mammals. Specific examples include Eagle MEM (Eagle Minimum Essential Medium), DMEM (Dulbecco’s Modified Eagle Medium), Ham’s F10 (Ham’s Nutrient Mixture F10) culture medium, Ham’s F12 (Ham’s Nutrient Mixture F12) culture medium, M199 culture medium, high performance culture medium 199, RPMI-1640 (Roswell Park Memorial Institute-1640) culture medium, and DMEM / F12 (Dulbecco’s Modified Eagle Medium / Ham’s Nutrient Mixture F12) culture medium. In the case of DMEM / F12 culture medium, the mixing ratio is not particularly limited. It is preferred that DMEM and F12 be mixed in a weight concentration ratio within a range of 6:4 to 4:6 as the components.

[0050] The specific composition of each of the above-mentioned culture media is known in the art. Culture media can be prepared based on the compositions described in appropriate documents (e.g., Kaech S. and Banker G., Nat. Protoc., 2006, 1(5): 2406-2415, in the case of standard cell culture media). Commercially available culture media from life science manufacturers (such as Thermo Fisher Scientific Inc. or FUJIFILM Wako Pure Chemical Industries Corporation) can be used.

[0051] The culture period varies depending on the type of cells to be cultured and may be a period of time during which the labeled mRNA introduced into the cells is expressed. The culture period is generally 12 to 72 hours, preferably 24 to 48 hours.

[0052] In the case of in vitro expression, as described above, a cell-free protein expression system is used to express the labeled polypeptide from each labeled mRNA. A "cell-free protein expression system" refers to a system that utilizes the biomolecule translation machinery included in the cell extract to express proteins from a gene of interest in a cell extract extracted from a cell. The original cells of the cell extract used in the cell-free protein expression system can be cells known in the art. For example, but not limited to, rabbit reticulocytes, wheat germ or Escherichia coli are appropriately used. Expression of labeled mRNA by a cell-free protein expression system can be carried out according to methods known in the art.

[0053] 1-3-3. Protein extraction steps

[0054] When the introduction step (S0310) is selected, the "protein extraction step" (S0330) is an optional step for extracting protein from cells after the expression step. This step is intended to extract the labeled polypeptide expressed from the labeled mRNA in the expression step. When the introduction step is selected, this step is preferably selected together, but is not limited.

[0055] In methods for extracting proteins from cells, the cells are typically lysed and the protein is then extracted from the cell lysate. Methods for lysing cells can be performed using the best known method appropriately selected according to the type of cell and the location of the labeled polypeptide of interest (in the cytoplasm, cell wall, or culture supernatant).

[0056] When the cells used in the introduction step are bacteria (such as Escherichia coli, insect cells, or cultured animal cells), suitable methods for lysing the cells include, but are not limited to, osmotic shock, freeze-thaw, surfactant extraction, or a combination of these methods. When cells have a cell wall (such as yeast or plant cells), suitable methods for lysing the cells include, but are not limited to, enzymatic digestion, ultrasonication, French press, homogenizer, glass bead method, or a combination of these methods.

[0057] Osmotic shock is a method of suspending cells in a hypotonic solution (such as sterile water) and using an osmotic pressure difference to destroy the cell membrane to lyse the cells. The freeze-thaw method is a method of rapidly cooling a cell suspension with liquid nitrogen and then thawing to destroy the cell membrane, thereby lysing the cells. The surfactant extraction method promotes the cleavage of hydrophobic proteins by adding a surfactant, thereby lysing the cells so that the protein can be cleaved, and it is the most common and most suitable method as a protein extraction method. The suitable surfactant to be used is a non-ionic or amphoteric surfactant, but is not limited to this. The enzymatic digestion method is a method of treating cells with an enzyme (lysozyme, cellulase or pectinase) specific to each cell, whereby the cell wall that is difficult to lyse is destroyed, thereby causing the cells to be lysed. The ultrasonic disruption method is a method of lysing cells by ultrasonically disrupting the cell membrane and cell wall. The French filter press method is a method of forcing a cell suspension through a hole under high pressure to break the cells under shear force. The homogenizer method is a method of mechanically homogenizing cells together with tissue or in a cell suspension to break them. The glass bead method is a method in which the cell wall or cell membrane is physically broken by collision or friction with glass beads.

[0058] Any of the above methods is known in the art, and based on the method, a more specific method can be performed.

[0059] In the present invention, the labeled polypeptide to be quantified is localized in the cytoplasm or culture supernatant, but is not limited thereto. Therefore, centrifuging the cell lysate prepared as described above and collecting the supernatant makes it possible to obtain a protein extract containing the labeled polypeptide of interest expressed in the cells.

[0060] 1-3-4. Quantification steps

[0061] "Quantitative step" (S0340) is a step in which an antibody against a common marker peptide of the labeled polypeptides is used to measure and quantify each of the expressed labeled polypeptides from the two or more differently labeled mRNAs in the expression step (S0320). This step is a necessary step that is highly characteristic and significant in the method for immunological evaluation according to the present invention. After this step, a quantitative value for each labeled polypeptide is obtained. This quantitative value can be a relative value or an absolute value as described above, but is preferably a value obtained by measurement and quantification based on the same reference according to the same quantitative method in the labeled polypeptide.

[0062] In the present invention, each labeled mRNA encoding two or more differently labeled polypeptides differs from one another in the base sequence of the target mRNA region contained in the labeled mRNA, but shares at least one of the labeled nucleotides attached to the termini of the target mRNA. Therefore, the labeled polypeptides expressed in each labeled mRNA during the expression step comprise the same labeled peptide. For quantification in this step, an antibody that specifically recognizes and binds to the labeled peptide, i.e., an anti-labeled peptide antibody, is used.

[0063] As used herein, "antibody" refers to a polypeptide comprising a framework region (FR) and a complementary determining region (CDR) both derived from an immunoglobulin and having the function of specifically binding to an antigen. As used herein, "antibody" includes not only full-length antibodies but also functional fragments (polypeptide fragments) having antigen-binding ability. Antibodies may be natural antibodies or artificial antibodies.

[0064] As used herein, "natural antibody" refers to an antibody produced in a living vertebrate or an antibody composed of the same amino acid sequence as the previous antibody and produced by an artificially produced antibody-producing cell (e.g., a hybridoma cell). Examples of natural antibodies include polyclonal antibodies and monoclonal antibodies. In terms of the specificity of the antibody for the labeled peptide, monoclonal antibodies are preferred, but are not limited thereto.

[0065] Polyclonal antibodies are a group of multiple immunoglobulins that recognize and bind to different epitopes of the same antigen. Polyclonal antibodies can be obtained from the serum of animals after immunizing them against a target molecule, which serves as an antigen.

[0066] A "monoclonal antibody" refers to a single clonal population of immunoglobulins. The immunoglobulins that make up a monoclonal antibody share common framework and complementarity-determining regions and are capable of recognizing and binding to the same epitope on the same antigen. Monoclonal antibodies can be obtained from hybridomas derived from a single cell.

[0067] In the case where the antibody is a polyclonal antibody or a monoclonal antibody, the immunoglobulin molecule can be of any class (e.g., any of IgG, IgA, IgE, IgM, IgD, and IgY). The antibody may comprise a single-chain antibody, such as a VHH antibody, which includes the VH region of the H chain and does not have an L chain, such as a single-chain antibody produced by camelids.

[0068] As used herein, an "artificial antibody" is an artificially constructed antibody. Examples include antibodies produced by introducing appropriate mutations into the amino acid sequence of the above-mentioned natural antibodies; and, in addition, single-chain antibodies that, in principle, do not exist in nature and are produced through structural modification. Specific examples of the latter artificial antibodies include recombinant antibodies. "Recombinant antibody" refers to a chimeric antibody, a humanized antibody, a synthetic antibody, or an antibody fragment.

[0069] A "chimeric antibody" is an antibody produced by combining the amino acid sequences of antibodies from different animals, and is obtained by replacing the variable region (V region) of an antibody with the variable region (V region) of another antibody. For example, an antibody corresponding to a chimeric antibody is an antibody in which the variable region (V region) is derived from a mouse and the C region is derived from a human, which is obtained by replacing the V region of a mouse monoclonal antibody with the V region of a human antibody.

[0070] A "humanized antibody" is a graft antibody obtained by replacing the complementary determining regions (CDRs; CDR1, CDR2, and CDR3) in the variable region (V region) of an antibody of a mammal other than a human (e.g., a suitable mouse) with the CDRs of a human monoclonal antibody. In chimeric and humanized antibodies, the V regions of the heavy and light chains are derived from antibodies of non-human animals (e.g., mice), or the complementary determining regions in the V regions of the heavy and light chains are derived from antibodies of non-human animals (e.g., mice). The framework regions (FRs; FR1, FR2, FR3, and FR4) in the C region or V region are derived from human antibodies, and the C regions of the heavy and light chains are derived from human antibodies. Therefore, the immune response to the antibody can be reduced in the human body.

[0071] "Synthetic antibody" refers to an antibody synthesized by chemical methods or recombinant DNA methods. Examples include monomeric polypeptide molecules obtained by artificially connecting one or more VL and one or more VH of a specific antibody by a connecting peptide consisting of an appropriate length and an appropriate sequence, and multimeric polypeptides of monomeric polypeptides. Specific examples of such polypeptides include single-chain Fv (scFv: a single-chain fragment of the variable region), diabodies, triabodies and tetrabodies. These synthetic antibodies are divalent to tetravalent antibody fragments with dimer to tetramer structures, respectively, based on the single-chain Fv structure. Diabodies and more multi-antibodies can be multispecific antibodies. "Multispecific antibody" refers to a multivalent antibody, that is, an antibody with multiple antigen binding sites in one molecule, in which each antigen binding site binds to a different epitope.

[0072] Examples of "antibody fragments" include Fab, F(ab')2 and Fv.

[0073] The antibody used in this step may be a modified antibody (modified labeled antibody). Specifically, in the case described below, where the anti-labeled peptide antibody is identified as a primary antibody, and a secondary antibody that specifically binds to the primary antibody is used for quantification of the labeled polypeptide in this step, the secondary antibody is preferably a modified antibody.

[0074] Modification of antibodies includes functional modification or labeling modification. Examples of functional modifications include glycosylation, acetylation, formylation, amidation, phosphorylation and pegylation. Examples of labeling modifications include fluorescent dyes (fluorescein, FITC, rhodamine, Texas red, Cy3 or Cy5), fluorescent proteins (e.g., PE, APC or GFP), enzymes (e.g., horseradish peroxidase, alkaline phosphatase or glucose oxidase), radioisotopes (e.g., 3 H. 14 C or 35 S), biotin or (streptavidin) avidin for labeling.

[0075] In this step, the expressed labeled polypeptide is quantified using immunoassay from the cell-free protein expression system solution obtained after the expression step or from the protein extract obtained after the protein extraction step.

[0076] An "immunological assay" is a method in which a target molecule is an antigen; an antibody that specifically binds to the target molecule is used to form an immune complex with the target molecule; and the target molecule is detected and quantified. In the immunological evaluation method according to the present invention, the labeled peptide included in the labeled polypeptide corresponds to the target molecule as described above. Therefore, the labeled polypeptide is detected and quantified using an anti-labeled peptide antibody.

[0077] Examples of immunological assays include enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, surface plasmon resonance assay (SPR method), quartz crystal microbalance (QCM) assay, radioimmunoassay (RIA), immunoturbidimetric assay (immunonephelometric assay), latex agglutination immunoassay, latex turbidimetric assay, particle aggregation assay, colloidal gold assay, capillary electrophoresis assay, protein immunoblotting assay, and immunohistochemical analysis (immunostaining) assay. Any of these assays is a known assay and, in principle, can be performed according to conventional methods in the art.For example, the methods described in the following can be referred to: Current protocols in Protein Sciences, 1995, John Wiley&Sons Inc.; Currentprotocols in Immunology, 2001, John Wiley&Sons Inc.; Green&Sambrook, Molecular Cloning, fourth edition, 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; JapanSociety of Clinical Pathology, “Immunoassayfor Clinical Test—Technology and Application”, TheJapanese Journal ofClinical Pathology, Special Issue, No. 53, 1983, The Clinical Pathology Press; EijiIshikawa et al., Eds., Enzyme Immunoassay, third edition, 1987, Igaku-Shoin; TsunehiroKitagawa et al., Eds., “Enzyme Immunoassay”, Proteins, Nucleic Acids, andEnzymes, Supp. No. 31, 1987, Kyoritsu Shuppan; Irie Minoru Ed., Radioimmunoassay, 1974, Kodansha Scientific; IrieMinoru Ed., A Sequel to Radioimmunoassay, 1979, Kodansha Scientific; Kazuhiro Nagata andHiroshi Handa, Eds., Real-TimeAnalytical and Experimental Method of Biomolecular Interactions, 1988, Springer-Veriag, Tokyo; and Toyosaka Moriizumi and Takamichi Nakamoto, SensorEngineering, 1997, Shokodo.

[0078] An enzyme immunoassay (ELISA) is a method in which a primary antibody bound to a target molecule is detected using a modified, labeled secondary antibody, and the target molecule is quantified based on the color optical density and fluorescence intensity produced by the modified label. Examples of this assay include methods in which an anti-labeled peptide antibody, which is a primary antibody bound to the labeled peptide, is captured by a labeled secondary antibody bound 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 are examples of this method.

[0079] A "surface plasmon resonance (SPR) assay" is a method for highly sensitively detecting and quantifying substances adsorbed on the surface of a thin metal film by utilizing the surface plasmon resonance phenomenon—in which the incident angle of a laser beam irradiated onto the thin metal film is changed so that the intensity of the reflected light is significantly attenuated at a specific incident angle (resonance angle). In the present invention, for example, an anti-labeled peptide antibody is solid-phased on the surface of the thin metal film; the rest of the thin metal film surface is blocked; then, a cell-free protein expression system solution or protein extract is run on the thin metal film surface; and based on the difference between the values ​​measured before and after the sample run, the labeled polypeptide can be detected and quantified. For example, detection and quantification by surface plasmon resonance assay can be performed using an SPR sensor commercially available from Biacore AB.

[0080] "Quartz crystal microbalance (QCM) assay" is a mass measurement method in which, when a substance is adsorbed on the surface of an electrode attached to a quartz crystal resonator, the resonant frequency of the quartz crystal resonator decreases according to the mass of the substance; and utilizing this phenomenon, a trace amount of adsorbate is quantitatively grasped according to the level of change in the resonant frequency. In the detection and quantification of this method, a commercially available QCM sensor can be used to detect and quantify the target molecule in the same manner as in the SPR assay. In the present invention, for example, the labeled polypeptide can be quantified based on the antigen-antibody reaction between an anti-labeled peptide antibody immobilized on the electrode surface and a labeled peptide included in the labeled polypeptide in the sample.

[0081] The anti-tag peptide antibody to be used in this step is determined based on the tag peptide included in the tagged polypeptide. For example, when the tag peptide is a Flag tag, the anti-tag peptide antibody to be used may be an anti-Flag antibody. Generally, when the tag peptide is an epitope tag, specific antibodies against various epitope tags are commercially available from various life science manufacturers and can be used.

[0082] Two or more different labeled polypeptides may each include two or more marker peptides that are partially identical. In the case of three or more different labeled polypeptides, each labeled polypeptide may have a different combination of marker peptides, making it possible to quantify labeled polypeptides in different combinations. For example, in one case, after three or more different labeled mRNAs are expressed, all labeled polypeptides expressed can be quantified based on the same reference using antibodies against a marker peptide common to all labeled polypeptides. After three or more different labeled mRNAs are expressed, specific two labeled polypeptides in the expressed labeled polypeptides can be quantified based on the same reference using antibodies against a marker peptide common to two labeled polypeptides.

[0083] 1-3-5. Analysis steps

[0084] The "analyzing step" (S0350) is a step of comparing each quantitative value of two or more different labeled polypeptides obtained in the quantification step (S0340) to analyze the expression level of the target mRNA included in each of the two or more different labeled mRNAs encoding each labeled polypeptide.

[0085] Comparison of quantitative values ​​obtained from two or more different labeled polypeptides is not particularly limited. As described above, these quantitative values ​​are measured and quantified using the same quantitative method and based on the same reference between the labeled polypeptides, and thus can be directly compared. Therefore, by comparing the quantitative values ​​of the labeled polypeptides, the magnitude of each value can be determined.

[0086] The magnitude of the quantitative value of the labeled polypeptide reflects the expression level of the target mRNA contained in the labeled mRNA encoding the labeled polypeptide. Therefore, the comparison results of the quantitative values ​​between two or more different labeled polypeptides are used to analyze the magnitude of the expression level of the target mRNA contained in each labeled mRNA, thereby being able to determine which target mRNA provides a high expression level. This makes it possible, for example, to select a target mRNA vaccine that can achieve the highest expression level in cells from a plurality of target mRNA vaccines consisting of different base sequences encoding the same amino acid sequence when screening mRNA vaccines.

[0087] 2. Methods for immunological evaluation of mRNA expression levels (2)

[0088] 2-1. Overview

[0089] The second aspect of the present invention is a method for immunological evaluation of mRNA expression levels in the same manner as the first aspect. In this aspect, the expression levels of two or more different target mRNAs are not compared and evaluated, but the expression level of the target mRNA is evaluated by comparison with a reference value. This method makes it possible to accurately evaluate the expression level of the target mRNA using readily available antibodies in the same manner as the first aspect even if a single target mRNA is used, without the need to prepare antibodies that specifically bind to each target mRNA, and without the influence of endogenous mRNA.

[0090] 2-2. Methods

[0091] Figure 3 The flow chart of the method for immunological evaluation according to the present aspect is shown in the same manner as the first aspect. That is, the method for immunological evaluation according to the present aspect also includes an expression step (S0320) and a quantitative step (S0340) as necessary steps, and an introduction step (S0310), a protein extraction step (S0330) and an analysis step (S0350) as optional steps. These steps are basically consistent with the details of the corresponding steps described in the first aspect. Therefore, the details identical with the first aspect are omitted. Here, the method and structural characteristics of the present aspect are specifically described below.

[0092] 2-2-1. Introduction steps

[0093] The "introduction step" (S0310) is a step of introducing a labeled mRNA in which a labeled nucleotide encoding a labeled peptide is linked to the end of the target mRNA into a cell. This step can be substantially the same as the introduction step (S0310) in the first aspect, except that the labeled mRNA to be introduced can be one.

[0094] 2-2-2. Expression steps

[0095] The "expression step" (S0320) is a step of allowing the labeled mRNA to be expressed. This step may also be substantially the same as the expression step (S0320) in the first aspect, except that the labeled mRNA to be expressed may be of one type.

[0096] 2-2-3. Protein extraction step

[0097] In the case where the introduction step (S0310) is selected, the "protein extraction step" (S0330) is an optional step of extracting protein from cells after the expression step. This step may be the same as the protein extraction step (S0330) in the first aspect.

[0098] 2-2-4. Quantification steps

[0099] 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 substantially the same as the quantification step (S0340) in the first aspect, except that the labeled mRNA to be expressed may be of a single type.

[0100] 2-2-5. Analysis steps

[0101] "Analysis step" (S0350) is a step of comparing the quantitative value of the labeled polypeptide obtained in the quantitative step with a reference value, and analyzing the expression level of the target mRNA based on the result of the comparison value. This step is the characteristic step that is most different from the immunological evaluation method in the first aspect. In the first aspect, the quantitative values ​​of two or more different labeled polypeptides are compared and analyzed with each other. In this step, the object for comparison with the quantitative value is the reference value. This step is very different from the first aspect because it analyzes the expression level of the target mRNA based on the comparison result with the reference value.

[0102] Reference values ​​are values ​​obtained by quantitatively labeling a reference polypeptide. The labeled reference polypeptide is a polypeptide expressed from a labeled reference mRNA, in which a labeled nucleotide is attached to the reference mRNA. If both the reference mRNA and the labeled nucleotide are unrestricted, the labeled reference polypeptide includes a large number of species. Thus, a large number of reference values ​​can exist. When performing this aspect, these reference values ​​previously obtained and stored in a database can optionally be used.

[0103] The reference value to be used in this step is a value obtained by quantitatively quantifying a labeled reference polypeptide using the same antibody as the antibody used to quantify the labeled polypeptide in the quantitative step (S0340). That is, in this step, it is preferred to select a reference value based on a labeled mRNA encoding the labeled polypeptide to be quantified in the quantitative step. For example, the base sequence of the reference mRNA is different from the base sequence of the target mRNA. The labeled nucleotide encodes a labeled peptide identical to the labeled polypeptide. The labeled nucleotide is measured and quantified based on the same reference as the labeled polypeptide. For example, a reference value that satisfies these conditions can be selected and obtained from the above-mentioned database.

[0104] Two or more reference values ​​may be used in this step to satisfy the conditions.

[0105] The comparison between the quantitative value and the reference value can be carried out according to the analysis step (S0350) in the first aspect. As mentioned above, the reference value is a value measured and quantified using the same method as the quantitative value based on the same reference. Therefore, direct comparison enables determination of the size of the quantitative value relative to the reference value.

[0106] In the same manner as in the first aspect, the magnitude of the quantitative value of the labeled polypeptide reflects the expression level of the target mRNA included in the labeled mRNA encoding the labeled polypeptide. Based on the comparison result with the reference value, the magnitude of the expression level of the target mRNA included in the labeled mRNA can be analyzed.

[0107] In this step, the reference value can be used as a "cutoff value", for example, for the classification of quantitative values. For example, when the reference value is based on the value of a reference mRNA that exhibits the minimum expected expression level for use as an mRNA vaccine, and when the reference value is lower than this quantitative value, it can be determined that the target mRNA is an inappropriate mRNA vaccine. For example, in the case where the overexpression of an mRNA vaccine can cause side effects to the human body, the upper limit reference value showing the expression level and the lower limit reference value showing the functional minimum expected expression level are used to verify whether the quantitative value is within the range from the lower limit reference value to the upper limit reference value. Therefore, the effect and safety of the mRNA vaccine can be determined.

[0108] In this step, the reference value can be used to determine a statistically significant difference from the quantitative value. Specifically, for example, the function of the target mRNA can be evaluated and determined based on whether the quantitative value is statistically significantly higher than the reference value.

[0109] As used herein, the phrase "statistically significant" means that the critical rate (significance level) of the value obtained 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)" represents the probability of assuming that the hypothesis is true by chance in the statistical distribution assumed in the statistical test. Therefore, a smaller p value means that the hypothesis is closer to truth. The phrase "statistically significant difference" means that when the difference between the measured value of the test target and the measured value of the population is statistically processed, the difference between the two is significant. As a test method for statistical processing, a known test method that can judge any significance can be appropriately used, and the method is not particularly limited. For example, the student's t-test method can be used.

[0110] Example

[0111] (Purpose)

[0112] In order to prove that the method according to the present invention is a method capable of grasping the actual expression level of a protein, the same protein EGFP was compared and examined by measuring fluorescence intensity and by using an immunostaining assay using an antibody against a marker peptide.

[0113] (method)

[0114] (1) Preparation of mRNA

[0115] The two mRNAs used in the examples are as follows: EGFP-GS-Flag mRNA synthesized using unmodified nucleic acid; and mEGFP-GS-Flag mRNA synthesized in a state where all U (uracil) in the EGFP-GS-Flag mRNA is replaced by a modified nucleic acid N1-methyl-pseudo-U. Each mRNA has a base sequence of SEQ ID NO: 11, and the Flag tag is connected to the 3' end of EGFP of SEQ ID NO: 10 via a GS linker of SEQ ID NO: 12. In some cases, SEQ ID Nos: 10 to 12 may be RNA, but are described as DNA sequences in the sequence listing.

[0116] Using a plasmid containing the base sequence of the EGFP-GS-Flag mRNA of SEQ ID NO: 11 as a template, a DNA template with a Poly A tail was produced using staggered thermal asymmetric PCR (Tailed PCR) technology. The DNA template was subjected to agarose gel electrophoresis. A band of the size of interest was cut out from the template and purified using a gel extraction kit (Qiagen NV). Utilizing unmodified nucleic acid and modified nucleic acid, mRNA was synthesized by in vitro transcription. For in vitro transcription, T7 RNA polymerase mixture (New England Biolabs, Inc.) was used. mRNA was treated with DNA enzyme (DNase) and then purified using Monarch RNA Cleanup Kit (New England Biolabs, Inc.). mRNA was capped using Vaccinia Capping Enzyme (New England Biolabs, Inc.) and mRNA Capping 2'-O-Methyltransferase (New England Biolabs, Inc.), and then purified again using the Monarch RNA Cleanup Kit (New England Biolabs, Inc.). The concentration of the mRNA was measured, and then the mRNA was used for introduction.

[0117] (2) Introduction of mRNA

[0118] HEK293 cells were seeded in a 96-well plate so that each well contained 20,000 cells. The cells were cultured overnight at 37°C under 5% CO2. The culture medium used was DMEM (Thermo Fisher Scientific Inc.) containing 10% FBS (Thermo Fisher Scientific Inc.).

[0119] The mixture was prepared by including 100 ng of each mRNA prepared in (1) and 0.2 μL of Lipofectamine Messenger MAX reagent (ThermoFisher Scientific Inc.) in each well of 10 μL Opti-MEM (Thermo Fisher Scientific Inc.). The resulting mixture was added to each cell of a 96-well plate to introduce into HEK293 cells. Here, the resulting mixture was introduced into three wells for each mRNA and used at N=3. A negative control in which no mRNA was introduced was provided in three wells in the same manner. Then, the cells were cultured in each culture dish at 37°C, 5% CO2 for 24 hours, and the mRNA introduced into each cell was allowed to express.

[0120] (3) Fluorescence measurement

[0121] Using portions of the three samples, the fluorescence intensity of EGFP in the cells was measured using a cell counter NC3000 (M&S TechnoSystems, Inc.).

[0122] (4) Immunostaining

[0123] Using parts from the three samples, immunostaining using anti-Flag antibodies was used to measure the expression level of the introduced miRNA. Each sample was fixed with 4% PFA, blocked with 3% BSA, and then used for detection using a 1 / 1000 dilution of a Flag antibody (Sigma-Aldrich Co. LLC). As a secondary antibody, a 1 / 2000 dilution of a fluorescently labeled anti-mouse IgG (H+L) antibody (Thermo Fisher Scientific Inc.) was used.

[0124] (result)

[0125] The results are as follows Figure 4 shown. Figure 4 A shows the measurement results of EGFP fluorescence intensity in each sample. Figure 4 B graphically represents the results of immunostaining assays using anti-Flag antibodies.

[0126] Figure 4 A is as follows: the fluorescence intensity of EGFP in each sample was measured; as a result, when EGFP-GS-Flag mRNA synthesized by a transfection method using unlabeled nucleic acid was introduced into HEK293 cells (lane: EGFP-GS-Flag mRNA), the expression of EGFP protein was verified compared to a mock mRNA not introduced (lane: no mRNA). In HEK293 cells into which mEGRP-GS-Flag mRNA synthesized using modified nucleic acid (lane: mEGFP-GS-Flag mRNA) was introduced, a significantly higher level of EGFP expression was verified compared to EGFP-GS-Flag mRNA synthesized using unlabeled nucleic acid.

[0127] Figure 4 B is as follows: the fluorescence intensity was measured using immunostaining assay with anti-Flag antibody for the same samples; Figure 4 Same results as in A.

[0128] The above results reveal that the measurement of the labeled polypeptide using the method for immunological evaluation according to the present invention reflects the expression level of the labeled polypeptide in the cell. Therefore, the results have demonstrated that the method for immunological evaluation of mRNA expression levels according to the present invention enables accurate quantification and comparison of the expression level of each labeled polypeptide using an antibody that specifically recognizes a common marker peptide of two or more different labeled polypeptides.

[0129] Citation List

[0130] Patent documents

[0131] Patent Document 1: Japanese Translation of PCT International Application Publication No. JP-T-2020-502255

[0132] Sequence Listing.

Claims

1. A method for immunological evaluation of mRNA expression levels, comprising: an expression step that allows expression of labeled mRNA consisting of two or more different target mRNAs and a labeled nucleotide encoding the same labeled peptide, the labeled nucleotide being attached to the end of each target RNA; and A quantification step of quantifying the labeled polypeptide expressed from each labeled mRNA in the expression step using the same antibody against the labeled peptide.

2. The method according to claim 1, further comprising: an analyzing step of comparing the quantitative values ​​of each labeled polypeptide obtained in the quantification step and analyzing the expression level of each target mRNA based on the comparison results.

3. The method according to claim 1 or 2, wherein the labeled mRNA is artificially synthesized in vitro. The method according to claim 3 , wherein the marker peptide is an epitope tag.

5. A method for immunological evaluation of mRNA expression levels, comprising: an expression step, which allows expression of a labeled mRNA consisting of a target mRNA and a labeled nucleotide encoding a labeled peptide, the labeled nucleotide being 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 analyzing 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 based on the comparison result, wherein the reference value is a value obtained by quantifying a labeled reference polypeptide expressed from a labeled reference mRNA using the antibody, the labeled reference mRNA comprising a reference mRNA consisting of a base sequence different from the target mRNA and the same labeled nucleotide linked to the reference mRNA. The method according to claim 5 , wherein the labeled mRNA and the labeled reference mRNA are artificially synthesized in vitro. The method according to claim 6 , wherein the marker peptide is an epitope tag.

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