JP2020200264A - Monoclonal antibodies that specifically recognize activated irf5 - Google Patents

Abstract

To provide established monoclonal antibodies capable of discriminating an active IRF5 whose serine at a specific position is phosphorylated from a non-active IRF5 whose serine at the specific position is not phosphorylated, as well as means for evaluating the activated state of IRF5.SOLUTION: The inventors have created a monoclonal antibody that specifically recognizes S446-phosphorylated active IRF5 and identified its CDR sequence by cloning genes of heavy chain and light chain variable regions from spleen cells of a rabbit in which polyclonal antibodies reactive to S446 phosphorylated IRF5 have been induced using the sequence of human IRF5 isoform b, preparing a phage library each displaying a scFv, and panning using a partial peptide of S446 phosphorylated IRF5 and the S446-phosphorylated IRF5 protein.SELECTED DRAWING: Figure 1

Description

The present invention relates to a monoclonal antibody or antibody fragment that specifically recognizes active IRF5, a method for analyzing the activation state of IRF5 using the antibody or antibody fragment, and a method for producing such a monoclonal antibody or antibody fragment. Regarding.

The immune system is originally a mechanism for eliminating foreign substances such as pathogens and cancer, but in autoimmune diseases, this mistakenly attacks itself and causes various symptoms. Systemic lupus erythematosus (SLE) is an intractable autoimmune disease without a silver bullet, and the number of patients is estimated to be 60 to 100,000 in Japan and an estimated 3.5 million worldwide. Although the survival rate of SLE patients has been improved by steroid therapy, there are various side effects such as immunocompromised diseases, so new treatment methods that can improve the quality of life and long-term prognosis are required.

The transcription factor interferon regulatory factor 5 (IRF5) plays an important role in the innate immune response (Non-Patent Document 1). When pathogens such as viruses and bacteria are recognized by the innate immune receptor Toll-like receptor (TLR), IRF5 is activated downstream by undergoing post-translational modification such as phosphorylation. Activated IRF5 translocates to the nucleus and induces genes for type I interferon and inflammatory cytokines (Non-Patent Documents 2 to 5). Although IRF5 is important for TLR-mediated innate immune response, it has been reported to be involved in the pathogenesis of SLE. From a human biology perspective, genome-wide association studies in various racial populations, including Japanese, have shown that IRF5 is a disease susceptibility gene whose polymorphism strongly correlates with the risk of developing SLE ( Non-Patent Document 6). In fact, it has also been reported that IRF5 is translocated to the nucleus in monocytes in the peripheral blood of SLE patients (Non-Patent Document 7). Furthermore, it has been found that IRF5 is necessary for the onset of pathological conditions in all SLE model mice studied so far, including the research by the inventors of the present application (Non-Patent Documents 8 and 9) (Non-Patent Document 10). ). And in recent years, the inventors of the present application have used a mouse model that excessive activation of IRF5 causes SLE-like pathology, and suppression of IRF5 prevents its onset, and thus IRF5 is a potential therapeutic target for SLE. (Non-Patent Document 9).

IRF5 has been reported to be associated with other autoimmune diseases such as Sjogren's syndrome and systemic scleroderma (Non-Patent Documents 11 and 12). In addition, the inventors of the present application have reported that IRF5 in microglia of the spinal cord causes neuropathic pain using a mouse model (Non-Patent Document 14).

From the results of previous studies, the activation of IRF5 is caused by a specific serine residue by an inhibitor of nuclear factor κ-B kinase β (IKKβ) (No. 446 in human IRF5 isoform b; S446, mouse IRF5 isoform 2, In 3, it is shown that the phosphorylation of the 445th serine residue) is important (Non-Patent Documents 4 and 5). The detection of phosphorylated IRF5 is carried out by a method established by the inventors of the present application using a phosphate group capturing molecule Phos-tag (Non-Patent Document 14) or a method using an anti-phosphorylated IRF5 polyclonal antibody (Non-Patent Document 4, Non-Patent Document 4, Although it is possible by 5 and 15), there is a problem that the phosphorylation site cannot be specified by the former method and it is difficult to obtain a certain result because the latter uses a polyclonal antibody. No anti-phosphorylated IRF5 monoclonal antibody that specifically recognizes the phosphorylation of the specific serine residues described above, which is important for IRF5 activation, has been reported so far.

Tamura, T. et al. The IRF family transcription factors in immunity and oncogenesis. Annu Rev Immunol 26, 535-584, doi: 10.1146 / annurev.immunol.26.021607.090400 (2008). Takaoka, A. et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434, 243-249, doi: 10.1038 / nature03308 (2005). Schoenemeyer, A. et al. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J Biol Chem 280, 17005-17012, doi: 10.1074 / jbc.M412584200 (2005). Lopez-Pelaez, M. et al. Protein kinase IKKβ-catalyzed phosphorylation of IRF5 at Ser462 induces its dimerization and nuclear translocation in myeloid cells. Proc Natl Acad Sci USA 111, 17432-17437, doi: 10.1073 / pnas.1418399111 (2014) .. Ren, J. et al. IKKβ is an IRF5 kinase that instigates inflammation. Proc Natl Acad Sci USA 111, 17438-17443, doi: 10.1073 / pnas.1418516111 (2014). Deng, Y. & Tsao, B.P. Genetic susceptibility to systemic lupus erythematosus in the genomic era. Nat Rev Rheumatol 6, 683-692, doi: 10.1038 / nrrheum.2010.176 (2010). Stone, R.C. et al. Interferon regulatory factor 5 activation in monocytes of systemic lupus erythematosus patients is triggered by circulating autoantigens independent of type I interferons. Arthritis Rheum 64, 788-798, doi: 10.1002 / art.33395 (2012). Savitsky, DA et al. Contribution of IRF5 in B cells to the development of murine SLE-like disease through its transcriptional control of the IgG2a locus. Proc Natl Acad Sci USA 107, 10154-10159, doi: 10.1073 / pnas.1005599107 (2010) ). Ban, T. et al. Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity. Immunity 45, 319-332, doi: 10.1016 / j.immuni.2016.07.015 (2016). Ban, T. et al. Regulation and role of the transcription factor IRF5 in innate immune responses and systemic lupus erythematosus. Int Immunol 30, 529-536, doi: 10.1093 / intimm / dxy032 (2018). Tang, L. et al. Association between IRF5 polymorphisms and autoimmune diseases: a meta-analysis. Genet Mol Res 13, 4473-4485, doi: 10.4238 / 2014. June.16.6 (2014). Eames, H. L. et al. Interferon regulatory factor 5 in human autoimmunity and murine models of autoimmune disease. Transl Res 167, 167-182, doi: 10.1016 / j.trsl. 2015.06.018 (2016). Masuda, T. et al. Transcription factor IRF5 drives P2X4R +-reactive microglia gating neuropathic pain. Nat Commun 5, 3771, doi: 10.1038 / ncomms4771 (2014). Sato G. R. et al. Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation. Bio-Protocol 7, doi: ARTN e2295, doi: 10.21769 / BioProtoc.2295 (2017). Zhao, Y. et al. Microbial recognition by GEF-H1 controls IKKε mediated activation of IRF5. Nat Commun, 10, 1349, doi: 10.1038 / s41467-019-09283-x (2019)

It is expected that the development of a method for inhibiting IRF5 will lead to a new treatment method for SLE. In addition, treatments targeting IRF5 may be applicable to non-SLE autoimmune diseases such as Sjogren's syndrome and systemic scleroderma, as well as diseases such as neuropathic pain. A method that can evaluate the activation status of IRF5 is required for selection of applicable cases and evaluation of prognosis of this treatment method. However, as mentioned above, no anti-phosphorylated IRF5 monoclonal antibody that specifically recognizes the phosphorylation of a specific serine residue important for IRF5 activation has been reported so far.

Therefore, an object of the present invention is to establish a monoclonal antibody capable of distinguishing an active IRF5 in which the above-mentioned specific serine residue is phosphorylated from an inactive IRF5 in which the serine residue is not phosphorylated. , The purpose of which is to provide a means for evaluating the activation state of IRF5.

The inventors of the present application aimed to establish a monoclonal antibody capable of specifically recognizing the active form of IRF5 in which the above-mentioned specific serine residue is phosphorylated, and utilize the sequence of human IRF5 isoform b to form the S446 phosphorylated form. A phage library that clones heavy and light chain variable region genes from rabbit spleen cells that have been able to induce polyclonal antibodies that are reactive against IRF5 and presents single chain fragment of variable regions (scFv). And panning with S446 phosphorylated IRF5 partial peptides and S446 phosphorylated IRF5 proteins, we succeeded in producing a monoclonal antibody that can specifically recognize active IRF5 phosphorylated by S446. The present invention was completed by determining the complementarity-determining region (CDR) sequence of the monoclonal antibody.

That is, the present invention is a monoclonal antibody or antibody fragment that binds to the active IRF5 protein and does not bind to the inactive IRF5 protein at a detectable level, and has the amino acid sequence CDR1 shown in SEQ ID NO: 2. The heavy chain variable region containing CDR2 of the amino acid sequence shown in SEQ ID NO: 3 and CDR3 of the amino acid sequence shown in SEQ ID NO: 4, CDR1 of the amino acid sequence shown in SEQ ID NO: 6, CDR2 of the amino acid sequence shown in SEQ ID NO: 7, and the sequence. Provided is a monoclonal antibody or antibody fragment containing a light chain variable region containing CDR3 of the amino acid sequence shown in No. 8.

The present invention also provides an activated state of the IRF5 protein, which comprises contacting the monoclonal antibody or antibody fragment of the present invention with a sample containing the IRF5 protein to immunologically detect the active IRF5 protein. Provides a method for analysis.

Furthermore, the present invention provides a detection kit for an active IRF5 protein containing the above-mentioned monoclonal antibody or antibody fragment of the present invention.

Furthermore, the present invention is a method for producing a monoclonal antibody or antibody fragment that binds to an active IRF5 protein and does not bind to an inactive IRF5 protein at a detectable level.
Immunization of non-human animals with phosphorylated IRF5 peptide, immune process;
From B cells of non-human animals after immunization, a cDNA encoding a heavy chain variable region and a cDNA encoding a light chain variable region were prepared to prepare a scFv gene or a Fab gene, which was incorporated into a plasmid vector for phage. A phage library preparation step, in which a library of vectors expressing scFv or Fab is prepared and a library of phages presenting each scFv or each Fab on the surface is prepared by the phage display method; and phosphorylated IRF5 peptide and activity. Positive selection using type IRF5 protein and negative selection using non-phosphorylated IRF5 peptide and inactive IRF5 protein concentrate phages with high specific binding to phosphorylated IRF5 peptide and active IRF5 protein. The IRF5 peptide is a partial sequence of the IRF5 protein, which is essentially composed of the partial sequence containing the amino acid sequence shown in SEQ ID NO: 24, and the phosphorylated IRF5 peptide is a partial sequence of SEQ ID NO: 24. Provided is a method in which the fourth corresponding serine residue in 24 is a phosphorylated IRF5 peptide.

The present invention provides for the first time a monoclonal antibody capable of distinguishing an active IRF5 protein from an inactive IRF5 protein. Since the monoclonal antibody or antibody fragment of the present invention can specifically recognize the phosphorylation of a specific serine residue important for the activation of IRF5, the activation state of IRF5 can be analyzed more accurately than in the prior art. Since it is monoclonal, the reproducibility is very high and stable detection results can be obtained. IRF5 activation is known to be involved in various autoimmune diseases such as systemic lupus erythematosus, Sjogren's syndrome, and systemic scleroderma, as well as various diseases such as neuropathic pain, infectious diseases, and cancer. ing. By analyzing the activated state of IRF5 according to the present invention, the severity and prognosis of IRF5-related diseases can be evaluated. In addition, when a drug that suppresses the activation of IRF5 is developed and established as a therapeutic drug for IRF5-related diseases, the activation state of IRF5 should be analyzed to select patients to whom the therapeutic drug should be applied. Can be done.

This is an outline of the process for producing an anti-active IRF5 monoclonal antibody. The base sequence of the VH chain (SEQ ID NO: 9) incorporated into the H chain vector (pEHX1.1 plasmid into which the constant region sequence of rabbit IgG has been inserted), and the amino acid sequence encoded by the sequence (SEQ ID NO: 10). The shaded amino acid sequence shows the signal sequence, and the bordered amino acid sequence shows CDR1, CDR2, and CDR3. Using the cloned scFv expression vector as a template, the VH chain is amplified by adding a signal sequence and a restriction enzyme recognition sequence by a polymerase chain reaction (PCR), and this is digested with the restriction enzymes HindIII and BspEI to form an H chain. Was inserted into the vector for. The base sequence of the L chain (SEQ ID NO: 11) incorporated into the vector for the L chain (pELX2.2), and the amino acid sequence encoded by the sequence (SEQ ID NO: 12). The shaded amino acid sequence shows the signal sequence, and the bordered amino acid sequence shows CDR1, CDR2, and CDR3. Using the cloned scFv expression vector as a template, the VL chain was amplified while adding the signal sequence and restriction enzyme recognition sequence by PCR, and then the κ chain constant region was added to prepare a VL-Cκ fragment, which was used as the restriction enzyme HindIII. It was digested with XbaI and inserted into the L chain vector. This is the result of evaluating the binding activity of the anti-pS446 type IRF5 monoclonal antibody. The binding activity of anti-pS446 type IRF5 monoclonal antibody (p-IRF5 mAb) and normal rabbit IgG (control IgG) to IRF5 peptide and solubilized IRF5 protein (both using non-phosphorylated or S446 phosphorylated form). Three antibody concentrations (0.1, 0.5 and 2.5 μg / mL) were evaluated by ELISA. It is an analysis result of endogenous phosphorylated IRF5 in a human immune cell line. Wild-type (WT) or IRF5-deficient (KO) CAL-1 cells were stimulated with untreated (-) or 3 μM R-848 for 2 hours and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IRF5 and phosphorylated forms. Protein expression of IRF5 (p-IRF5) was analyzed by immunoblotting (IB). Results of analysis of endogenous phosphorylated IRF5 in human primary immune cells. PBMC isolated from peripheral blood of healthy volunteers was stimulated with 3 μM R-848 for 2 hours, and the protein expression of GAPDH, IRF5 and phosphorylated IRF5 (p-IRF5) was analyzed by immunoblotting (IB). It is the analysis result of endogenous phosphorylated IRF5 in mouse primary immune cells. Wild-type (WT) and IRF5-deficient (Irf5 ^-/- ) mouse-derived spleen cells were stimulated with 3 μM R-848 or 0.15 μM CpG-B oligodeoxynucleotides (ODN) for 2 hours, GAPDH, IRF5. And the protein expression of phosphorylated IRF5 (p-IRF5) was analyzed by immunoblotting (IB). It is the analysis result of the phosphorylated IRF5-positive cell population in human primary immune cells. Human PBMCs were stimulated with 3 μM R-848 for 2 hours and IRF5 phosphorylation in each cell type was analyzed by flow cytometry. (A) B cell, monocyte (Mo), pDC and cDC gating methods are shown. The left panel shows the percentage of cells after doublet removal. (B) Phosphorylation of IRF5 in each cell type was analyzed using this antibody (anti-p-IRF5) or normal rabbit IgG. It is the analysis result of phosphorylated IRF5 in SLE model mouse. C57BL / 6J (B6) mice were used as wild-type mice, and Lyn-deficient (Lyn ^-/- ) mice and NZB / W F1 (BWF1) mice were used as SLE model mice (female, 18-20 weeks old). After pooling 2-3 mouse spleen cells into one, CD11c ⁺ cells were isolated and the protein expression of IRF5 and phosphorylated IRF5 (p-IRF5) was analyzed by immunoblotting (IB). Data for two pools (# 1 and # 2) are shown for each strain. Alignment of amino acid sequences of representative isoforms of human IRF5 and mouse IRF5. Alignment of amino acid sequences of representative isoforms of human IRF5 and mouse IRF5 (continuation of FIG. 10-1). The partial region surrounded by the frame is the region adopted for the IRF5 partial peptide in the examples, and S indicated by outline characters is a serine residue important for activation of IRF5.

In the present invention, the active IRF5 protein means an IRF5 protein in which a specific serine residue important for activation is phosphorylated, and an inactive IRF5 protein means a phosphorylated specific serine residue. Means the IRF5 protein that is not. This particular serine residue is the 446th residue in the amino acid sequence of human IRF5 isoform b (SEQ ID NO: 17). FIG. 10 shows the alignment of amino acid sequences of representative IRF5 isoforms in humans and mice. S indicated by outline characters in FIG. 10-2 is the specific serine residue, and is present at the following positions in each amino acid sequence.

Human:
isoform b (NP_001092097, SEQ ID NO: 17): 446th
isoform d (NP_001092099, SEQ ID NO: 26): 462nd
isoform e (NP_001229381, SEQ ID NO: 28): 360th
variant 4 (AAR90326, SEQ ID NO: 30): 436th mouse:
isoform 1 (NP_001239311, SEQ ID NO: 32): 454th
isoform 2 (NP_036187, SEQ ID NO: 34): 445th
isoform 3 (NP_001298012, SEQ ID NO: 36): 445th

The term "serine residue corresponding to serine No. 446 of human IRF5 isoform b (SEQ ID NO: 17)" refers to the specific serine residue described above in human IRF5 isoform b and other IRF5 proteins. It is a word. Therefore, the term "IRF5 protein in which the serine residue corresponding to serine No. 446 of human IRF5 isoform b (SEQ ID NO: 17) is phosphorylated" refers to human IRF5 isoform b in which serine No. 446 is phosphorylated. Human IRF5 isoform d phosphorylated with No. 462 serine, human IRF5 isoform e phosphorylated with No. 360 serine, human IRF5 variant 4 phosphorylated with No. 436 serine, mouse phosphorylated with No. 454 serine Includes IRF5 isoform 1, mouse IRF5 isoform 2 phosphorylated with serine # 445, and mouse IRF5 isoform 3 phosphorylated with serine # 445. In humans and mice, various isoforms other than those illustrated in FIG. 10 are known, and various IRF5s in animals other than humans and mice are also known. The amino acid sequences of these IRF5 proteins are also registered in databases such as GenBank and are easily available. In IRF5 proteins other than those exemplified in FIG. 10, the serine residue corresponding to serine No. 446 of human IRF5 isoform b uses the amino acid sequence of the IRF5 protein and the amino acid sequence of SEQ ID NO: 17 as a known algorithm such as Clustal W. It can be easily identified by aligning with.

The monoclonal antibody of the present invention is a monoclonal antibody that binds to the active IRF5 protein and does not bind to the inactive IRF5 protein at a detectable level. The definitions of active IRF5 and inactive IRF5 are as described above. Such binding is called specific binding to active IRF5. An antibody having such binding property is also referred to as an "anti-active IRF5 antibody".

The anti-active IRF5 monoclonal antibody of the present invention has a heavy chain variable region containing CDR1 of the amino acid sequence shown in SEQ ID NO: 2, CDR2 of the amino acid sequence shown in SEQ ID NO: 3, and CDR3 of the amino acid sequence shown in SEQ ID NO: 4, and a sequence. It includes CDR1 of the amino acid sequence shown in No. 6, CDR2 of the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region containing CDR3 of the amino acid sequence shown in SEQ ID NO: 8.

As long as the monoclonal antibody of the present invention has specific binding property to active IRF5, the sequence of the entire variable region may be any sequence. The CDR is a region that determines the specificity of the antibody against the antigen, and if it has the heavy chain CDR sequence shown in SEQ ID NOs: 2 to 4 and the light chain CDR sequence shown in SEQ ID NOs: 6 to 8, it becomes active IRF5. The arrangement of the framework region is not particularly limited because it can be said that the specific binding property of the above can be exhibited. As an example of the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody of the present invention, the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5, respectively, can be mentioned, and the scope of the present invention includes these sequences. It is not limited. There are no restrictions on the arrangement of the constant region.

The antibody fragment of the present invention may be any fragment as long as it has specific binding property to active IRF5. Specific examples include, but are not limited to, Fab fragments, F (ab') ₂ fragments, and single-chain variable regions or single-chain antibodies (scFv). As is well known, Fab fragments and F (ab') ₂ fragments can be obtained by treating monoclonal antibodies with proteolytic enzymes such as papain and pepsin. The method of producing scFv is also well known. Briefly, mRNA is extracted from cells (hybridoma, etc.) that produce an antibody having specific binding property to active IRF5, and cDNA is prepared by reverse transcription, which is specific to immunoglobulin H chain and L chain. PCR is performed using primers to amplify H-chain genes and L-chain genes, these are linked with a linker, an appropriate restriction enzyme site is added, and the vector is introduced into a plasmid vector. Escherichia coli, yeast, animal cells, etc. ScFv can be prepared by transforming a host cell of the above and recovering scFv from the host cell. When scFv having specific binding property to active IRF5 is prepared as an antibody fragment of the present invention by a production method using a phage display method described later, scFv is obtained from a candidate clone whose specific binding property has been confirmed. Since the expression vector can be recovered, the scFv gene may be replaced with another suitable vector and introduced into the host cell as needed, and scFv may be expressed and recovered.

According to the anti-active IRF5 monoclonal antibody of the present invention, the activated state of the IRF5 protein (how much level of active IRF5 in which the above-mentioned specific serine residue is phosphorylated, that is, how much IRF5 is present) Is it activated?) Can be analyzed. A method for analyzing the activated state of an IRF5 protein comprises contacting the monoclonal antibody or antibody fragment of the present invention with a sample containing the IRF5 protein to immunologically detect the active IRF5 protein. The term "detection" includes qualitative and quantitative detection, and quantitative detection includes relative quantification, absolute quantification and semi-quantitative.

Immunoassay techniques for immunologically detecting proteins of interest are well known in the art. When the immunoassay method is classified based on the reaction type, there are a sandwich method, a competitive method, an agglutination method and the like, and when classified based on a label, there are enzyme immunoassay, radioimmunoassay, fluorescence immunoassay and the like. Any of these immunoassays may be used to analyze the activation status of the IRF5 protein. Specific examples of methods that can be preferably adopted include chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and chemiluminescent immunoassay. In addition to the sandwich method, Western blotting, immunoprecipitation, etc., flow cytometry for identifying and analyzing target cells (cells having active IRF5 in the case of the present invention) using a chemiluminescent monoclonal antibody can be mentioned. However, it is not limited to these.

Reagents required for each immunoassay method are also well known, and immunoassay of active IRF5 protein can be performed using a conventional immunoassay kit, except that the antibody used is the monoclonal antibody or antibody fragment of the present invention. it can. That is, the present invention also provides a detection kit for an active IRF5 protein containing the above-mentioned antibody or antibody fragment of the present invention. Reagents contained in the kit other than the antibody or antibody fragment of the present invention may be the same as those of a known ordinary immunoassay kit.

The sample containing IRF5 may be a biological sample isolated from an animal such as a human or a mouse. Since IRF5 is expressed in various immune cells such as monocytes, B cells, and dendritic cells, blood samples such as peripheral blood (for example, peripheral blood mononuclear cell samples) are examples of biological samples containing IRF5. , Spleen cell sample, kidney cell sample, lymph node cell sample, bone marrow cell sample and the like.

The biological sample containing IRF5 may be a sample isolated from a patient with or at risk of having a disease associated with IRF5 activation. Various autoimmune diseases such as systemic lupus erythematosus (SLE), Sjogren's syndrome, and systemic scleroderma (Non-Patent Documents 9 to 12 etc.) as diseases associated with IRF5 activation (hereinafter referred to as "IRF5-related diseases") ), Neuropathic pain (Non-Patent Document 14 etc.), Infectious diseases (Barnes et al. J Biol Chem 276, 23382-23390, doi: 10.1074 / jbc.M101216200 (2001) etc.), Cancer (Yanai et al. Proc Natl Acad Sci USA 104, 3402-3407, doi: 10.1073 / pnas.0611559104 (2007), etc.) are known. The severity and prognosis of IRF5-related diseases can be assessed by analyzing the activation status of the IRF5 protein using samples isolated from such patients. In addition, when a drug that suppresses the activation of IRF5 is developed in the future and a therapeutic drug for IRF5-related diseases is developed, the therapeutic drug can be obtained by analyzing the activated state of the IRF5 protein according to the present invention. You can choose which patients to apply. In addition, various model animals such as SLE model mice and neuropathy pain model mice are known, and biological samples containing IRF5 may be samples isolated from such model animals, or pets. It may be a sample isolated from a non-human animal that is not a model animal, such as an animal.

A monoclonal antibody or antibody fragment that specifically binds to active IRF5 can be prepared by a method including the following steps.

A step of immunizing a non-human animal with a phosphorylated IRF5 peptide (immunization step).
From B cells of non-human animals after immunization, cDNA encoding the heavy chain variable region and cDNA encoding the light chain variable region are prepared to prepare the scFv gene or Fab fragment gene, which is incorporated into a plasmid vector for phage. A step of preparing a library of vectors expressing scFv or Fab, and preparing a library of phages presenting each scFv or each Fab on the surface by a phage display method (phage library preparation step).
Positive selection using phosphorylated IRF5 peptide and active human IRF5 protein, and negative selection using non-phosphorylated IRF5 peptide and inactive human IRF5 protein to phosphorylated IRF5 peptide and active human IRF5 protein. A step of concentrating phages having a high specific binding property (panning step).

<Immune process>
Immunize non-human animals (rabbits, mice, rats, etc.) with the phosphorylated IRF5 peptide. An IRF5 peptide is a peptide essentially consisting of a partial sequence of an IRF5 protein containing a serine residue corresponding to serine # 446 (S446) of human IRF5 isoform b (SEQ ID NO: 17). The phosphorylated IRF5 peptide is a peptide in which the serine residue corresponding to the S446 is phosphorylated in the IRF5 peptide. The non-phosphorylated IRF5 peptide used in the subsequent step is an IRF5 peptide in which the serine residue corresponding to the S446 is not phosphorylated, for example, an IRF5 peptide containing no phosphorylated residue. obtain.

As an example of an IRF5 peptide, a peptide having a size of 7 to 40 residues, which is essentially composed of an IRF5 protein partial sequence containing LQISNPD (SEQ ID NO: 24), can be mentioned. LQISNPD (SEQ ID NO: 24) is the region of amino acids 443 to 449 in the amino acid sequence of human IRF5 isoform b shown in SEQ ID NO: 17, and the fourth S of LQISNPD is S446 of human IRF5 isoform b. Corresponds to. Therefore, in this example, the phosphorylated IRF5 peptide is an IRF5 peptide in which the fourth S of LQISNPD is phosphorylated.

Examples of more preferably usable IRF5 peptides include peptides with a size of 13-40 residues, essentially consisting of an IRF5 protein partial sequence containing SIRLQISNPDLKD (SEQ ID NO: 23). SIRLQISNPDLKD (SEQ ID NO: 23) is the region of amino acids 440 to 452 in the amino acid sequence of human IRF5 isoform b shown in SEQ ID NO: 17, and the 7th S of SIRLQISNPDLKD is S446 of human IRF5 isoform b. Corresponds to. Therefore, in this example, the phosphorylated IRF5 peptide is the IRF5 peptide in which the 7th S of SIRLQISNPDLKD is phosphorylated.

In both the phosphorylated form and the non-phosphorylated form, the IRF5 peptide may be in a form in which an appropriate residue is added to the end for convenience of modification such as biotinylation and binding to other proteins. For example, a Lys residue is generally added to the peptide terminal for biotinylation, and a Cys residue (as used by the MBS method) for binding to a carrier protein. The term "peptide essentially consisting of a partial sequence of IRF5 protein" includes a small number (1 to number) not derived from the sequence of IRF5 protein for the purpose of processing the peptide, in addition to the peptide consisting of a partial sequence of IRF5. Contains peptides having a structure in which residues (eg, 1 to 5, 1 to 4, 1-3, 1-2, or 1) are added to the C-terminus or N-terminus of the IRF5 partial sequence. To.

Phosphorylated IRF5 peptides and non-phosphorylated IRF5 peptides can be prepared by conventional chemical synthesis. Phosphorylated IRF5 peptides are relatively short peptides, so when immunizing non-human animals, they are usually bound to carrier proteins such as keyhole limpet hemocyanin, bovine thyroglobulin, and bovine serum albumin as the immunogen. Use. In addition, it is desirable to use a phosphorylated IRF5 peptide used for immunity with sufficiently high purity. A peptide having a purity of 85% or higher, for example 87% or higher, particularly 90% or higher, can sufficiently induce an antibody against a phosphorylated peptide in the body of a non-human animal.

The number of immunizations with the phosphorylated IRF5 peptide is not particularly limited, but it may be, for example, 6 times or more, 7 times or more, or 8 times or more because it becomes easier to achieve sufficient antibody induction by increasing the number of immunizations.

<Phage library production process>
B cells of non-human animals immunized with the phosphorylated IRF5 peptide can be collected, for example, from the spleen or peripheral blood. When using small non-human animals such as mice and rabbits, it is common to use spleen cells as B cells. MRNA is extracted from spleen cells or peripheral blood lymphocytes, cDNA is synthesized by reverse transcription reaction, and cDNA encoding the region containing the heavy chain variable region (VH cDNA) and cDNA encoding the region containing the light chain variable region (VH cDNA) VL cDNA) may be comprehensively amplified by PCR. If you want the phage to present the Fab region, you can comprehensively amplify the cDNA encoding the heavy chain variable region and heavy chain constant region 1 and the cDNA encoding the light chain variable region and light chain constant region by PCR. Good. When scFv is desired to be presented to phage, the cDNA encoding the heavy chain variable region and the cDNA encoding the light chain variable region may be comprehensively amplified, and the sequence encoding the constant region is unnecessary. Primers for comprehensively amplifying the variable region of an antibody are known (eg, Marks, JD et al. J Mol Biol 222, 581-597 (1991); Welschof, M. et al. J Immunol Methods 179, 203-214 (1995); Welschof, M. et al. Methods Mol Med 13, 593-603 (1998); Orlandi, R. et al. Proc Natl Acad Sci USA 86 3833-3837 (1989); Marks, JD et al. Eur J Immunol 21 985-991 (1991); Campbell, MJ et al. Mol Immunol 29, 193-203 (1992); Barbas III, CF et al. Proc Natl Acad Sci USA 88, 7978-7982 (1991) ).

The amplified VH cDNA and VL cDNA are then ligated by conventional assembly PCR or Fusion PCR to prepare DNA encoding a scFv or Fab fragment (scFv gene or Fab fragment gene). When amplifying VH cDNA and VL cDNA, PCR is performed using a primer to which DNA corresponding to an appropriate linker peptide sequence is added, and the obtained VH cDNA amplification fragment and VL cDNA amplification fragment are subjected to assembly PCR or Fusion PCR. It may be connected. As a result, an scFv gene or a Fab fragment gene in which VH cDNA and VL cDNA are randomly linked via a DNA encoding a linker is obtained. The scFv gene or Fab fragment gene linked in the order of 5'- [VH cDNA]-[linker DNA]-[VL cDNA] -3'and 5'-[VL cDNA]-[linker DNA]-[VH cDNA] ] -Both the scFv gene and the Fab fragment gene linked in the order of -3'may be prepared.

Next, the prepared scFv gene or Fab fragment gene is incorporated into a plasmid vector for phage to prepare a vector library (scFv gene library or Fab fragment gene library) expressing the scFv or Fab fragment, and then the phage display method. Creates a library of phages that present each scFv or each Fab on the surface. The phage vector contains all the phage genes necessary for forming phage particles, and includes a phage vector capable of forming phage particles independently and a phage vector containing a g3p gene but not containing other phage protein genes. There are two types of phagemid vectors that require helper phage for particle formation, but the phagemid vector can be preferably used. By introducing a scFv or Fab fragment gene library prepared using a phagemid vector into Escherichia coli and severely infecting Escherichia coli with helper phage, individual vectors of the scFv or Fab fragment gene library are packaged, and the vector becomes A library of phages that present the expressed scFv or Fab on the surface can be prepared.

<Panning process>
In the panning of the phage library, a positive selection using a phosphorylated IRF5 peptide and an active IRF5 protein and a negative selection using a non-phosphorylated IRF5 peptide and an inactive IRF5 protein are performed. Not only immunogenic peptides, but also IRF5 proteins are used in the selection. In addition to the positive selection based on the binding property to the phosphorylated type (active type), the negative selection is performed to screen out the phages that do not bind to the non-phosphorylated type (inactive type). As a result, phage clones having high specific binding to both phosphorylated IRF5 peptide and active human IRF5 protein can be efficiently concentrated.

In the panning step, the target protein (phosphorylated and non-phosphorylated IRF5 peptide, and active and inactive human IRF5 protein) and the solid phase are modified with a specific binding partner molecule, and the solid phase is modified with a specific binding partner molecule via the specific binding partner molecule. It is preferable to use an immobilized target protein. Biotin and streptavidin can be preferably used as the specific binding partner molecule. Magnetic beads can be preferably used as the solid phase. In the positive selection, phages bound to the target protein are magnetically separated and recovered. In the negative selection, the phages bound to the target protein may be magnetically separated and removed, and the phages not bound to the target protein may be recovered.

The recovered phage is lysed, the packaged vector is recovered and introduced into Escherichia coli again, and the helper phage is coinfected to form phage particles again. Positive selection and negative selection are performed again on these phage particles. By repeating these operations and performing the selection for a plurality of rounds, phage clones that specifically and strongly bind to phosphorylated (active) peptides and proteins can be sufficiently concentrated.

The phosphorylated and non-phosphorylated IRF5 peptides used in the panning step may differ from the phosphorylated IRF5 peptides used in the immune step in any additional residue. The amino acid sequence other than any additional residue, that is, the amino acid sequence of the portion corresponding to the partial sequence of the IRF5 protein is preferably the same as that of the phosphorylated IRF5 peptide used in the immune step, but only a few residues. Differences (eg, the portion derived from the IRF5 protein is several residues longer than the peptide used in the immune process) may be present.

As the active IRF5 protein, for example, IKKβ and IRF5 may be forcibly expressed in an appropriate cell line, and the IRF5 protein phosphorylated by IKKβ may be recovered. When human IRF5 isoform b (SEQ ID NO: 17) is used, human IKKβ (NP_001547) having the amino acid sequence shown in SEQ ID NO: 19 may be used. If biotinylated IRF5 is forcibly expressed in a cell line, phosphorylated (active) IRF5 protein can be easily recovered and purified from cell lysates by streptavidin. The inactive IRF5 protein may be recovered and purified by forcibly expressing only IRF5 in an appropriate cell line. The inactive IRF5 protein may be a protein that has not undergone any phosphorylation. The technique for expressing biotinylated proteins in cell lines is a well-established technique. The cDNA of IRF5 to which a biotinylated tag (a special peptide that is biotinylated in the cell) is added and the cDNA of the biotin ligase that recognizes this biotinylated tag may be expressed in the same cell. By the action of biotin ligase, biotin binds to the biotinylated tag fused to IRF5, resulting in the production of biotinylated IRF5 in the cell.

Phages having a candidate scFv expression vector or Fab expression vector can be obtained by concentrating phages through a selection of multiple rounds in the panning step. However, in order to further narrow down the candidates, the following clone selection step is performed after the panning step. You may. Further, when it is desired to prepare an IgG antibody as an antibody that binds to an active IRF5 protein and does not bind to an inactive IRF5 protein at a detectable level, the candidate scFv or Fab may be IgGized. If you want to prepare a Fab as an antibody fragment that binds to an active IRF5 protein but does not bind to an inactive IRF5 protein at a detectable level, you can Fab the candidate scFv, leave the candidate Fab fragment intact, or It may be used with appropriate modifications.

<Clone selection process>
In the clone selection step, the scFv expression vector is recovered from the phage obtained by the panning step, the vector is introduced into Escherichia coli to prepare a clone of Escherichia coli expressing scFv, and the obtained clone is converted into an active IRF5 protein. The reactivity and the sequence of the variable region of Escherichia coli are examined, and clones having high specific binding to the active IRF5 protein are selected. Screening for Escherichia coli clones may be performed by, for example, ELISA using an active IRF5 protein as an antigen, or ELISA using a phosphorylated IRF5 peptide as an antigen. In addition, since there may be multiple clones with the same or very similar variable regions, the nucleotide sequences of VH and VL are determined, duplicate clones are confirmed, and clones are grouped. May be good. Through these operations, clones with high specific binding to the active IRF5 protein can be selected and candidates can be further narrowed down.

<IgG process>
The technique of IgGation for producing an IgG antibody from a desired VH chain and VL chain is well known.
When a scFv-presenting phage library was prepared and panned, the scFv expression vector was recovered from the candidate phage or E. coli clone, and the polynucleotides encoding VH and VL were amplified by PCR using this as a template. By Fusion PCR, the variable region is a constant region (for VH, IgG heavy chain constant regions 1 to 3 (CH1 to CH3), for VL, light chain variable region (CL), and CL is a constant region of κ chain. H chain expression by preparing a polynucleotide fragment to which a region (Cκ) or a constant region of a λ chain (Cλ) is added and incorporating it into a plasmid vector, or by sequentially incorporating a variable region and a constant region into a plasmid vector. A vector and an L chain expression vector can be constructed respectively. Alternatively, an H chain expression vector incorporating CH1 to CH3 and an L chain expression vector incorporating CL (Cκ or Cλ) are also widely known, and commercially available products are also available. Vectors may be used. The desired IgG antibody can be obtained by co-expressing the constructed H chain expression vector and L chain expression vector in an appropriate host cell such as CHO cells or HEK293 cells. Alternatively, a necessary region may be excised from the H chain expression vector and the L chain expression vector and linked to construct an IgG antibody expression vector, which may be expressed in a host cell. The IgG antibody expressed in the host cell can be recovered and purified by a conventional method using, for example, an affinity column on which Protein A or the like is immobilized.
When a Fab-presented phage library is prepared and panned, only the VH and VL regions may be amplified and IgG-ized by using the Fab expression vector recovered from the candidate phage or E. coli clone as a template. Alternatively, the constant region may be included in the amplification, and the insufficient constant region may be added for IgG formation.

<Fab conversion process>
The technique for converting scFv to Fab is also well known, and a vector for expressing the Fab region portion of an IgG heavy chain in which CH1 is incorporated is also widely known, and a commercially available product also exists. An H-chain Fab region expression vector can be prepared by inserting a PCR amplification fragment of VH into such a known vector, or by preparing a polynucleotide fragment in which CH1 is added to VH by Fusion PCR and incorporating it into a plasmid vector. The desired Fab fragment can be obtained by co-expressing this expression vector and the L chain expression vector constructed as described above in an appropriate host cell. Alternatively, a required region may be cut out from the H chain Fab region expression vector and the L chain expression vector and ligated to construct a Fab expression vector, which may be expressed in a host cell. Fabs can be recovered and purified from host cells by a conventional method using an affinity column on which Protein G or the like is immobilized.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples. The abbreviations in the examples mean the following.
APC: allophycocyanin allophycocyanin
CD: Differentiation cluster of differentiation
CDRs: complementarity-determining regions
cDC: classical dendritic cell
CHO: Chinese hamster ovary
EDTA: ethylenediaminetetraacetic acid
ELISA: Enzyme-linked immunosorbent assay
FACS: Fluorescent-activated cell sorting
FBS: fetal bovine serum
GAPDH: glyceraldehyde-3-phosphate dehydrogenase
HEK: human embryonic kidney
HLA: human leukocyte antigen
HPLC: High-performance liquid chromatography
HRP: Horseradish peroxidase
IB: Immunoblotting
Ig: Immunoglobulin immunoglobulin
IKKβ: inhibitor of nuclear factor κ-B kinase β
IRF5: interferon regulatory factor 5
mAb: monoclonal antibody
NZB / W: New Zealand Black / White
ODN: oligodeoxynucleotides
PAGE: polyacrylamide gel electrophoresis
PBMC: Peripheral blood mononuclear cell
PBS: Phosphate buffered saline
PCR: polymerase chain reaction
pDC: Plasmacytoid dendritic cell
PE: phycoerythrin
RIPA: Radioimmunoprecipitation assay
RPMI: Roswell Park Memorial Institute
scFv: Single-chain variable fragment
SDS: sodium dodecyl sulfate
SLE: Systemic lupus erythematosus
TLR: Toll-like receptor
TMB: tetramethylbenzidine

In the following experiments, human IRF5 isoform b was used as IRF5. Therefore, the serine residue important for activation is described as S446, and the active IRF5 is described as pS446 type IRF5. The amino acid sequence (RefSeq ID: NP_001092097) of human IRF5 isoform b is shown in SEQ ID NO: 17, and the nucleotide sequence encoding this (NM_001098627) is shown in SEQ ID NO: 16.

1. 1. Creation of monoclonal antibody that specifically recognizes pS446 type IRF5
Aiming to establish a monoclonal antibody that can recognize pS446-type IRF5 by distinguishing it from inactive IRF5 in which S446 is not phosphorylated, using pS446-type IRF5 partial peptide as an immunogen, the production of polyclonal antibodies and mesenteric lymph nodes Attempts were made to produce monoclonal antibodies by the method and the spleen method. Methods (1) to (4) were carried out by entrusting antibody production to a total of three companies.

The pS446-type IRF5 partial peptide includes the sequence of residues 440 to 452 in the amino acid sequence of human IRF5 shown in SEQ ID NO: 17 (SIRLQISNPDLKD, SEQ ID NO: 23), and corresponds to serine No. 446. A phosphorylated peptide having a structure in which a serine residue was phosphorylated (SIRLQI (pS) NPDLKDC (SEQ ID NO: 20) or SIRLQI (pS) NPDLKDK (biotin) (SEQ ID NO: 21)) was chemically synthesized. A peptide in which a carrier protein was bound to the peptide of SEQ ID NO: 20 having cysteine introduced at the C-terminal was used as an immunogen. Phosphorylated peptides, phosphorylated and non-phosphorylated, were synthesized and used for monoclonal antibody screening and polyclonal antibody purification.

Method (1): Rat mesenteric lymph node method A phosphorylated peptide (purity 80%) was immunized twice in a rat, and a hybridoma was prepared using the mesenteric lymph node. The hybridoma screening was performed by ELISA using a phosphorylated peptide and a non-phosphorylated peptide (SEQ ID NO: 21) as antigens.

Method (2): Mouse spleen method Phosphorylated peptides (purity 80%) were immunized to mice 6 times to prepare hybridomas using the spleen. The hybridoma screening was performed in the same manner as in method (1).

Method (3): Rabbit polyclonal antibody production 1
Rabbits were immunized with a phosphorylated peptide (purity 80%) 5 times and serum was collected. After affinity purification of the antibody from the serum using the phosphorylated peptide, the antibody binding to the non-phosphorylated peptide was absorbed and removed using the non-phosphorylated peptide.

However, none of the methods (1) to (3) gave an antibody capable of specifically recognizing pS446 type IRF5.

Then, as described in the following method (4), the phosphorylated peptide was resynthesized to try again to produce a rabbit polyclonal antibody.

Method (4): Rabbit polyclonal antibody production 2
Rabbits were immunized with a phosphorylated peptide (purity 90%) eight times and serum was collected. After affinity purification of the antibody from the serum using the phosphorylated peptide, the antibody binding to the non-phosphorylated peptide was absorbed and removed using the non-phosphorylated peptide.

As a result, by the method (4), a polyclonal antibody capable of detecting the signal of pS446 type IRF5 by Western blotting (WB) could be obtained. Differences between method (3) and method (4) include the purity of the peptide and the number of immunizations. It is considered that one or both of the fact that the high-purity peptide was used as the immunogen and the number of immunizations was high contributed to the acquisition of the desired polyclonal antibody.

However, although the polyclonal antibody obtained by the method (4) can detect the signal of pS446 in WB, a non-specific signal is also generated, and there is a problem in specificity. Therefore, when a monoclonal antibody was produced by the antibody phage display method using the spleen of a rabbit immunized according to the method (4) and producing the above polyclonal antibody (method (5)), an anti-pS446 type IRF5 monoclonal antibody was attempted. We have succeeded in producing an antibody (hereinafter sometimes referred to as "this antibody").

Method (5): Monoclonal antibody production by antibody phage display method The outline of the production process is shown in FIG. In addition to the selection using phosphorylated and non-phosphorylated peptides derived from the human IRF5 partial region, the selection using pS446 and non-pS446 (S446 non-phosphorylated) human IRF5 recombinant proteins was also performed. We have succeeded in producing a monoclonal antibody that specifically recognizes pS446 type IRF5. Rabbit immunity-spleen collection was outsourced to Immuno-Biological Laboratory Co., Ltd. The work from the synthesis of the antigen peptide and the peptide for panning, the preparation of the scFv gene library to the purification of the IgG-ized antibody was outsourced to Medical Biology Research Institute, Inc. The inventors of the present application prepared IRF5-expressing cells used for evaluating the binding activity and specificity of a candidate Escherichia coli clone (scFv).

A phosphorylated peptide [SIRLQI (pS) NPDLKDC (SEQ ID NO: 20), purity 90.1%] bound to a carrier protein was used as an immunogen, and rabbits were immunized eight times and then the spleen was collected.

After RNA was extracted from the spleen, cDNA was synthesized by reverse transcription reaction, and PCR was performed using this as a template to prepare cDNA encoding the VH chain (VH cDNA) and cDNA encoding the VL chain (VL cDNA). Using the linker DNA encoding the linker peptide sequence, VH cDNA and VL cDNA were randomly ligated by assembly PCR, and the scFv gene (5'- [VH cDNA]-[linker DNA]-[VL cDNA] -3', 5'-[VL cDNA]-[Linker DNA]-[VH cDNA] -3') was prepared. The scFv gene was integrated into a phagemid vector to prepare a scFv gene library.

After transforming the scFv gene library into Escherichia coli, the helper phage was heavily infected to prepare a phage library (scFv antibody phage library) in which various scFv were presented on the surface. Two types of scFv gene libraries were prepared from the spleens of two immunized rabbits, and finally two types of scFv antibody phage libraries were prepared.

In the panning step, biotinylated phosphorylated peptide [SIRLQI (pS) NPDLKDK (biotin) (7th serine in SEQ ID NO: 21 is phosphorylated), purity 90.9%] and biotinylated non-phosphorylated peptide [SEQ ID NO: SIRLQISNPDLKDK] (biotin) (SEQ ID NO: 21), purity 99.9%] was used for positive and negative selections, as well as positive and negative selections using pS446-type IRF5 recombinant protein and S446 non-phosphorylated IRF5 recombinant protein. Phosphants presenting scFv antibodies that specifically react with both peptides and pS446 recombinant proteins were enriched. The pS446 type IRF5 recombinant protein is HEK293T in which biotinylated IRF5 and IKKβ (RefSeq ID: NP_001547 (SEQ ID NO: 19) and the polynucleotide sequence encoding this (the sequence of CDS of NM_001556) shown in SEQ ID NO: 18) are forcibly expressed. It was obtained by purification with streptavidin from cell lysate. The S446 non-phosphorylated IRF5 recombinant protein was obtained by purification with streptavidin from the lysate of HEK293T cells in which only biotinylated IRF5 was forcibly expressed. For forced expression of biotinylated IRF5, a plasmid vector containing the cDNA of IRF5 with a biotinylated tag of 17 residues and the cDNA of biotin ligase that recognizes this biotinylated tag was introduced into HEK293T cells, and the biotinylated IRF5 was added. Biotinylated IRF5 was produced by expressing biotin ligase and biotin ligase in cells.

After preparing an Escherichia coli colony plate and cloning the scFv antibody, the reactivity of 192 clones was evaluated and the sequence analysis / classification of the H chain variable region was performed. Four clones were selected based on the evaluation results and subjected to IgG conversion. As VH and VL fragments, the scFv expression vector was used as a template and amplified by PCR while adding a signal sequence and a restriction enzyme recognition sequence, respectively. VL subsequently prepared a VL-Cκ fragment by fusion-PCR to which Cκ (constant region of the κ chain, SEQ ID NO: 13) was added. Each of these gene fragments was treated with two types of restriction enzymes, and the H-chain vector (pEHX1.1, Toyobo) and the L-chain vector (pELX2.2, Toyobo) into which the rabbit CH1, 2, and 3 genes had been incorporated in advance Each fragment was incorporated. As the secretory signal sequence, MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 14) was used for the H chain, and MRLLAQLLGLLMLWVPGSSG (SEQ ID NO: 15) was used for the L chain. The H-chain vector and the L-chain vector were sequenced, and it was confirmed that the correct gene fragment could be inserted (Fig. 2, SEQ ID NO: 9, and Fig. 3, SEQ ID NO: 11).

The H-chain vector and the L-chain vector were digested with two types of restriction enzymes, and the H-chain vector and the L-chain vector were ligated to prepare an IgG antibody expression vector. This was introduced into CHO cells, and stable expression cells were obtained by puromycin selection. For the most reactive clone, stable expression cells were cloned. The culture supernatant was collected and purified on an affinity column using Protein A. The purified IgG obtained above was used as an anti-pS446 type IRF5 monoclonal antibody (this antibody) in the following experiments.

The CDR sequence of this antibody was as follows.
Heavy chain variable region: SEQ ID NO: 1
Heavy chain CDR1: DYIMI (SEQ ID NO: 2)
Heavy chain CDR2: TISSRGTTHYASWAKG (SEQ ID NO: 3)
Heavy chain CDR3: GISGSGYYNV (SEQ ID NO: 4)
Light chain variable region: SEQ ID NO: 5
Light chain CDR1: QASENIYSGLA (SEQ ID NO: 6)
Light chain CDR2: RASTLES (SEQ ID NO: 7)
Light chain CDR3: QCATFVSTTYSNG (SEQ ID NO: 8)

2. 2. Evaluation of binding activity of this antibody and detection method of pS446 type IRF5 by this antibody
ELISA
The binding property of the antibody to the antigen was evaluated by the indirect method. For peptide antigens, biotinylated phosphorylated peptide or biotinylated non-phosphorylated peptide is diluted with PBS-T [1x PBS (pH 7.2), 0.05% Tween 20] to 1 μg / mL and Nunc Immobilizer. It was immobilized by incubating each well of Streptavidin 12X8 Strip (Thermo Fisher Scientific) at 4 ° C. for 1 hour. In the case of solubilized protein antigens, HEK293T cells forcibly expressing biotinylated IRF5 and IKKβ or HEK293T cells forcibly expressing only biotinylated IRF5 are used in RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, Nunc lysate obtained by dissolving in 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, cOmplete Mini EDTA-free protease inhibitor cocktail (Roche), PhosSTOP phosphatase inhibitor cocktail (Roche)] Immobilizer Streptavidin 12X8 Strip was sensitized by incubating at 4 ° C. for 1 hour in addition to each well. PBS-T was used to wash the wells and dilute the antibody. The reaction of the primary antibody (anti-pS446 type IRF5 monoclonal antibody or normal rabbit IgG) and the secondary antibody (anti-rabbit IgG-HRP label, MBL, diluted 5000 times) was carried out at room temperature for 1 hour. For color development, TMB was used as a substrate and reacted at room temperature for 3 minutes.

Cell culture Human plasma cell-like dendritic cell line CAL-1 cells ¹ , human peripheral blood mononuclear cells (PBMC), mouse spleen cells and mouse spleen CD11c ⁺ cells are in RPMI medium containing 10% FBS [RPMI 1640 medium]. (Contains L-glutamine / HEPES, Nakaraitesk), 10% FBS, 100 U / mL penicillin G potassium (Meiji), 0.1 mg / mL streptomycin sulfate (Meiji), 50 mM 2-mercaptoethanol (Sigma-Aldrich), 0.1 The cells were cultured in a CO ₂ incubator (saturated water vapor, 37 ° C., 5% CO ₂ conditions) using mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco)]. CpG-B ODN (CpG ODN 1668: 5'-TCCATGACGTTCCTGATGCT-3'(SEQ ID NO: 22), phosphorothioate skeleton, HPLC purification) used for cell stimulation was purchased from Eurofin Genomics, and R-848 was purchased from Enzo Life Sciences.

Isolation of human PBMCs Human PBMCs were isolated by density gradient centrifugation. Put 15 mL of Lymphoprep (STEMCELL Technologies), which is a specific gravity solution, in a SepMate-50 tube (STEMCELL Technologies), and dilute the peripheral blood [peripheral blood (heparin-added blood) collected from healthy volunteers) in an equal volume of 2 % FBS-containing PBS was added and diluted] was layered and centrifuged (1200 xg, 4 ° C, 20 minutes). Of the separated layers, the PBMC layer was recovered, an equal amount of PBS containing 2% FBS was added, and centrifugation (300 xg, 4 ° C, 8 minutes) was performed. The cell pellet was washed once with PBS containing 2% FBS, suspended in RPMI medium containing 10% FBS, and a stimulation experiment was performed immediately. Analysis using human specimens (peripheral blood) has been deliberated and approved by the Yokohama City University Medical Research Ethics Committee, and has been deliberated and approved by the "Ethical Guidelines for Medical Research for Humans" and "Ethics for Human Genome / Gene Analysis Research". We followed the guidelines.

mouse
The production of Irf5 gene-deficient (Irf5 ^-/- ) and Lyn gene-deficient (Lyn ^-/- ) mice has been reported ^2,3 . The NZB / W F1 mouse was purchased from Nippon SLC Co., Ltd. Wild-type mice used the C57BL / 6J strain. Regarding the use of laboratory animals, after deliberation and approval by the Animal Experiment Committee of Yokohama City University, the use was carried out in compliance with the "Rules for Conducting Animal Experiments at Yokohama City University".

Isolation of mouse spleen cells The spleen was removed from euthanized mice, injected with 1.5 mL of enzyme mixture [1.67 U / mL Liberase DL (Roche), 0.2 mg / mL DNase I (Roche)], and then dissected. It was chopped with scissors and incubated in a CO ₂ incubator (saturated water vapor, 37 ° C, 5% CO 2) for 30 minutes. The enzyme-treated cell suspension and shredded spleen were transferred to a cell strainer (Corning, 70 μm) and ground with a dropper of a 2.5 mL syringe (Terumo) while washing with 10 mL PBS containing 2 mM EDTA. The cell suspension was centrifuged (300 xg, 4 ° C, 5 minutes) and the supernatant was removed. Suspend the cells in 1 mL of erythrocyte lysate (155 mM NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM EDTA) and allow to stand at room temperature for 1 minute, then add 10 mL of RPMI medium containing 10% FBS and centrifuge. (300 xg, 4 ° C, 5 minutes). The supernatant was removed, the cell pellet was suspended in RPMI medium containing 10% FBS, and then transferred to a new tube through a cell strainer. The isolated spleen cells were immediately used in the stimulation experiment.

Isolation of mouse spleen CD11c ⁺ cells
Mouse spleen cells were rapidly isolated without enzyme treatment to avoid degradation of the active IRF5 protein in SLE model mice. Isolation of CD11c ⁺ cells from spleen cells was performed using anti-mouse CD11c antibody microbeads (Miltenyi Biotec) according to the instruction manual. Briefly, anti-CD16 / 32 antibody (BioLegend, clone93) was used to block Fc receptors, anti-CD11c antibody microbeads were reacted, and then autoMACS Pro Separator (Miltenyi Biotec) was used in the POSSEL_D program to CD11c ^+. Cells were isolated.

Immunoblotting cells were lysed with RIPA buffer to prepare lysates. SDS-PAGE was performed based on the Laemmli method, and the gel after electrophoresis was transferred to a membrane using a semi-dry blotting device. Membrane washes were performed with TBS-T [25 mM Tris-HCl (pH 7.4), 3 mM KCl, 140 mM NaCl, 0.05% Tween 20], blocking and antibody dilution with TBS-T containing 5% skim milk. .. In addition to this antibody (0.2 μg / mL), the primary antibody is anti-GAPDH antibody (Abcam, clone 6C5; 0.05 μg / mL), anti-IRF5 antibody (Abcam, ab21689; 0.2 μg / mL) or anti-IRF5 antibody (Abcam, clone). EPR17067; 1.0 μg / mL), anti-mouse IgG-HRP (GE Healthcare; 20,000-fold dilution) or anti-rabbit IgG-HRP (GE Healthcare; 20,000-fold dilution) was used as the secondary antibody. The chemiluminescent reaction was detected by ImageQuant LAS 4000 mini (GE Healthcare) using ECL Prime (GE Healthcare) or ImmunoStar LD (Wako Pure Chemical Industries) as a substrate.

Flow cytometry Intracellular staining was performed using this antibody. After collecting the cells and centrifuging (300 xg, 4 ° C, 5 minutes) to remove the supernatant, the cell pellet is suspended in a blocking buffer for cell surface staining (1 x PBS, 10% normal human serum, 2% FBS). It became cloudy and allowed to stand on ice for 10 minutes. A mixture of antibodies against cell surface markers (excluding PE-Cy7 labeled antibody) was added and allowed to stand on ice for 15 minutes. The mixture was stirred by tapping and allowed to stand for another 15 minutes (antibody reaction for a total of 30 minutes). Add 2% FBS-containing PBS and centrifuge (300 xg, 4 ° C, 5 minutes) to remove the supernatant, suspend the cell pellet in 2% FBS-containing PBS, and then warm to an equivalent volume of 37 ° C. BD Cytofix Buffer (BD Biosciences) was added, and the mixture was shaken at 37 ° C. for 10 minutes. After removing the supernatant by centrifugation (400 xg, 4 ° C, 5 minutes), BD Phosflow Perm Buffer III (BD Biosciences) was added while breaking the cell pellet with a vortex, and the cells were allowed to stand on ice for 30 minutes. PBS containing 2% FBS was added, and then the supernatant was removed by centrifugation (400 xg, 4 ° C, 5 minutes). This washes were performed again (2 washes in total). The cell pellet was suspended in intracellular blocking buffer (TBS-T containing 5% skim milk) and shaken at room temperature for 30 minutes. The primary antibody diluted with the intracellular staining blocking buffer was added, and the mixture was shaken at room temperature for 1 hour. After removing the supernatant by centrifugation (400 xg, 4 ° C., 5 minutes), the cell pellet was suspended in TBS-T and shaken at room temperature for 5 minutes. Centrifugation and supernatant removal were performed again (a total of 2 washes). A secondary antibody diluted with an intracellular staining blocking buffer or an antibody for PE-Cy7-labeled cell surface staining was added to the cell pellet, and the mixture was shaken at room temperature for 1 hour. After two washes, the cell pellet was suspended in PBS containing 2% FBS and then transferred to a FACS tube while passing through a mesh. The analysis was performed with a BD FACSCanto II flow cytometer (BD Biosciences). Antibodies for cell surface staining are APC-labeled anti-CD14 antibody (BioLegend, clone M5E2), APC-Cy7-labeled anti-CD1c antibody (BioLegend, clone L161), Brilliant Violet 421-labeled anti-CD16 antibody (BioLegend), Alexa Fluor 488-labeled anti-CD303. Antibodies (BioLegend, clone 201A), Brilliant Violet 510-labeled anti-CD19 antibody (BioLegend, clone HIB19) and PE-Cy7-labeled anti-HLA-DR antibody (BioLegend, clone L243) were used. The primary antibody for intracellular staining is this antibody or normal rabbit IgG (Cell Signaling Technology) at a final concentration of 1 μg / mL, and the secondary antibody is PE-labeled Affini Pure Donkey Anti-Rabbit IgG (H + L) (Jackson Immuno Research). It was used at a concentration of 3 μg / mL.

Results Evaluation of antibody binding activity The binding activity of this antibody to peptide antigens or protein antigens was evaluated by ELISA. As shown in Fig. 4, this antibody hardly reacts at any antibody concentration with the non-phosphorylated IRF5 peptide [corresponding to the 440th to 452nd amino acids of the IRF5 protein (RefSeq ID: NP_001092097, human isoform b)]. In contrast, the phosphorylated peptide (corresponding to the phosphorylated serine residue at position 446) reacted strongly and was concentration-dependent (Fig. 4, left). Similar results were obtained in the evaluation of binding activity to non-phosphorylated or S446 phosphorylated IRF5 proteins (Fig. 4, right). From the above results, it was shown that this antibody has a binding activity specific to the S446 phosphorylated form for both the peptide and protein antigens of IRF5.

In CAL-1 cells ¹ is evaluated human plasmacytoid dendritic cell lines endogenous phosphorylation IRF5 in human immune cell lines, ⁴ IRF5 in innate immune response through TLR have been reported to be activated .. Immunoblotting was used to analyze the phosphorylation of endogenous IRF5 when CAL-1 cells were stimulated with TLR7 and R-848, a ligand for TLR8. Anti-GAPDH antibody, anti-IRF5 antibody, and this antibody were used as the primary antibody. As a result, IRF5 phosphorylation was significantly induced by R-848 stimulation (p-IRF5 panel in Fig. 5). On the other hand, this induction was not detected in IRF5-deficient CAL-1 cells (KO). From the above results, it was shown that this antibody is an antibody that specifically recognizes the phosphorylation of endogenous IRF5 during TLR stimulation of human immune cell lines.

Evaluation of Endogenous Phosphorylated IRF5 in Human Primary Immune Cells Human peripheral blood mononuclear cells (PBMCs) include immune cells that express IRF5, such as monocytes and B cells. As a result of isolating PBMC from the peripheral blood of healthy subjects and analyzing the phosphorylation of IRF5 during R-848 stimulation by immunoblotting, IRF5 phosphorylation was also induced by R-848 stimulation in human PBMC (Fig. 6). .. It can be said that the expression level of the entire IRF5 protein did not change before and after the stimulation, and the result of the increase in the IRF5 protein by the stimulation was not seen. From the above results, it is suggested that this antibody can evaluate the phosphorylation of endogenous IRF5 during TLR stimulation of human primary immune cells.

Evaluation of endogenous phosphorylated IRF5 in mouse primary immune cells Mouse spleen cells contain immune cells that express IRF5, such as dendritic cells and B cells, and these cells contain IRF5-dependent immunity via TLR. Responses have been shown to ^{provoke 3,5} . Immunoblotting analysis of endogenous IRF5 phosphorylation when wild-type mouse spleen cells were stimulated with TLR7 and TLR8 ligands (R-848) or TLR9 ligands (CpG-B ODN) showed that both stimuli were present. It significantly induced phosphorylation of IRF5 (Fig. 7). On the other hand, this induction was not detected in spleen cells derived from IRF5-deficient mice. From the above results, it was shown that this antibody is an antibody that specifically recognizes the phosphorylation of endogenous IRF5 during TLR stimulation of mouse immune cells. In addition, this result indicates that this antibody can specifically bind not only to the human active IRF5 protein but also to the mouse active IRF5 protein.

Analysis of Phosphorylated IRF5-Positive Cell Population in Human Primary Immune Cells There are no reports of single-cell level analysis of IRF5 phosphorylation in human primary immune cells. PBMC was isolated from the peripheral blood of normal subjects, and the phosphorylation of IRF5 in each cell type when stimulated with R-848 was analyzed by flow cytometry using this antibody. As a result, plasmacytoid dendritic cells (pDC), It was found that IRF5 is phosphorylated in most cells of classical dendritic cells (cDC) and monocytes (Mo) (Fig. 8). The degree of phosphorylation of IRF5 in R-848-stimulated B cells was weaker than that of the above cell types. These results suggest that it is possible to analyze the phosphorylated IRF5-positive cell population in human primary immune cells by using this antibody.

Analysis of Phosphorylated IRF5 in SLE Model Mice The inventors have shown that IRF5 is in an activated state in CD11c ⁺ cells (mainly dendritic cells) of Lyn-deficient mice, which is one of the SLE model mice. ⁶ shown in the past in a way that does not use an anti-phosphorylated antibody as immunoblotting. IRF5 phosphorylation in spleen CD11c ⁺ cells of 18-20 week old female Lyn-deficient mice was analyzed by immunoblotting using this antibody. As a result, IRF5 was significantly phosphorylated compared to wild-type mice. (Fig. 9). Similar results were obtained with the widely used SLE model mouse, the NZB / W F1 mouse. From the above results, it is suggested that the active IRF5 in SLE model mice can be analyzed by using this antibody.

Literature
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3. Takaoka, A. et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434, 243-249, doi: 10.1038 / nature03308 (2005).
4. Steinhagen, F. et al. IRF-5 and NF-κB p50 co-regulate IFN-β and IL-6 expression in TLR9-stimulated human plasmacytoid dendritic cells. Eur J Immunol 43, 1896-1906, doi: 10.1002 / eji.201242792 (2013).
5. Savitsky, DA, Yanai, H., Tamura, T., Taniguchi, T. & Honda, K. Contribution of IRF5 in B cells to the development of murine SLE-like disease through its transcriptional control of the IgG2a locus. Proc Natl Acad Sci USA 107, 10154-10159, doi: 10.1073 / pnas.1005599107 (2010).
6. Ban, T. et al. Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity. Immunity 45, 319-332, doi: 10.1016 / j.immuni.2016.07.015 (2016) ).

Claims (13)

A monoclonal antibody or antibody fragment that binds to the active IRF5 protein and does not bind to the inactive IRF5 protein at a detectable level, and is the amino acid shown in CDR1 and SEQ ID NO: 3 of the amino acid sequence shown in SEQ ID NO: 2. The heavy chain variable region containing CDR2 of the sequence and CDR3 of the amino acid sequence shown in SEQ ID NO: 4, CDR1 of the amino acid sequence shown in SEQ ID NO: 6, CDR2 of the amino acid sequence shown in SEQ ID NO: 7, and the amino acid sequence shown in SEQ ID NO: 8. A monoclonal antibody or antibody fragment comprising a light chain variable region containing CDR3 of. The active IRF5 protein is an IRF5 protein in which a serine residue corresponding to serine No. 446 of human IRF5 isoform b (SEQ ID NO: 17) is phosphorylated, and the inactive IRF5 protein has the serine residue. The monoclonal antibody or antibody fragment according to claim 1, which is an IRF5 protein that is not phosphorylated. The monoclonal antibody or antibody fragment according to claim 1 or 2, wherein the sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 1, and the sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 5. Activation of the IRF5 protein, which comprises contacting the monoclonal antibody or antibody fragment according to any one of claims 1 to 3 with a sample containing the IRF5 protein to immunologically detect the active IRF5 protein. How to analyze the state. The method according to claim 4, wherein the sample is a sample isolated from a patient having or may have an autoimmune disease. The method of claim 5, wherein the autoimmune disease is systemic lupus erythematosus, Sjogren's syndrome, or systemic scleroderma. A detection kit for an active IRF5 protein, which comprises the monoclonal antibody or antibody fragment according to any one of claims 1 to 3. A method for producing a monoclonal antibody or antibody fragment that binds to an active IRF5 protein but does not bind to an inactive IRF5 protein at a detectable level.
Immunization of non-human animals with phosphorylated IRF5 peptide, immune process;
From B cells of non-human animals after immunization, prepare a cDNA encoding a region containing a heavy chain variable region and a cDNA encoding a region containing a light chain variable region, and prepare a single chain antibody (scFv) gene or a Fab fragment gene. To prepare a library of vectors expressing scFv or Fab by incorporating this into a plasmid vector for phage, and to prepare a library of phage that presents each scFv or each Fab on the surface by the phage display method. Library preparation step; and positive selection with phosphorylated IRF5 peptide and active IRF5 protein, and negative selection with non-phosphorylated IRF5 peptide and inactive IRF5 protein, phosphorylated IRF5 peptide and active form The IRF5 peptide comprises a panning step that concentrates phages with high specific binding to the IRF5 protein, said IRF5 peptide containing a serine residue corresponding to serine No. 446 of human IRF5 isoform b (SEQ ID NO: 17). A method, which is a peptide essentially consisting of a partial sequence of a protein, wherein the phosphorylated IRF5 peptide is a peptide in which a serine residue corresponding to the above-mentioned No. 446 serine is phosphorylated. The IRF5 peptide is a peptide with a size of 7 to 40 residues consisting essentially of an IRF5 protein partial sequence containing LQISNPD (SEQ ID NO: 24), and the phosphorylated IRF5 peptide is the fourth serine in LQISNPD. The method of claim 8, wherein the residue is a phosphorylated IRF5 peptide. The method of claim 9, wherein the IRF5 peptide is a peptide sizing of 13-40 residues, essentially consisting of an IRF5 protein partial sequence comprising SIRLQISNPDLKD (SEQ ID NO: 23). The method according to any one of claims 8 to 10, wherein in the immunization step, a non-human animal is immunized with a phosphorylated IRF5 peptide to which a carrier protein is bound. The method according to any one of claims 8 to 11, wherein the phosphorylated IRF5 peptide used for immunization of non-human animals has a purity of 85% or more. The method according to any one of claims 8 to 12, wherein the number of times of immunization with the phosphorylated IRF5 peptide to the non-human animal is 6 times or more.