JP2015189715A - Monoclonal antibodies against pb2 subunit of rna-dependent rna polymerase of influenza virus - Google Patents

Abstract

Disclosed is a monoclonal antibody against RNA-dependent RNA polymerase of influenza virus, which does not neutralize influenza virus.
A monoclonal antibody or antigen-binding fragment thereof that does not neutralize the influenza virus by reacting with an antigen antibody with the PB2 subunit of RNA-dependent RNA polymerase of influenza virus is provided.
[Effects] Monoclonal antibodies can be used not only to confirm the expression of influenza virus RNA-dependent RNA polymerase, but also do not inhibit the growth of influenza viruses. Useful as a tool for.
[Selection] Figure 1

Description

  The present invention relates to a monoclonal antibody against the PB2 subunit of influenza virus RNA-dependent RNA polymerase (hereinafter sometimes referred to as “iRdRP”).

  New influenza and avian influenza H5N1 and H7N9 are worried about a global pandemic, and specific measures are needed on a global scale, including Japan. Japan has also launched a full-scale countermeasure against the new influenza virus since last fiscal year, and pre-pandemic vaccines that have already been formulated based on avian influenza (H5N1, H7N9) that are highly likely to be mutated to new influenza. We stockpile Tamiflu and Relenza, which are virus agents. According to a report from the Ministry of Health, Labor and Welfare, if a pandemic occurs in Japan, it is estimated that 32 million people will be infected and a maximum of 640,000 people will die. From this point of view, iRdRP, which causes almost no mutation, has attracted attention as an important drug discovery target from various research institutions including pharmaceutical companies around the world.

  Very few monoclonal antibodies against iRdRP are currently on the market. If there is a monoclonal antibody against iRdRP that does not neutralize influenza virus, that is, does not inhibit the growth of influenza virus, it can be used not only for iRdRP expression confirmation, but also after binding the antibody Can be cultured and propagated as is, and the complex of iRdRP and anti-influenza virus drug candidate can be detected. However, such monoclonal antibodies are not known.

Fujimoto Y et al., Vet J. 2013 Nov; 198 (2): 487-93.doi: 10.1016 / j.tvjl.2013.09.019. Epub 2013 Sep 21 MacDonald LA et al., Virology. 2012 Apr 25; 426 (1): 51-9.doi: 10.1016 / j.virol.2012.01.015. Epub 2012Feb 8. Hatta M et al., Arch Virol. 2000; 145 (9): 1947-61. Ochoa M et al., Virus Res. 1995 Aug; 37 (3): 305-15. Barcena J et al., J Virol. 1994 Nov; 68 (11): 6900-9.

  Accordingly, an object of the present invention is to provide a novel monoclonal antibody against iRdRP that does not neutralize influenza virus.

  As a result of intensive studies, the present inventors have succeeded in producing a novel monoclonal antibody against the PB2 subunit of iRdRP, which does not neutralize influenza virus, and completed the present invention.

  That is, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that reacts with an antigen antibody with the PB2 subunit of RNA-dependent RNA polymerase of influenza virus and does not neutralize the influenza virus.

  The present invention provides for the first time a monoclonal antibody against iRdRP that does not neutralize influenza virus. The monoclonal antibody of the present invention can be used not only for confirming the expression of iRdRP, but also does not inhibit the growth of influenza virus, so it is useful as a tool for molecular biological research of influenza virus and development of anti-influenza virus agents. .

It is a figure which shows the result of the western blotting method using the monoclonal antibody of this invention performed in the Example of this invention. It is a figure which shows the result of the immunoprecipitation method using the monoclonal antibody of this invention performed in the Example of this invention. It is a figure which shows the result of the binding experiment using the monoclonal antibody of this invention performed in the Example of this invention.

  As described in detail in the Examples below, the monoclonal antibody of the present invention is a genetically engineered preparation of a partial region on the C-terminal side of the PB2 subunit of iRdRP (hereinafter simply “PB2”), which is used to immunize mice. Then, a hybridoma was prepared by a conventional method. Among the obtained hybridomas, those that produced monoclonal antibodies having affinity for PB2 but not inhibiting the growth of influenza virus were screened, and the obtained hybridomas were cultured. The monoclonal antibody was recovered from the supernatant.

  As specifically described in the following examples, the base sequences of the genes encoding the variable regions of the heavy and light chains of the monoclonal antibodies obtained in the following examples and the amino acid sequences encoded by them were determined. SEQ ID NO: 1 is the base sequence of the gene encoding the H chain variable region, SEQ ID NO: 2 is the amino acid sequence encoded by this gene, SEQ ID NO: 3 is the base sequence of the gene encoding the L chain variable region, SEQ ID NO: 4 is The amino acid sequence encoded by this gene is shown.

  In the amino acid sequence shown in SEQ ID NO: 2, the region from the 18th glycine to the 25aa tryptophan from the N-terminal (hereinafter referred to as “18aa”), that is, GYTFTDYW is CDR1 (complementary in the variable region of the H chain) Sex-determining region 1 (complementarity-determining region 1)) sequence, 43 aa isoleucine to 50 aa threonine, ie, CDR2 sequence in variable region of IDTSDSYT heavy chain, 89 aa alanine to 97 aa tyrosine region, That is, ARWYDEIDY is the CDR3 sequence in the variable region of the heavy chain.

  In the amino acid sequence shown in SEQ ID NO: 4, the region from 19aa lysine to 28aa tyrosine, that is, KSVSTSGYSY is the CDR1 sequence in the light chain variable region, the region from 46aa leucine to 48aa serine, ie, LVS The CDR2 sequence in the light chain variable region, the region from 85aa glutamine to 93aa serine, ie, QHIRELTRS is the CDR3 sequence in the light chain variable region.

  As is well known, the corresponding epitope and affinity of an antibody are determined by the complementarity determining region (CDR). Therefore, a monoclonal antibody having the same properties as the monoclonal antibody of the present invention, that is, a monoclonal antibody that reacts with PB2 as an antigen and does not inhibit the growth of influenza virus, is a heavy chain of a gene of a monoclonal antibody that does not have such properties. In addition, each CDR1 to CDR3 region in the region encoding the variable region of the L chain can be generated by converting each of the above CDR1 to CDR3 regions. In addition, although each CDR1-CDR3 region may be individually converted into each of the CDR1-CDR3 regions described above, a region including each of these regions may be converted. For example, the entire variable region of the H chain is arranged. It may be converted into a gene having the base sequence shown by No. 2, and the entire variable region of the L chain may be converted into a gene having the base sequence shown by SEQ ID No. 4. In this case, the recombination operation becomes easier. The monoclonal antibody of the present invention can be produced by culturing a hybridoma having such a recombinant gene by a conventional method and recovering the monoclonal antibody of the present invention from the culture supernatant or ascites by a conventional method. Usually, the variable region (CH1) including CDR1 to CDR3 regions of IgH (heavy chain) is transferred to, for example, human IgG1 expression vector pFUSE-CHIg-hG1 (trade name, InvivoGen), and IgL (light chain). For example, pFUSE2-CLIg-hk (trade name, InvivoGen), which is a human IgL-kL expression vector, is transferred to the variable region including the CDR1 to CDR3 regions. These two genes can be expressed in CHO cells and the antibody can be recovered. This is a conventional method called chimerization. In addition, since the nucleotide sequence of the variable region has been clarified as shown in SEQ ID NO: 1 and SEQ ID NO: 3, it can be easily prepared by a conventional method such as a genetic engineering technique. Of course, the monoclonal antibody of the present invention can also be produced by the same method as in Examples described later.

The present invention provides not only the monoclonal antibodies described above, but also antigen-binding fragments thereof. The antigen-binding fragment is a fragment that maintains the binding property of this antibody, such as a Fab fragment or F (ab ′) ₂ fragment, and can be obtained from a monoclonal antibody by a known method. Single chain antibodies are also taken to be encompassed by antigen-binding fragments. Further, for example, a humanized antibody obtained by converting only the constant region of the monoclonal antibody of the present invention into another constant region includes the antigen-binding fragment of the monoclonal antibody of the present invention, and thus is included in the scope of the present invention.

  Since the monoclonal antibody of the present invention or the antigen-binding fragment thereof reacts with PB2 by antigen-antibody, it can be subjected to various known immunoassays to detect PB2. The immunoassay method itself is well known and can be used for various well-known methods such as sandwich method, aggregation method, competition method, western blot method, immunoprecipitation method, immunostaining method and the like. In addition, antibody labeling methods are well known, and the monoclonal antibody of the present invention can be bound with a label such as a fluorescent label, an enzyme label, a chemiluminescent label, a biotin label, a radiolabel, and the like by a conventional method.

  Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

1. Construction of PB2 protein expression system The PB2 gene of influenza RNA polymerase was amplified by PCR using cDNA (influenza / Puerto Rico / 8/1934, H1N1) incorporated into pET14b (available from Addgene) as a template. The gene region expressing PB2 protein 530-759 was treated with restriction enzymes BamHI and NotI, and ligated with a commercially available pET28a vector (Novagen). SD (Shine Dargarno) sequence, starting ATG, histidine tag and TEV (tobacco etch virus) protease cleavage site DNA sequence are added between the XbaI and BamHI sites of the commercially available pET28a vector, and the N-terminal side of the expressed PB2 protein An expression system plasmid was constructed so that a histidine tag and a TEV protease cleavage site were added to.

2. Expression of PB2 protein Using the prepared plasmid DNA pET28a-PB2 (530-759), E. coli BL21 (DE3) codonplus RILP (trade name, manufactured by Merck) for protein expression was transformed with 40 μg / ml of antibiotic kanamycin. The cells were cultured at 37 ° C. for 16 hours on an LB plate medium containing 35 μg / ml of chloramphenicol. The cultured E. coli colonies were shake-cultured in 1 L of LB liquid medium containing 50 μg / ml kanamycin and 35 μg / ml chloramphenicol. The culture was started at 37 ° C., and cooled on ice for 1 hour when the OD ₆₀₀ value reached 0.6. After cooling, a final concentration of 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added, and shake-cultured at 15 ° C. for 16 hours to express the protein. The culture solution was centrifuged at 5,000 rpm for 15 minutes to separate the culture supernatant and the cells, and only the cells were collected.

3. Purification of PB2 protein Mass cultured cells are suspended in a cell disruption buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 500 mM urea, 25 mM imidazole, 10 mM 2-mercaptoethanol). The product was crushed using (trade name Vibra cell). The crushing conditions were set to Amplitude: 80, Pulse: 1 second, Time: 10 minutes, and this was performed for 2 cycles. The crushed liquid was centrifuged at 19,000 rpm for 30 minutes, and the supernatant was recovered.

  The supernatant was filtered with a 0.45 μm filter and equilibrated with Ni-NTA A buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 500 mM urea, 25 mM imidazole, 10 mM 2-mercaptoethanol). Ni-NTA superflow resin (QIAGEN) was mixed to adsorb the protein to the resin. The resin / protein mixture was mixed by inverting at 4 ° C. for 30 minutes, and then the resin was washed with 400 ml of Ni-NTA A buffer. After washing, 60 ml of Ni-NTA B buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 500 mM urea, 500 mM imidazole, 10 mM 2-mercaptoethanol) was added to elute the protein from the resin. In order to remove the histidine tag from the PB2 protein, 3 ml of 21 μm TEV protease was added to 60 ml of the eluate from Ni-NTA resin and reacted at 20 ° C. for 12 hours. After the reaction, the protein was dialyzed against 2 L of Ni-NTA A buffer for 12 hours. Again, the dialyzed sample was mixed with Ni-NTA resin equilibrated with Ni-NTA A buffer, and the flow-through fraction from the resin was collected to purify the protein. The flow-through fraction was dialyzed against Q column A buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT) for 12 hours. The dialyzed protein solution was purified by Hi Trap Q-column (GE Healthcare) equilibrated with Q column A buffer. Purification was performed using AKTA purifier (GE Healthcare), and the flow-through fraction from the Q column was collected. The purified protein solution is dialyzed against 2 L of the final buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT) for 12 hours and concentrated using a centrifugal concentration tube centricon YM-10 (MILLIPORE). did.

4). Preparation of hybridoma PB2 3-1.6 The RNA-dependent RNA polymerase PB2 subunit (530-759) protein derived from the above-prepared and purified influenza virus H1N1 was emulsified with TiterMax Gold (CytRx), and BALB / c mice (8 weeks old) , Female) was injected subcutaneously and immunization was started. After a total of 4 immunizations at 2-week intervals, blood was collected, and the cell lysate expressed in 293T cells was fused with an immunoblot (PB2 subunit (530-759) and myc3 tag fused to SDS. The antibody titer was confirmed by electrophoresis by PAGE and electrophoresis of a cell lysate of 293T cells into which no gene had been introduced for control.

After preparation of spleen cells from immunized mice in which an increase in antibody titer was confirmed, the method by Galfre et al. (Galfre, G. and Milstein, C .: Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol., 73: 3- 46, 1981), fusion with mouse myeloma cells P3 / NSI / 1-Arg-1 (ATCC TIB-18) was performed using polyethylene glycol. The ratio of spleen cells / myeloma cells to be mixed was spleen cells: myeloma = 10: 1. This was layered on feeder cells (peritoneal exudate cells) that had been put in a 96-well culture plate the day before and cultured in a carbon dioxide incubator (37 ° C., 5% CO ₂ ). On the next day of culture, cells were cultured using 20% FCS (fetal calf serum) and HAT (hypoxanthine, aminopterin, thymidine) (ICN) RPMI1640 medium (Nissui) and selection was started. Two weeks later, after collecting the culture supernatant of wells containing cells grown in HAT selective medium, ELISA using the RNA-dependent RNA polymerase PB2 subunit (530-759) protein derived and purified from influenza virus H1N1 ( Antibody production was confirmed and the antibody titer was evaluated by enzyme-linked immunosorbent assay. A fused cell (hybridoma) producing an ELISA positive antibody was further cloned three times by limiting dilution to obtain a hybridoma producing a monoclonal antibody. Hereinafter, this hybridoma is referred to as “hybridoma PB2 3-1.6”, and the monoclonal antibody produced by the hybridoma is referred to as “PB2 3-1.6 antibody”.

5. Determination of monoclonal antibody subtype The antibody class of the obtained monoclonal antibody (PB2 3-1.6 antibody) was evaluated by enzyme immunoassay using IsoStrip mouse monoclonal antibody isotyping kit (Roche). Was confirmed.

6). Western blotting The obtained monoclonal antibody (PB2 3-1.6 antibody) was subjected to Western blotting. Specifically, it was performed as follows. The cell lysate was electrophoresed on SDS-polyacrylamide gel, and then transferred to a PVDF (polyvinylidene fluoride) membrane (Millipore) by the semi-dry method. After blocking the protein-transferred membrane with PBS containing 5% nonfat dry milk and 0.1% Tween-20, each specific antibody solution is bound to the transfer membrane as a primary antibody, and excess with PBS containing 0.1% Tween-20. The primary antibody was washed off well. Further, a secondary antibody labeled with HRP (horseradish peroxidase) was bound to the transfer membrane, and the excess secondary antibody was thoroughly washed away with PBS containing 0.1% Tween-20. Using a fluorescent reagent that emits light when decomposed (Western Lightning Chemiluminescence reagent, Perkin Elmer) as a substrate for HRP, it is exposed to X-ray film (Fuji Film) by fluorescence from the target protein band on the transfer film. And detected.

  The results are shown in FIG. 1 (in FIG. 1, M is a molecular weight marker). As shown in FIG. 1, in Western blotting, the PB2 3-1.6 antibody recognized the PB2 subunit (530-759) protein expressed in cells stronger than the existing myc tag antibody (9E10). That is, it was revealed that the PB2 3-1.6 antibody can be used in Western blotting.

7). Immunoprecipitation method The obtained monoclonal antibody (PB2 3-1.6 antibody) was subjected to immunoprecipitation. Specifically, it was performed as follows. Each specific antibody was bound to protein A sepharose beads (GE Healthcare Japan) and the excess antibody was washed off well. The beads to which the specific antibody was bound were added and bound to each cell lysate, and the proteins other than the protein that specifically bound to the beads were thoroughly washed out. The beads were suspended in 2 × SDS sample buffer and treated at 100 ° C. for 5 minutes to prepare a sample. The sample was electrophoresed on SDS-polyacrylamide gel, and then transferred to a PVDF (polyvinylidene fluoride) membrane (Millipore) by a semi-dry method. After blocking the protein-transferred membrane with PBS containing 5% nonfat dry milk and 0.1% Tween-20 (trade name), each specific antibody solution is bound as a primary antibody to the transfer membrane, and contains 0.1% Tween-20 Excess primary antibody was washed off thoroughly with PBS. Further, a secondary antibody labeled with HRP (horseradish peroxidase) was bound to the transfer membrane, and the excess secondary antibody was thoroughly washed away with PBS containing 0.1% Tween-20. Using a fluorescent reagent that emits light when decomposed (Western Lightning Chemiluminescence reagent, Perkin Elmer) as a substrate for HRP, it is exposed to X-ray film (Fuji Film) by fluorescence from the target protein band on the transfer film. And detected.

  The results are shown in FIG. 2 (in FIG. 2, M is a molecular weight marker). As shown in Fig. 2, PB2 3-1.6 antibody recognizes PB2 subunit (530-759) protein expressed in cells as strongly as or better than existing myc antibody (9E10) and is capable of immunoprecipitation. there were. In other words, it was revealed that the PB2 3-1.6 antibody is a monoclonal antibody that can recognize and immunoprecipitate the PB2 subunit (530-759).

8). Binding experiments Antigen protein PB2 was bound to PB2 3-1.6 monoclonal antibody obtained by isolation and purification of immunoglobulin G (IgG) from the ascites of mice transplanted with hybridomas, and the total molecular weight of the complex was measured. Went. As the measuring instrument, an analytical ultracentrifuge (AUC) was used to measure the molecular weight.

  The results are shown in FIG. As shown in FIG. 3, it was clarified that the PB2 3-1.6 monoclonal antibody and the antigen protein PB2 were strongly bound.

9. Inhibition of influenza virus growth
It was tested whether the PB2 3-1.6 monoclonal antibody inhibited the growth of influenza virus. This was done as follows. The PB2 3-1.6 monoclonal antibody isolated and purified as immunoglobulin G (IgG) from the ascites of the mouse transplanted with the hybridoma was further purified as a Fab fragment using an ImmunoPure Fab Preparation kit (Pierce). Then, using the Alexa Fluor 488 Protein Labeling Kit (Molecular Probe), the PB2 3-1.6 monoclonal antibody purified as a Fab fragment was labeled with Alexa Fluor 488. The control antibody G196 having the same isotype was similarly performed.

  Fab labeled with Alexa Fluor 488 to Madin Darby Canine Kidney (MDCK) cell line (purchased from JCRB cell bank. JCRB9029) using bead method (McNeil, PL. Methods Cell Biol., 29: 153-173, 1989). Fragments were introduced. 30 minutes later, influenza virus A / Aichi / 2/68 (H3N2) was infected. After 30 minutes, excess virus was washed away, and after 7 hours, a sprouting virus on the cell surface was detected using a polyclonal antibody (homemade) that recognizes influenza virus A / Aichi / 2/68 (H3N2).

  As a result, similar to the control antibody G196 having the same isotype, it was revealed that the PB2 3-1.6 monoclonal antibody does not inhibit the growth of influenza virus.

10. Gene analysis of monoclonal antibody (PB2 3-1.6 antibody) RNA was extracted from the obtained hybridoma PB2 3-1.6, and then reverse-transcribed with an oligo-dT primer to prepare cDNA. Using this cDNA as a template, PCR is performed using the following H chain primer set and L chain primer set, nucleotides encoding the H chain variable region and amino acid sequences thereof, and nucleotides encoding the L chain variable region and amino acids thereof The sequence was analyzed. The base sequence of each primer set was as follows.

<H chain primer set>
VH1-1S: 5'-gg ggatcc ag gts mar ctg cag sag tcw gg-3 '(SEQ ID NO: 5)
s = g + c, m = a + c, r = a + g, w = a + t
IgG2-1AS: 5'-gg gaattc ctt gac cag gca tcc tag agt ca-3 '(SEQ ID NO: 6)

<L chain primer set>
VK-1S: 5′-gg ggatcc gay att gtg mts acm car wct mca -3 ′ (SEQ ID NO: 7)
y = c + t, m = a + c, s = g + c, r = a + g, w = a + t
CK-2AS: 5'-gg gaattc gaa gat gga tac agt tgg tgc-3 '(SEQ ID NO: 8)

  The nucleotide sequence of the gene encoding the H chain variable region is SEQ ID NO: 1, the amino acid sequence encoded by this gene is SEQ ID NO: 2, the nucleotide sequence of the gene encoding the L chain variable region is SEQ ID NO: 3, The amino acid sequence encoded by the gene is shown in SEQ ID NO: 4.

Claims (5)

  A monoclonal antibody or an antigen-binding fragment thereof that reacts with an antigen antibody with the PB2 subunit of RNA-dependent RNA polymerase of influenza virus and does not neutralize the influenza virus.   The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of CDR1 in the variable region of the heavy chain is GYTFTDYW, the amino acid sequence of CDR2 is IDTSDSYT, and the amino acid sequence of CDR3 is ARWYDEIDY.   The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the variable region of the heavy chain is represented by SEQ ID NO: 2.   The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the amino acid sequence of CDR1 in the variable region of the light chain is KSVSTSGYSY, the amino acid sequence of CDR2 is LVS, and the amino acid sequence of CDR3 is QHIRELTRS. .   The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the amino acid sequence of the variable region of the L chain is represented by SEQ ID NO: 4.