Rabbit monoclonal antibodies with asialogm1 binding activity

Abstract

To provide anti-ASGM1 monoclonal antibodies of which administration to mice and rats reduces the NK activity in vivo and / or to provide IgG type anti-ASGM1 monoclonal antibodies useful for immunoassay to detect NK cells.SOLUTION: An IgG type rabbit anti-ASGM1 monoclonal antibody that exhibits very strong in vivo inhibition of NK activity when administered to a mouse can be obtained with high efficiency by immunizing an animal not belonging to Rodentia with ASGM1 and preparing a monoclonal antibody from the lymphocytes of the immunized animal.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a rabbit monoclonal antibody reactive to asialoGM1 (hereinafter sometimes referred to as "ASGM1"), an anti-ASGM1 monoclonal antibody that has a strong inhibitory effect on NK activity in vivo when administered to mice and rats, and a method for obtaining said antibody. [Background technology]

[0002] Natural killer cells (hereinafter sometimes referred to as "NK cells") are involved in innate immunity and play an important role in removing virus-infected cells and cancer cells. NK cells can attack various cancer cells spontaneously without prior immunity.

[0003] ASGM1 is a glycolipid present on the surface of mouse and rat NK cells and is considered a marker for NK cells. Rabbit antisera and polyclonal antibodies against ASGM1 have been obtained and are used in NK cell detection assays by immunological techniques such as flow cytometry and immunohistochemistry.

[0004] In addition, antisera and polyclonal antibodies against ASGM1 are known to have an inhibitory effect on NK activity in mice and rats. "NK activity" refers to the cytotoxic activity of NK cells, and the value of the activity can be measured by coexisting NK cells with target cells for NK cells such as YAC-1 cells, quantifying the labeled substances released from target cells that have been labeled with radioactive substances in advance, or measuring the activity of lactate dehydrogenase (LDH) leaked from damaged target cells.

[0005] For example, when a different organ or cancer cell is transplanted into an immunodeficient mouse such as a SCID mouse, the rate of adhesion of the transplanted organ or cell is reduced due to NK activity, but administration of anti-ASGM1 rabbit antiserum to these mice suppresses NK activity and improves the rate of adhesion. For this reason, rabbit antiserum and polyclonal antibodies against ASGM1 have been used in NK cell research and in suppressing NK activity in model animals.

[0006] In these assays and NK cell research, rabbit antisera and polyclonal antibodies against ASGM1 have been used in the past. However, antisera and polyclonal antibodies have problems such as cross-reactivity with glycolipids with related molecular structures, such as monosialoganglioside (GM1), as well as differences between antibody lots, and there have been concerns about the continuity of production and supply.

[0007] On the other hand, in order to solve the above problems, attempts have been made to produce anti-ASGM1 monoclonal antibodies that have the effect of suppressing NK activity, mainly using mice.

[0008] However, in mice and rats, ASGM1 is naturally present on the surface of normal tissues and cells, and therefore ASGM1 is an autoantigen for mice and rats, and it was not possible to obtain IgG-type anti-ASGM1 antibodies with high binding affinity from these animals. Therefore, conventional anti-ASGM1 monoclonal antibodies were limited to mouse IgM, not IgG. In general, IgM antibodies have lower affinity than IgG antibodies, so the lack of IgG-type monoclonal antibodies was also an issue in detecting ASGM1. In addition, as described in detail below, these conventionally obtained IgM-type anti-ASGM1 mouse monoclonal antibodies could not be said to actually exhibit NK activity suppression effects when administered to mice and rats.

[0009] Conventionally, an example of an anti-ASGM1 mouse monoclonal antibody is an IgM monoclonal antibody produced from an autoimmune mouse immunized with ASGM1 (Patent Document 1). Patent Document 1 describes that when the four obtained antibody clones were reacted with mouse splenocytes in vitro together with complement, the antibody was only able to reduce the antibody titer of splenocytes to 75-38% of that of untreated cells.

[0010] The same group also reported that when they produced five clones of IgM monoclonal antibodies from autoantibody mice immunized with ASGM1 and reacted the antibodies with complement in an in vitro experimental system, they were only able to reduce the antibody concentration to a maximum of 26.2% of the control when the ascites was diluted 3-fold (the antibody concentration in the ascites was reported to be 1.0-1.9 mg / mL, which corresponds to 333-633 μg / mL) (Non-Patent Document 1).

[0011] Additionally, a mouse IgM monoclonal antibody against ASGM1 has been reported (Non-Patent Document 2). This monoclonal antibody reacts with ASGM1, but also cross-reacts with ASGM3 and GM1. It has also been shown that when this antibody is allowed to act on mouse splenocytes in vitro together with complement, NK activity can be suppressed. It has been shown that the effect was observed at a 1 / 60 dilution of ascites containing the monoclonal antibody (the antibody concentration in this ascites is described as 1.8 mg / mL, which corresponds to 30 μg / mL).

[0012] Other reports include the production of IgM-type anti-ASGM1 monoclonal antibodies, but no evidence of any inhibitory effect on NK activity (Non-Patent Document 3), and the presence or absence of any inhibitory effect on NK activity (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Patent Publication No. 62-029599 [Non-patent literature]

[0014] [Non-Patent Document 1] Preparation of monoclonal antibodies against a glycolipid asialo GM1. S Shimada, D Iwata. 1987, Microbiol Immunol, 31, 923-933. [Non-Patent Document 2] A monoclonal antibody with reactivity to asialo GM1 and murine natural killer cells. FR Solomon, TJ Higgins. 1987, Mol. Immunol., 24, 57-65 [Non-Patent Document 3] Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer. S Watarai, S Handa, T Tadakuma, T Yasuda. 1987, J. Biochem., 102, 59-67. [Non-Patent Document 4] Production and characterization of a mouse monoclonal antibody to the glycolipid asialo-GM1. F Jacquemart, G Millot, C Goujet-Zalc, G Mahouy, B Zalc. 1988, Hybridoma, 7, 323-331. [Non-Patent Document 5] From rabbit antibody repertoires to rabbit monoclonal antibodies. Justus Weber, Haiyong Peng, Christoph Rader. Exp Mol Med, 2017, 24, 49, e305. Summary of the Invention [Problem to be solved by the invention]

[0015] As mentioned above, there have been reports of mouse IgM monoclonal antibodies against ASGM1, but their NK activity inhibitory effect was not observed or was only demonstrated in in vitro experimental systems.

[0016] To suppress NK activity in model animal experimental systems, such as improving the take-up rate of tumor tissue transplanted into mice, it is useful to have an antibody that is effective when administered to the animal in question (i.e., in vivo); however, no anti-ASGM1 monoclonal antibody that can reduce NK activity in vivo when administered to mice or rats has been reported.

[0017] Furthermore, to specifically detect NK cells in immunological assays such as immunohistochemical staining and flow cytometry, a monoclonal antibody with higher specificity than a polyclonal antibody is desirable. In addition, it is considered useful to use an IgG-type monoclonal antibody with higher affinity; however, no IgG-type anti-ASGM1 monoclonal antibodies have been reported to date.

[0018] Therefore, the objective of the present invention is to obtain an anti-ASGM1 monoclonal antibody that can reliably reduce NK activity in vivo when administered to mice and rats, and / or an IgG type anti-ASGM1 monoclonal antibody that is useful in immunological assays to detect NK cells. [Means for solving the problem]

[0019] In view of the above-mentioned problems, the inventors conducted extensive research into methods for producing monoclonal antibodies that have a strong inhibitory effect on NK activity, and as a result, they discovered that by producing monoclonal antibodies from lymphocytes of a non-rodent animal that has been immunized with ASGM1, it is possible to obtain with extremely high efficiency IgG-type rabbit anti-ASGM1 monoclonal antibodies that exhibit a strong inhibitory effect on NK activity in vivo when administered to mice, and thus completed the present invention. Effect of the Invention

[0020] The anti-ASGM1 monoclonal antibody of the present invention is the first anti-ASGM1 monoclonal antibody to be shown to reduce NK activity in vivo when administered to mice and rats. Even when administered to mice and rats at low doses, it shows a sufficiently strong NK activity suppressing effect. Furthermore, it is a rabbit IgG type anti-ASGM1 monoclonal antibody.

[0021] For these reasons, the anti-ASGM1 monoclonal antibody of the present invention has extremely superior characteristics when used to suppress NK activity in model animal experimental systems or in immunological assays to detect NK cells, compared to conventionally used antisera and polyclonal antibodies and conventionally obtained IgM-type mouse anti-ASGM1 monoclonal antibodies.

[0022] Furthermore, according to the method for producing a monoclonal antibody of the present invention, it is possible to obtain an excellent anti-ASGM1 monoclonal antibody having the above-mentioned properties with extremely high efficiency. [Brief description of the drawings]

[0023] [Figure 1] Figure 1 shows the reaction of anti-ASGM1 monoclonal and polyclonal antibodies to immobilized ASGM1 and glycolipids with related structures. In the figure, ■: ASGM1, □: ASGM2, ▲: GM1, △: GM2, ◇: GM3, ●: GD1a, ◯: GD1b. [Diagram 2] Figure 2 shows the reaction of anti-ASGM1 monoclonal and polyclonal antibodies to ASGM1 and glycolipids with related structures developed by silica gel TLC. The results of orcinol staining of the TLC sheet developed under the same conditions are shown on the left side of the figure (Orcinol). Lane 1; GD1a, 2; GD1b, 3; GM1, 4; GM2, 5; GM3, 6; ASGM1, 7; ASGM2, 8; Lactosylceramide (ASGM3), arrow: indicates the position of ASGM1 development. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates to a novel anti-ASGM1 monoclonal antibody that exhibits an inhibitory effect on NK activity in vivo when administered to mice or rats, and a method for obtaining the same.

[0025] "In vivo" means "inside a living body" and refers to a test in which a test substance is administered directly into the living body of an experimental animal such as a mouse to detect the drug's reaction in the living body or within cells. In contrast, "in vitro" means "in a test tube" and refers to a test in which a drug reaction is detected by creating an environment similar to that inside the body in a test tube or incubator using human or animal tissue.

[0026] The antibody of the present invention has binding activity (reactivity) with ASGM1. The reactivity with ASGM1 can be evaluated by the ELISA method described in Example 1, for example.

[0027] The antibody of the present invention is derived from an animal other than rodents. Specific examples of animals include cows, horses, sheep, goats, pigs, camels, alpacas, dogs, rabbits, pigeons, and chickens, with rabbits being particularly preferred.

[0028] The antibody of the present invention is preferably an IgG antibody, which has higher affinity than conventionally obtained IgM antibodies and is useful in various assays.

[0029] Furthermore, the antibody of the present invention has an effect of suppressing NK activity in vivo when administered to mice or rats. In the present invention, "having an effect of suppressing NK activity in vivo" means that when the antibody is administered to mice or rats according to the method described in Example 5, the NK activity of splenocytes 3 days after administration is 20% or less of that of a control without administration. The method of administration to mice or rats may be any known method, and although intraperitoneal administration or intravenous administration is common, other methods are also possible, and any method of administration is included in the present invention. The type of mouse or rat is also not limited.

[0030] Furthermore, when the antibody of the present invention is administered at 100 μg per mouse according to the method described in Example 5, the NK activity of splenocytes 3 days after administration is 20% or less, preferably 10% or less, more preferably 5% or less, and even more preferably 0% of that of a no-administration control.

[0031] Similarly, when the antibody of the present invention is administered at 20 μg per mouse according to the method described in Example 5, the NK activity of splenocytes 3 days after administration is 20% or less, preferably 10% or less, more preferably 5% or less, and even more preferably 0% of that of a non-administered control.

[0032] Similarly, when the antibody of the present invention is administered at 4 μg per mouse according to the method described in Example 5, the NK activity of splenocytes 3 days after administration is 20% or less, preferably 10% or less, more preferably 5% or less, and even more preferably 0% of that of a non-administered control.

[0033] The antibody of the present invention preferably reacts with ASGM1 and does not substantially react with ASGM2, ASGM3, GM2, GM3, GD1a, and GD1b. Here, "does not substantially react" means that, when comparing the antibody concentrations required for a reaction at one point (e.g., absorbance 1.0) in a reaction range that is linear with respect to the antibody concentration in an antigen solid-phase ELISA method, there is a difference of 200 times or more between the concentration required for a certain reaction with ASGM1 and the concentration required for a certain reaction with other antigens.

[0034] Furthermore, it is more preferable that the antibody of the present invention reacts with ASGM1 and does not substantially react with ASGM2, ASGM3, GM2, GM3, GD1a, and GD1b.

[0035] If there is cross-reactivity with the above-mentioned ASGM2, ASGM3, GM1, GM2, GM3, GD1a, and GD1b, it will also react with unintended antigens, which may result in unintended effects when administered to mice or rats, or an unintended reduction in the inhibitory effect on NK activity. However, the antibody of the present invention can be said to have excellent properties that do not cause these problems.

[0036] The antibodies of the invention can be produced by isolating monoclonal antibodies from lymphocytes of a non-rodent animal immunized with ASGM1, which can be obtained from the spleen, lymph nodes, blood, or other organs.

[0037] As the immunogen, ASGM1 isolated and purified from bovine brain or the like by a known method, as well as various tissues and cells containing ASGM1 can be used. Alternatively, ASGM1 can be obtained by treating isolated and purified GM1 with formic acid or the like to remove sialic acid and convert it into ASGM1.

[0038] The type of animal to be immunized should preferably be avoided from animals expressing ASGM1 in normal tissues, such as rodent mice and rats. Preferably, non-rodent animals that do not express ASGM1 in normal tissues and show strong immune responses to ASGM1 are used. Specific examples include cows, horses, sheep, goats, pigs, camels, alpacas, dogs, rabbits, pigeons, and chickens, with rabbits being particularly preferred.

[0039] As means for producing monoclonal antibodies, in addition to traditional methods such as the hybridoma method, methods using recently developed molecular biology techniques such as the phage display method and the single cell picking method can be used.

[0040] For example, in the single cell picking method, lymphocytes are introduced one by one into tiny wells arranged in a microchamber, and the target antibody is detected after cultivation, allowing lymphocytes secreting the target antibody to be isolated one by one. This method does not require the creation of hybridomas, and can produce rabbit or human monoclonal antibodies. (Non-Patent Document 5)

[0041] It can be confirmed that the prepared monoclonal antibody reacts with asialoGM1 and has an inhibitory effect on NK activity in mice and rats in vivo, for example, by the procedures described in Example 1 and Example 5, respectively. EXAMPLES

[0042] Examples are presented below, but the present invention is not limited to these examples.

[0043] (Example 1) Measurement of anti-ASGM1 antibodies by ELISA In this Example, anti-ASGM1 antibody measurement was carried out according to the following measurement method unless otherwise specified.

[0044] (Measurement method) 50 μL of ethanol solution containing 1 μg / mL of commercially available ASGM1 was added to each well of a 96-well ELISA plate and dried under vacuum. After blocking and washing each well with a 1% gelatin solution, 100 μL of the measurement sample was added and reacted at room temperature for 1 hour. After washing three times, 100 μL of peroxidase-labeled anti-rabbit IgG solution was added and reacted at room temperature for 1 hour. After washing four times, 100 μL of the substrate 3,3',5,5'-tetramethylbenzidine (TMB)-hydrogen peroxide solution was added and the color reaction was carried out at room temperature for 30 minutes. After adding 2M sulfuric acid to stop the reaction, the 450 nm absorbance of each well was measured using a plate reader.

[0045] (Example 2) Preparation of rabbit monoclonal antibody ASGM1 was mixed with bovine serum albumin (BSA) at a weight ratio of 1:1, suspended, and mixed with Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA) at a volume ratio of 1:1 to form an emulsion to prepare an immunogen. Japanese white rabbits were immunized subcutaneously on the back three times every two weeks with the immunogen equivalent to 2, 1, or 1 mg of ASGM1 per rabbit. The increase in the antibody titer in the blood was confirmed by the ELISA of Example 1. 10-12 days after the third immunization, the spleen was removed, and erythrocytes were removed with a hypotonic solution to obtain splenic lymphocytes.

[0046] A single cell picking system (AS ONE) was used to isolate anti-ASGM1 antibody-producing cells. A total of 8x10^5 rabbit splenic lymphocytes were placed on a microchamber on which ASGM1 had been immobilized in advance and cultured one cell at a time, and the secreted anti-ASGM1 antibodies were detected by a fluorescent immunoassay using Alexa Fluor 488-labeled anti-rabbit IgG. A total of 40 clones of anti-ASGM1 antibody-producing cells were collected using a cell picking system, and RNA was extracted from each clone. RT-PCR was performed from this RNA to amplify the heavy chain variable region (VH) and light chain variable region (VL) of the antibody gene.

[0047] DNA in which constant regions were added to the amplified heavy chain variable region (VH) and light chain variable region (VL) was transfected into human embryonic kidney 293 (HEK293 cells), and 40 clones that produced antibodies were collected from the culture supernatant. The anti-ASGM1 antibodies in the culture supernatant were measured by the ELISA described in Example 1, and five antibody-positive clones were selected.

[0048] The H-chain and L-chain genes of each of the five selected clones were inserted into an expression vector (pCEC rabbit). HEK293 cells were transfected with this vector and then cultured in serum-free medium (HE200 medium, Gmep Co., Ltd.) to produce monoclonal antibodies in the culture supernatant.

[0049] We confirmed that monoclonal antibodies were produced in the culture supernatant and had binding activity to ASGM1, and established five rabbit monoclonal antibody clones (hereafter referred to as GA109, GA115, GA116, GA131, and GA134, respectively). Sequence analysis revealed that all five of these clones were different.

[0050] The antibody was purified from the culture supernatant by chromatography using a Protein A column to obtain a purified monoclonal antibody.

[0051] (Example 3) Examination of reaction specificity in ELISA The reaction specificity of the antibody was examined by ELISA using glycolipids, which have a related molecular structure and may cross-react.

[0052] An ethanol or ethanol-chloroform solution containing 1 μg / mL of ASGM1, ASGM2, ASGM3, GM1, GM2, GM3, GD1a, and GD1b (structures shown in Table 1 below), which are glycolipids having molecular structures related to ASGM1, was prepared. 50 μL of the solution was added to wells of a 96-well ELISA plate and dried in vacuum. After blocking and washing each well with a 1% gelatin solution, 100 μL of measurement samples in which rabbit monoclonal antibodies GA109, GA115, GA116, GA131, and GA134 or rabbit polyclonal antibodies were serially diluted were added to each well and reacted at room temperature for 1 hour. After washing three times, 100 μL of peroxidase-labeled anti-rabbit IgG solution was added and reacted at room temperature for 1 hour. After washing four times, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB)-hydrogen peroxide solution, which is a substrate, was added and the color reaction was carried out at room temperature for 30 minutes. The reaction was stopped by adding 2M sulfuric acid, and the absorbance of each well was measured at 450 nm using a plate reader. The results are shown in Figure 1.

[0053] [Table 1]

[0054] As a result, all five clones reacted strongly with ASGM1, whereas none of the monoclonal antibodies reacted with ASGM2, GM2, GD1a, or GD1b.

[0055] From the ELISA results, the antibody concentration at which the reaction intensity (absorbance) for each antigen became 1 was determined, and the ratio to the reaction for ASGM1 was calculated (Table 2).

[0056] [Table 2]

[0057] The GA115 and GA131 monoclonal antibodies showed cross-reactivity with GM1. The GA109 monoclonal antibody showed a reactivity ratio with ASGM1 of 240:1, and had virtually no reactivity with GM1. GA116 and GA134 also showed virtually no reactivity with GM1.

[0058] The polyclonal antibody reacted not only to GM1 but also to ASGM2 and GM3.

[0059] In addition to the polyclonal antibody, the GA109 and GA115 monoclonal antibodies also reacted with the GM3 solid phase. However, the molecular structure of GM3 is significantly different from that of ASGM1 (Table 1), and we were unsure why they reacted with the GM3 solid phase, so we conducted further investigations.

[0060] (Example 4) Reaction specificity in thin layer chromatography (TLC)-immunostaining To examine in more detail the reactivity of monoclonal antibodies GA115 and GA109 to GM3 in ELISA, we decided to examine the reactivity of the antibodies by thin-layer chromatography (TLC)-immunostaining.

[0061] 1μg of each glycolipid was dropped onto a TLC sheet (Polygram / Macherey-Nagel, Germany) cut to 100mm x 100mm, and developed with a developing solution of chloroform / methanol / water / acetic acid = 60:40:6:0.2 volume at room temperature for 30 minutes, and then dried. This TLC sheet was sprayed with 2mg / mL orcinol (5-Methylresorcinol)-2M sulfuric acid solution and heated at 110℃ for 5 minutes to color the glycolipid.

[0062] Immunostaining was performed as follows. The TLC sheet developed under the same conditions as above was blocked with a 1% gelatin, 1% polyvinylpyrrolidone solution, and then reacted with 50 ng / mL anti-ASGM1 monoclonal antibody or 1,000-fold diluted polyclonal antibody at room temperature for 1 hour. After washing with PBS containing Tween 20, peroxidase-labeled anti-rabbit IgG (Jackson Lab., 1:40,000) was reacted at room temperature for 1 hour. After washing with PBS containing Tween 20, TMB substrate solution (TMB solution for Western Blotting, Nakarai Chemical) was reacted. This visualizes the bound antibody in blue.

[0063] In the above TLC, each glycolipid (GD1a, GD1b, GM1, GM2, GM3, ASGM1, ASGM2, ASGM3) was developed at a different position (Figure 2, Orcinol).

[0064] The reactions of each monoclonal and polyclonal antibody are shown in Figure 2. All five clones (GA109, GA115, GA116, GA131, and GA134) reacted strongly with the ASGM1 spots (Figure 2, lane 6). GA131 showed a reaction to GM1 (Figure 2, lane 3). In addition, all antibodies showed a relatively small reactive spot in the lane where GM3 was spotted (Figure 2, lane 5). GM3 had a higher mobility (upper side) than ASGM1 (Figure 2, Orcinol, lane 5), but the spots reacted with each antibody were not GM3 itself but were developed with the same mobility as ASGM1 (Figure 2, arrow).

[0065] From these findings, it was considered that the GM3 preparation used contained ASGM1 as an impurity, and that the GA109 and GA115 antibodies reacted with the impurity ASGM1.

[0066] Therefore, it was revealed that the GA131 monoclonal antibody cross-reacts with GM1, whereas the other four clones (GA109, GA115, GA116, and GA134) react specifically with ASGM1.

[0067] Example 5: Suppression of NK activity by administration of monoclonal antibodies to mice The in vivo NK activity suppression effect when each antibody was administered to mice was confirmed using the following measurement system.

[0068] Each monoclonal antibody (0, 0.8, 4, 20, 100μg) or polyclonal antibody (equivalent to 0, 0.8, 4, 20μL) was administered intraperitoneally to BALB / c mice. Two days later, 200μg of Poly I:C was administered intraperitoneally to activate NK cells. One day later (day 3), the spleen was removed from the mouse and red blood cells were removed with hypotonic solution to obtain splenic lymphocytes. These lymphocytes (1x10^6 cells / well) and YAC-1 cells (mouse lymphoma cells) (2x10^4 cells / well) (E:T ratio = 50:1) were seeded in a 96-well plate. Wells seeded with only each lymphocyte and wells seeded with only YAC-1 served as controls. These were cultured for 4 hours in a 5% CO2, 37℃ incubator.

[0069] Since NK cells attack YAC-1 cells, the cytotoxic activity of NK cells (NK activity) can be measured by measuring the activity of lactate dehydrogenase (LDH) leaked from damaged YAC-1 cells. LDH activity was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega).

[0070] After 4 hours of culture, 50 μL of the culture supernatant was transferred to another 96-well ELISA plate. 50 μL of LDH substrate solution was added to the supernatant and reacted at room temperature for 30 minutes. 50 μL of reaction stop solution was added, and the absorbance at 492 nm was measured using a plate reader.

[0071] Assays were performed in 4 wells per condition, and the average was used. Cytotoxicity (NK activity) was calculated using the following formula, and negative values ​​were regarded as 0%.

[0072] (formula) % Cytotoxicity = (ER-ESR-TSR) / (TMR-TSR) x 100

[0073] ER: Experimental Response (Absorbance of wells seeded with YAC-1 + lymphocytes: Lymphocyte cytotoxicity) ESR: Effector Spontaneous Response (Absorbance in wells seeded with lymphocytes only: spontaneous cytotoxicity of lymphocytes) TSR: Target Spontaneous Response (Absorbance in wells seeded with YAC-1 alone: ​​spontaneous cell damage of YAC-1) TMR: Target Maximum Response (Absorbance when a surfactant is added to a well in which only YAC-1 is seeded, and all YAC-1 is killed: LDH activity when all YAC-1 is killed)

[0074] The results are shown in Table 3. For all five anti-ASGM1 monoclonal antibody clones (GA109, GA115, GA116, GA131, and GA134), the cytotoxic activity (NK activity) of the splenocytes of the administered mice was reduced in a dose-dependent manner, and at 100 μg, the activity was reduced to zero for all five clones.

[0075] The ratio was calculated assuming the cytotoxic activity of the control without antibody administration (0 μg) to be 100%, and is shown at the bottom of Table 2. The dose per mouse at which this ratio was 20% or less was 4 μg for GA109, GA115, GA116, and GA134, and 20 μg for GA131. In contrast, PoAb was 20% or less at an administration of 4 μL.

[0076] These results demonstrated that all five anti-ASGM1 rabbit monoclonal antibody clones (GA109, GA115, GA116, GA131, and GA134) had a strong inhibitory effect on NK activity in vivo.

[0077] [Table 3]

[0078] (Consideration) ASGM1 is present on the surface of normal tissues and cells in mice, rats, and humans, but not in rabbits. Therefore, it was thought that ASGM1 has strong antigenicity in rabbits, and rabbit antibodies with strong reactivity to ASGM1 could be obtained. Based on this, we thought that by producing rabbit monoclonal antibodies against ASGM1, it might be possible to obtain monoclonal antibodies that have strong reactivity to ASGM1 and also have strong NK activity inhibitory effects.

[0079] However, although it was previously thought that anti-ASGM1 antibodies were involved in the NK activity inhibitory effect of rabbit polyclonal antibodies, this was a prediction based on test results using antisera or crudely purified polyclonal antibodies, and there was no conclusive evidence that anti-ASGM1 antibodies alone exhibited the NK activity inhibitory effect without being affected by other components contained in antisera, etc.

[0080] In addition, it was unclear whether the anti-ASGM1 antibodies that inhibited NK activity were mainly IgG or IgM. Furthermore, there was no evidence that anti-ASGM1 rabbit monoclonal antibodies had the effect of inhibiting NK activity, so even if anti-ASGM1 rabbit monoclonal antibodies were obtained, it was doubtful whether they would have the effect of inhibiting NK activity.

[0081] In achieving this invention, it was shown that anti-ASGM1 rabbit IgG antibody has a strong inhibitory effect on NK activity in mice, and it was confirmed that the antibody alone has the effect of inhibiting NK activity.Furthermore, it was shown that by obtaining anti-ASGM1 rabbit IgG antibody, the obtained monoclonal antibody will have a very high probability of having a strong inhibitory effect on NK activity (in this result, for all five antibodies obtained).

Claims

1. A monoclonal antibody having binding activity to asialoGM1 and derived from an animal not belonging to the order Rodentia.

2. The monoclonal antibody according to claim 1, wherein the non-rodent animal is a rabbit.

3. The monoclonal antibody according to claim 1 or 2, which is an IgG monoclonal antibody.

4. The anti-asialo GM1 monoclonal antibody according to claim 1, which exhibits an inhibitory effect on NK activity when administered to mice or rats.

5. The monoclonal antibody according to claim 4, which, when administered to mice at 100 µg / animal, reduces the NK activity of splenocytes three days after administration to 20% or less of that of a control without administration.

6. The monoclonal antibody according to claim 4, which, when administered to mice at 20 µg / animal, reduces the NK activity of splenocytes three days after administration to 20% or less of that of a control without administration.

7. The monoclonal antibody according to claim 4, which, when administered to mice at 4 µg / animal, reduces the NK activity of splenocytes three days after administration to 20% or less of that of a control without administration.

8. 8. The monoclonal antibody according to claim 1, which reacts with ASGM1 and does not substantially react with ASGM2, ASGM3, GM2, GM3, GD1a and GD1b.

9. 8. The monoclonal antibody according to claim 1, which reacts with ASGM1 and does not substantially react with ASGM2, ASGM3, GM1, GM2, GM3, GD1a and GD1b.

10. A method for suppressing NK activity in mice or rats by administering a monoclonal antibody according to any one of claims 1 to 9.

11. A method for producing a monoclonal antibody which reacts with asialo GM1 and has an in vivo NK activity suppressing effect in mice or rats, by immunizing a non-rodent animal with asialo GM1 and producing a monoclonal antibody from the spleen cells of the animal.

12. The method for producing a monoclonal antibody according to claim 11, wherein the non-Rodent animal is a rabbit.

13. The method for producing a monoclonal antibody according to claim 12, wherein the monoclonal antibody is a rabbit IgG monoclonal antibody.

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