RU2745374C1 - Monoclonal anti-idiotypic antibody ai-k11b, having antigenic properties of morphine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to antibodies imitating morphine-specific antigenic properties and capable of binding to opioid receptors.

EFFECT: obtained monoclonal antibody AI-K11B binds to opiate receptors and induces, during immunization, antibodies specifically recognizing derivatives of morphine.

3 cl, 6 tbl, 3 ex, 4 dwg

Description

The invention relates to the field of biotechnology, in particular to antibodies that mimic morphine-specific antigenic properties and are capable of binding to opioid receptors.

Currently, according to the Federal Statistical Office of the Ministry of Health of Russia, among the registered drug addicts, the overwhelming majority are patients with dependence on opiates (78.4%) [Kirzhanova V.V., Grigorova N.I. On the disease of substance abuse disorders in 2014 // Questions of narcology, 2015; 4: 19-28]. In this regard, an active search for effective ways of treating and preventing opiate addiction is being carried out in our country. Treatment and prevention of drug addiction are closely related to the development of new effective methods of immunotherapy using highly specific vaccines for psychoactive substances (PAS).

The world is widely used vaccination against drug addiction using conjugates of high molecular weight compounds, such as cholera toxin or snail hemocyanin, and surfactants [Bonese K.F., Wainer V.N., Fitch F.W, Rothberg R.M., Schuster C.R. Changes in heroin self-administration by a rhesus monkey after morphine immunization // Nature, 1974; 252 (5485): 708-710; Anton B., Salazar A., Flores A., Matus M., Marin R., Hernandez J., Leff P. Vaccines against morphine / heroin and its use as effective medication for preventing relapse to opiate addictive behaviors // Human Vaccines, 2009; 5 (4): 214-229]. With this method of vaccination, antibodies are induced that are specific not only to drugs, but also to endogenous opioid peptides. Thus, a vaccine is proposed that contains opium alkaloids as an antigen, which causes the formation of antibodies to heroin and morphine in the body. Antibodies form a complex with heroin injected into the body, and thus its penetration into the brain is reduced (CN 1196955 A, 28.10.1998). This approach contradicts the Federal Law No. 3-FZ of 01/08/1998 "On Narcotic Drugs and Psychotropic Substances" in force in the Russian Federation, which prohibits the treatment of drug addiction with narcotic drugs. One of the most promising areas is the creation of a vaccine based on secondary anti-idiotypic antibodies - Ab2. A known method of vaccination against drug addiction using monoclonal anti-idiotypic (MAi) antibodies to cocaine [Schabacker DS, Kirschbaum KS, Segre M. Exploring the feasibility of an anti-idiotypic cocaine vaccine: analysis of the specificity of anticocaine antibodies (Ab1) capable of inducing Ab2b anti-idiotypic antibodies // Immunology, 2000; 100: 48-56].

Since the metabolites of heroin in humans are not only 6-monoacetylmorphine, morphine and morphine-6-glucuronide, but also morphine-3-glucuronide [Berzina AG, Gamaleya NB. et al. Methodological approaches to the development of a vaccine for the treatment of opiate dependence // Narcology, 2015; 11 (167): 25-31], for the treatment and prevention of dependence on these opiates, as well as for the development of methods of specific diagnostics, mouse monoclonal antibodies (mAb) 3K11 were originally obtained, which recognize 3-Ocarboxymethyl morphine ester conjugated with BSA [patent for the invention of the Russian Federation No. 2702002].

The technical problem solved by the authors was to obtain mAi antibodies to mAb 3K11, capable of specifically binding to opioid receptors and, upon vaccination of animals, to induce the production of third-generation antibodies Ab3 to morphine derivatives.

The technical result was achieved by creating mAi antibody AI-K11B, which _{binds with high affinity K D} 3.2 nM to the idiotype of antibody 3K11, induces, during immunization, the production of antibodies that specifically recognize morphine derivatives, that is, exhibits antigenic properties of morphine, and binds to opioid receptors.

Anti-idiotypic monoclonal antibody AI-K11B contains the variable region of the heavy chain according to SEQ ID NO: 3 and the variable region of the light chain according to SEQ ID NO: 4, as well as the hypervariable regions of the variable region of the heavy chain according to SEQ ID NO: 5-7 and the hypervariable regions of the variable region light chain according to SEQ ID NO: 8-10.

The resulting mAi antibody AI-K11B binds to the opioid receptors of human glioblastoma cells of the T98G line. AI-K11B during rabbit immunization induces the production of Ab3 antibodies that bind 6-hemisuccinyl (GM), 3-O-carboxymethyl (CMM) and 2-p-carboxy-phenylazomethyl (FAM) morphine esters conjugated to BSA using bifunctional reagent 1 -ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride according to the methods described in the article: Berzina A.G., Gamaleya N.B., Ulyanova L.I, Shestakov K.A., Ulyanova M.A., Kapanadze K D., Stankova N.V., Revyakin A.O., Fokin Yu.V., Krotov G.I., Rodchenkov G.M. Methodological approaches to the development of a vaccine for the treatment of opiate dependence // Narcology, 2015; 11 (167): 25-31.

The antibody production technology included immunization of animals with an antigen representing mAb 3K11 conjugated with snail lymph hemocyanin (KLH) according to the method described in: Hermanson G.T. Bioconjugate Techniques. ISBN 9780123705013, Academic Press Inc., 2008.

To select positive producers of mAi antibodies during enzyme-linked immunosorbent assay (ELISA), mAb 3K11 was used as the antigen sorbed on the plates; at the last stage of the analysis, the detection of specific antibodies was carried out with the conjugate 3K11-horseradish peroxidase.

The properties and structure of the antibody are illustrated by the following graphic materials:

FIG. 1 shows the results of electrophoresis in 4-20% polyacrylamide gel of the mouse monoclonal antibody AI-K11B;

a - restoring conditions;

b - markers of molecular weight (kD);

c - non-reducing conditions.

FIG. 2 shows the curve of competitive suppression of the binding of the mAb 3K11 - horseradish peroxidase conjugate with the morphine derivative KMM-BSA by antibodies AI-K11B.

FIG. 3 shows the sensograms of binding of 20, 40, and 80 nM AI-K11B to the CM5 chip, on which antibodies to mouse IgG are adsorbed, after passing 80 nM 3K11B. The abscissa is the time in seconds, the ordinate is the response (RU).

FIG. 4 demonstrates the effect of AI-K11B and morphine on the intensity of DNA synthesis (cpm), reflecting the proliferative activity in cultures of the human glioblastoma cell line T98G under conditions of free action and combined with naloxone.

The essence and industrial applicability of the invention are illustrated by the following examples:

Example 1. Obtaining antigens for immunization and screening of hybridomas, immunization of mice and selection of AI-K11B hybridoma.

Example 1.1. Antigen production. For immunization, a conjugate of snail lymph hemocyanin (KLH) with monoclonal antibodies 3K11 (3K11-KLH), prepared according to the previously described method [Hermanson Greg T., Bioconjugate Techniques. ISBN 0-12-342336-8, Academic Press Inc., 1996]. When setting ELISA used antibodies 3K11, conjugated with horseradish peroxidase according to the described method [ibid]. The prepared conjugates of mAb 3K11 were aliquoted and stored at -80 ° C.

Example 1.2. Immunization of mice and screening for hybridoma. Balb / c mice were immunized with the preparation obtained as described in Example 1.1. with 3K11-KLH. For this, the antigen emulsified in complete Freund's adjuvant (CFA) at a dose of 10 μg per mouse was injected into the plantar aponeurosis of the hind limbs. On the 30th day after immunization, the animals were injected intravenously with 5 μg of mAb 3K11 in physiological saline. On the 4th day after injection, the animals were sacrificed by cervical dislocation and splenocytes and lymphocytes of the inguinal and abdominal lymph nodes were isolated. The resulting cells were mixed with mouse myeloma cells of the SP 2/0 line in a ratio of 2: 1. Cells were hybridized in a 50% solution of polyethylene glycol (PEG) with a molecular weight of 1500 Da for 1.5 min, followed by 4-fold addition of RPMI-1640 medium after 1 min in a volume equal to the volume of the PEG solution. After fusion, the cells were washed twice with culture medium and plated in 96-well culture plates with daily peritoneal macrophages (5 × 10 ³ cells per well) at the rate of 5 × 10 ⁴ myeloma cells per well. The selection of hybrids was carried out in RPMI-1640 medium supplemented with 10% fetal bovine serum (Sigma, USA) containing: 4⋅10 ^-4 M hypoxanthine, 4⋅10 ^-7 M aminopterin and 1.6⋅10 ^-5 M thymidine [Pandey S. Hybridoma technology for production of monoclonal antibodies // Int. J. Pharm. Sci. Res., 2010; 1: 88-94].

Antibodies were screened by ELISA using mAb 3K11 as an antigen for adsorption on the solid phase. The bound antibodies were then detected with the 3K11 horseradish peroxidase conjugate (Sigma, USA) prepared according to the manufacturer's protocol.

The primary selection of positive clones was carried out by the binding of secreted antibodies to mAb 3K11; mAb 6G1, which bind GSM-BSA, and the IgG fraction of mouse immunoglobulins were used as a negative control. For this, one of the antibodies was sorbed in the wells of Corning polystyrene plates (Sigma, USA) in a landing buffer (20 mM borate buffer, pH 8.0, containing 0.15 M NaCl): mAb 3K11, 6G1, or mouse IgG with a concentration of 1.5 μg / ml for 20 hours in a humid chamber at room temperature. At the end of the sorption, the plates were washed with a wash buffer (landing buffer containing 0.05% Tween-20). Wells were filled with 100 μl of wash buffer containing 10% mouse serum and 50 μl of culture medium. The plates were incubated for 1 hour with stirring at 37 ° C. Then a solution of 3K11 horseradish peroxidase conjugate at a concentration of 100 ng / ml was added to the washed plate. The plates were incubated for 1 hour with stirring at 37 ° C, after which they were thoroughly washed and stained with a tetramethylbenzidine substrate solution (XEMA, Russia).

Competitive ELISA was performed to confirm the specificity of mAi antibodies from selected positive clones to mAb 3K11. In the wells of Corning polystyrene plates (Costar), 5 μg / ml KMM-BSA in 20 mM borate buffer with pH 8.0 containing 0.15 M NaCl was added and incubated for 20 hours in a humid chamber. At the end of the sorption, the plates were washed with a washing buffer (20 mM borate buffer, pH 8.0, containing 0.15 M NaCl and 0.05% Tween-20). Pre-mAi antibodies at a concentration of 0; 6.25; 12.5; 25; fifty; 100 and 200 ng / ml were incubated for 30 min at room temperature with 3K11-horseradish peroxidase conjugate at a concentration of 200 ng / ml, and then 100 μl of the resulting solutions were added to each well. The results of the competitive analysis are shown in FIG. 2.

Based on the screening performed, clone sAI-K11B was selected, which produces specific antibodies to mAb 3K11. The properties of the AI-K11B monoclonal antibody are shown in Table 1. Data on the isotype of the AI-K11B monoclonal antibody and specificity to mouse IgG, mAb 3K11 and mAb 6G1 are presented.

As a result of subsequent cultivation, cryopreservation and control of cell purity, the hybridoma strain sAI-K11B, a producer of the monoclonal antibody AI-K11B, was obtained.

Example 2. Study of the properties of the monoclonal antibody AI-K11B.

Example 2.1. Cleaning mAb AI-K11V.

Antibody AI-K11B was isolated from the ascites fluid of Balb / c mice with intraperitoneally inoculated sAI-K11B hybridoma by gel permeation chromatography on an HW-65S sorbent (TosoHaas, Japan) according to the instructions.

Example 2.2. Study of the molecular properties of the AI-K11V antibody.

The calculation, carried out according to the results of electrophoresis in 4-20% polyacrylamide gel with sodium dodecyl sulfate, showed that the molecular weight of the main part of the AI-K11B antibody preparation under non-reducing conditions is about 800 kDa, i.e. is represented by a pentamer of an IgM class immunoglobulin (Fig. 1c), while under reducing conditions the antibody dissociates into heavy (70 kDa) and light (25 kDa) chains (Fig. 1a). The isotype of the AI-K11B antibody, established using the Mouse monoclonal antibody isotyping reagents kit (Sigma, USA), is IgM. The preparations of the purified AI-K11B antibody were used to study its biological properties.

Example 2.3. Investigation of the biological properties of the AI-K11V monoclonal antibody.

Example 2.3.1. The affinity of the interaction of the anti-idiotypic antibody AI-K11B with the idiotype of the antibody 3K11 was assessed by surface plasmon resonance using a BiaCore X100 analyzer and standard CM5 chips (GE Healthcare, Austria). A chip with antibodies to mouse IgG was obtained using a Mouse Antibody Capture Kit (GE Helthcare - BR100838) according to the manufacturer's instructions: antibodies to mouse IgG were immobilized on an optical CM5 sensor using the amide immobilization method. The chip was activated with an equimolar mixture of N-ethyl-N'-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide (0.2 M), then a solution of antibodies to mouse IgG (20 μg / ml) in 10 mM sodium was injected at a rate of 5 μl / min for 18 minutes -acetate buffer, pH 5.0. Then free groups of the chip were blocked with 1 M ethanolamine, pH 8.5.

The study of the interaction of AI-K11B with 3K11 was carried out in HBS-P + buffer (150 mM NaCl, 10 mM Hepes-NaOH, pH 7.5, 0.05% polyoxyethylene sorbitan) according to the following scheme: a chip with antibodies to mouse IgG was equilibrated with HBS-P buffer 1 minute. Then antibodies 3K11 with a concentration of 80 nM were passed over the chip for 1.5 minutes. The chip was washed for 3 minutes and AI-K11V was applied for 1 minute. The chip was washed again for 3 minutes and desorption was performed for 1 minute with 10 mM Gly-HCl, pH 1.7. After 3 cycles with a sequential increase in the concentration of AI-K11B 20, 40 and 80 nM, the equilibrium dissociation constant (K _D ) of antibodies 3K11 and AI-K11B was calculated. The results are shown in Table 2 and FIG. 3.

Example 2.3.2. The study of the biological activity of monoclonal antibodies AI-K11B was carried out on the model of the proliferative response of the transplanted human glioblastoma cell line T98G in vitro. On the surface of the cells of this line, the presence of a large number of μ- and κ-opioid receptors and a small amount of δ-opioid receptors was revealed [Lazarczyk M., Matyja E., Lipkowski A.W. A comparative study of morphine stimulation and biphalin inhibition of human glioblastoma T98G cell proliferation in vitro // Peptides, 2010; 31: 1606-1612].

FIG. 4 shows the data on the stimulation of morphine and AI-K11B DNA synthesis in cell culture. Shown is the neutralizing effect of naloxone when administered together.

As can be seen from the data obtained, mAb of clone sAI-K11B, like morphine, has the ability to enhance DNA synthesis in the culture of the human glioblastoma cell line T98G. This effect is blocked by the action of naloxone, which, when administered together, completely eliminates the stimulating effect. It should be noted that the blockade of the action of morphine by naloxone occurs at the level of opioid receptors present on cultured cells. Therefore, mAb AI-K11B is also capable of binding to opiate receptors.

Example 2.3.3. Study of the immunogenic properties of MAi AI-K11V on rabbits of the Soviet Chinchilla breed. Table 3 schematically shows a method for immunizing a rabbit with mAi with AI-K11B antibodies to obtain Ab3 antibodies.

The determination of the specificity of Ab3 in the blood serum of rabbits was carried out by ELISA. The following antigens were used: KMM-BSA, FAM-BSA, GSM-BSA, BSA as a negative control, which were immobilized on the bottom of the wells of ELISA plates by incubation of 10 μg / ml protein in 20 mM borate buffer, pH 8.0, containing 0 , 15 M NaCl (landing buffer), for 20 hours at room temperature. At the end of the sorption, the plates were washed with washing buffer (planting buffer containing 0.05% Tween-20), and 100 μl of the tested rabbit Ab3 antibodies were added to the wells. The plates were incubated for 1 hour with stirring at 37 ° C. Then, solutions of horseradish peroxidase conjugate with goat anti-species antibodies against rabbit or mouse immunoglobulins (Sigma, USA) with a concentration of 0.5 μg / ml were added to the three times washed plate. The plates were incubated for 1 hour with stirring at 37 ° C, after which they were thoroughly washed and stained with a tetramethylbenzidine substrate solution (XEMA, Russia).

It was found that the Ab3 antibodies of a rabbit vaccinated with mA and the AI-K11B antibody are specific to all tested morphine derivatives (Table 4).

To determine the temporal dynamics of antibody formation, blood was taken from the ear vein of rabbits on days 16, 28, 31, 37, and 50 after the last immunization. The level of Ab3 antibodies in the blood serum of rabbits was determined by ELISA. In this variant of the analysis, a mixture of KMM-BSA, FAM-BSA, and GSM-BSA conjugates was immobilized at the bottom of the wells of ELISA plates by incubating a solution containing 5 μg / ml of each conjugate in the landing buffer for 20 hours. At the end of the sorption of the antigen, the plates were washed with a washing buffer, and 100 μl of the tested rabbit sera were added to the wells, which were preliminarily prepared in the form of tenfold serial dilutions. Rabbit Ab1 antibodies isolated by immunoaffinity chromatography and immunized with KMM-BSA and GSM-BSA conjugates were used as a standard for determining the concentration of specific antibodies in the sera of immune animals. All further stages of the analysis coincided with the final stages in determining the specificity of antibodies. The Ab3 content in the serum was determined from the calibration curve of the dependence of the OD ₄₅₀ intensity on the concentration of antibodies, taking into account the dilution. The results of the dynamics of Ab3 production in rabbits immunized with AI-K11B are presented in Table 5.

Example 3. Synthesis and sequencing of DNA encoding the variable portions of the light and heavy chains of the AI-K11B monoclonal antibody.

RNA was isolated from sAI-K11B hybridoma cells, on the template of which DNA fragments encoding the variable regions of the heavy (VH) and light (VL) chains of the antibody were amplified using sets of synthetic primers (Immunogenetics Information System http://www.imgt.org) ... The resulting DNA fragments were cloned into the pAL-TA vector (Evrogen) and sequenced using external primers (M13). The sequences of DNA fragments encoding VH and VL are shown in SEQ ID NO: 1 and SEQ ID NO: 2, the calculated amino acid sequences VH and VL are shown in SEQ ID NO: 3 and SEQ ID NO: 4. Analysis of the amino acid sequences of the variable regions of heavy and light chains of monoclonal antibody 3K11 were produced according to Cabot (Kabat E.A., Wu T.T., Perry H., Gottesman K. and Foeller C. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91: 3242), which made it possible to isolate the CDR regions that determine the complementarity of the antibody to the antigen (Table 5).

Monoclonal antibody AI-K11B according to the present invention can serve as a basis for creating chimeric and humanized antibodies suitable for creating a vaccine and drugs that block the biological activity of morphine.

The applicants ask to consider the submitted materials of the application "Monoclonal anti-idiotypic antibody AI-K11B, possessing the antigenic properties of morphine" for the grant of a Russian patent for an invention.

Claims (3)

1. Monoclonal antibody AI-K11B against antibodies to morphine derivatives, possessing antigenic properties of morphine and containing hypervariable regions of the heavy chain with the sequences SEQ ID NO: 5-7 and hypervariable regions of the light chain with the sequences SEQ ID NO: 8-10. 2. The monoclonal antibody of claim 1, having a heavy chain variable region sequence according to SEQ ID NO: 3 and a light chain variable region sequence according to SEQ ID NO: 4. 3. The monoclonal antibody of claim 2, encoded by DNA comprising the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

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