RU2703494C1 - Monoclonal antibody 6g1 to morphine derivatives - Google Patents

Abstract

FIELD: biochemistry.SUBSTANCE: invention relates to biochemistry, particularly to a monoclonal antibody to 6-hemisuccinyl ether of morphine, capable of blocking the biological activity of morphine.EFFECT: invention allows effective binding with morphine derivatives and inhibit biological activity of the drug.3 cl, 2 dwg, 2 tbl, 3 ex

Description

The invention relates to the field of biotechnology, in particular to antibodies that bind morphine derivatives and neutralize their biological activity.

Currently, according to the federal statistical observation of the Ministry of Health of Russia, among the registered drug addicts, the vast majority are opiate addicts (78.4%) [Kirzhanova VV, Grigorova NI About the disease of narcological disorders in 2014 // Issues of Addiction, 2015, 4: 19–28]. In this regard, our country is actively seeking effective ways to treat and prevent drug addiction. The possibility of creating an anti-idiotypic vaccine for opiate addiction immunotherapy is being studied. Research is being carried out in the field of improving the immunochemical methods for detecting psychoactive substances [Lyubavina I.A., Zinchenko A.A., Lapenkov M.I., Nikolaeva T.L. An express method for the detection of morphine in aqueous samples using immunochromatography using monoclonal antibodies labeled with colloidal gold // Bioorganic chemistry, 2005, 31: 108-112].

Treatment and prevention of drug dependence are closely related to the development of new effective methods of immunodiagnostics and immunotherapy using highly specific antibodies to psychoactive substances (surfactants). The creation of immunodiagnostic and therapeutic drugs based on poly- and monoclonal antibodies against surfactants can form the basis of highly effective biotechnological production.

Since the main active metabolites of heroin in humans are 6-monoacetylmorphine, morphine and morphine-6-glucuronide [Berzina A.G., Gamaleya NB, Ulyanova L.I., Shestakov K.A., Ulyanova MA, 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], for the treatment and prevention of dependence on these opiates, as well as for the development of specific diagnostic methods, it is necessary, first of all, to obtain antibodies reacting with both these drugs and their active derivatives. Such antibodies can be obtained by immunization of laboratory animals with protein-conjugated derivatives of morphine on hydroxyl residues at carbon atoms at 6 and 3 positions of the phenanthrene ring of the morphine molecule, for example, 6-hemisuccinyl morphine ester (GSM) and 3-O-carboxymethyl morphine ester (KMM) [Berzina A.G., Ulyanova L.I., Gamaleya N.B., Stankova N.V., Kapanadze G.D. The study of the immune response in mini pigs of the Svetlogorsk population to two derivatives of morphine // Biomedicine, 2018, 2: 15-21].

The closest in technical essence to the claimed invention are antibodies 9 B1 to morphine [Pozharski E., Wilson M.A., Hewagama A., Shanafelt A.V., Petsko G., Ringe D .. Anchoring a Cationic Ligand: The Structure of the Fab Fragment of the Anti-morphine Antibody 9B1 and its Complex with Morphine, J. Mol. Biol, 2004, 337: 691-697], for which the morphine-binding paratope of this antibody was determined and it was shown that the specific interaction of 9 B1 with morphine is mainly determined by the topological and electrostatic complementarity of objects, as well as hydrophobic interactions. A comparative analysis of the sequence of the hypervariable regions (CDR) of the heavy and light chains showed the absence of homology between antibodies 9 B1 and 6G1.

The technical problem solved by the authors was to expand the range of antibodies that can specifically bind to morphine derivatives and inhibit the biological activity of the drug. It was important that the antibodies did not affect endogenous opioid peptides, which play an important role in the physiology of the body.

The technical result was achieved by the creation of a monoclonal antibody (MAT) 6G1, which specifically and with high affinity binds to molecules of morphine derivatives and does not bind to human endogenous opioid peptides, such as endorphin, enkephalin, etc. It recognizes morphine 6-hemisuccinyl ester conjugated as with bovine serum albumin (BSA) and lysozyme do not bind to CMM and the 2-p-carboxy-phenylazomethyl morphine derivative (FAM) and blocks the biological activity of morphine.

Monoclonal antibody 6G1 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 (CDR) 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. A comparative analysis of the CDR sequence of the heavy and light chains showed the absence of homology between the antibody of the closest analogue and the antibody 6G1.

The technology for producing antibodies included immunization of animals with an antigen, which is a derivative of morphine: GSM and KMM conjugated with bovine serum albumin (BSA) using a bifunctional reagent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, which were obtained by methods described in the article [Berzina A.G., Gamaleya NB, 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 addiction // Narcology, 2015, 11 (167): 25-31]. As antigens for the selection of positive producers of monoclonal antibodies during ELISA, GSM and KMM conjugates with lysozyme were used, as well as a conjugate of another morphine derivative, 2-p-carboxy-phenylazomethyl (FAM).

The resulting murine monoclonal antibody (MAT) recognizes GSM, conjugated with both BSA and lysozyme, does not bind to CMM and FAM and blocks the biological activity of morphine.

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

In FIG. 1 shows the results of murine MAT 6G1 polyacrylamide gel electrophoresis (4-20% polyacrylamide gel):

a - non-reducing conditions;

b - molecular weight markers (kD);

c - restoring conditions;

In FIG. 2 demonstrates the lack of competition of MAT 6G1 with endogenous opioid peptides.

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

Example 1. Obtaining antigens for immunization and screening with hybrid-immunization, immunization of mice and selection of hybridoma sG1.

Example 1.1. Getting antigens. For immunization, GSM was used conjugated with BSA using a bifunctional reagent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride according to the procedure described in the article [Berzina A.G., Gamaleya NB. and other Methodological approaches to the development of a vaccine for the treatment of opiate addiction // Narcology, 2015, 11 (167): 25-31]. As antigens for adsorption on the solid phase, ELISA was performed using GSM, KMM and FAM conjugated with lysozyme according to the described methods [ibid.]. The prepared conjugates of morphine derivatives were aliquoted and stored at -80 ° C.

Example 1.2 Immunization of mice and hybridoma screening. Balb / c mice were immunized with the GSM-BSA preparation emulsified in Freund's complete adjuvant (PAF), as described in Example 1.1., At a dose of 10 μg per mouse in plantar hind limb aponeurosis. After 4 weeks, animals were injected intravenously with 5 μg of antigen in saline. On the fourth day after injection, the animals were sacrificed by cervical dislocation and lymphocytes of the inguinal and abdominal lymph nodes were isolated. The obtained lymphocytes were mixed with SP 2/0 myeloma cells in a 2: 1 ratio. Hybridization was carried out with a 50% solution of polyethylene glycol (PEG) with a molecular weight of 1,500 Da for 1.5 min, then RPMI-1640 medium was added 4 times with an interval of 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 cells per well) at the rate of 5-10 ⁴ myeloma cells per well. Hybrid breeding was performed in RPMI-1640 medium supplemented with 10% bovine fetal serum containing 10 ^-4 M hypoxanthine, 4⋅10 ^-7 M aminopterin and 1.6⋅10 ^-5 M thymidine.

Antibody screening was performed using the indirect ELISA, using conjugates of morphine derivatives with lysozyme as antigens for adsorption on the solid phase. When setting the test used antiviral immunoperoxidase conjugates of the company Sigma-Aldrich.

The primary selection of positive clones was performed by binding monoclonal antibodies secreted by hybridomas with antigens in ELISA. For this, 5 μg / ml of GSM-lysozyme was adsorbed into the wells of Corning polystyrene plates (Costar) in 20 mM borate buffer with a pH of 8.0 containing 0.15 M NaCl for 20 hours in a humid chamber. At the end of the adsorption, the plates were washed with wash buffer (20 mM borate buffer, pH 8.0, containing 0.15 M NaCl and 0.05% Tween-20). Then, 100 μl of washing buffer containing 1 mg / ml BSA and 50 μl of culture media containing MAT were added to each well. The plates were incubated for 1 hour at 37 ° C with stirring. At the end of the incubation, antibodies that were not bound to the antigen were washed with washing buffer and a solution of horseradish peroxidase conjugated goat immunoglobulins to mouse immunoglobulins (Sigma-Aldrich, USA) was added to the wells according to the attached instructions. The plates were incubated for 1 hour at 37 ° C and stirring, after which they were thoroughly washed and stained with a solution of tetramethylbenzidine substrate (Khema, Russia). The culture medium of the clones that showed specific binding to antigens was re-tested in a similar experiment for KMM-lysozyme, GSM-lysozyme, FAM-lysozyme and unmodified lysozyme.

Based on the screening, sG1 clone was selected that produced specific antibodies to the morphine derivative of the GSM-lysozyme, but did not recognize KMM- and FAM-lysozyme. The properties of monoclonal antibodies 6G1 are shown in Table 1.

As a result of subsequent intraperitoneal propagation, cryopreservation and control of cell purity, a hybridoma strain sG1, a producer of monoclonal antibody 6G1, was obtained.

Example 2. The study of the properties of monoclonal antibodies 6G1.

Example 2.1 Cleaning MAT 6G1. Antibody 6G1 was isolated from ascitic fluid of Balb / c mice with an intraperitoneal inoculated sG1 hybridoma using affinity chromatography on MabSelect-Sepharose sorbent (General Electrics, USA) according to the instructions.

Example 2.2 The study of the molecular properties of antibodies 6G1. According to the results of electrophoresis in a 4-20% polyacrylamide gel with sodium dodecyl sulfate, the molecular weight of the 6G1 antibody under non-reducing conditions corresponds to the expected 170 kDa for native mouse IgG (Fig. 1a), under reducing conditions, the antibody dissociates into heavy (55 kDa) and light (29 kDa) ) chains (Fig. 1c). The isotype of antibody 6G1, established using the kit "Mouse monoclonal antibody isotyp-ing reagents" (Sigma, USA), - IgG2a. Preparations of purified 6G1 antibody were used to study its biological properties.

Example 2.3 The study of the biological properties of MAT 6G1.

Example 2.3.1. The study of the biological activity of MAT 6G1 was carried out on an in vitro model of the proliferative reaction of lymphocyte cultures isolated from the peripheral blood of 6 healthy volunteers using a density gradient of ficollaverographin (density 1.077 g / cm). Table 2 presents the results of evaluating the neutralizing effect of naloxone and MAT 6G1 on morphine stimulation of DNA synthesis in a lymphocyte culture when they are co-administered with morphine.

Analysis of the data shown in Table 2 showed the effect of MAT 6G1 on the specific activity of morphine. As can be seen from the data obtained, MAT 6G1 have the ability to completely block morphine amplification of DNA synthesis in cultures of human blood lymphocytes. This effect is similar to that of naloxone, which when co-administered with morphine completely eliminates the stimulating effect of opiate on lymphocytes. It should be noted, however, that the mechanism of the neutralizing effect of naloxone is different from the mechanism of action of the primary MAT 6G1. The blockade of the naloxone effect of morphine is carried out at the level of μ-opioid receptors present on cultured human lymphocytes. Primary blockade of MAT 6G1 is not related to their effect on lymphocyte receptors, since such antibodies themselves do not have an effect on proliferation. The blockade in this case is due to the binding of the drug to antibodies in the reaction mixture, as a result of which the effect of morphine on μ-opioid lymphocyte receptors is reduced.

Note: Comparison on one basis with spontaneous proliferation - SP (P _SP ) and morphine (P _M ) was carried out using the nonparametric Wilcoxon test (W) for two dependent groups without taking into account the nature of the distribution option [Glants S. Biomedical statistics, Practice. 1999, 459 pp.]. In parentheses is the percentage change from the average values of SP and M (Δ% and Δm%, respectively).

Example 2.3.2. Study of cross-linking of MAT 6G1 with certain endogenous opioid peptides. To test the cross interaction of MAT 6G1 with endogenous opioid peptides, a competitive enzyme-linked immunosorbent assay was performed. As a competitor used β-endorphin, Leu- and Met-enkephalins. For this purpose, 5 μg / ml GSM-BSA in 20 mM borate buffer with pH 8.0 containing 0.15 M NaCl was added to the wells of Corning polystyrene plates (Costar) and kept for 20 hours in a humid chamber. At the end of the adsorption, the plates were washed with wash buffer (20 mM borate buffer, pH 8.0, containing 0.15 M NaCl and 0.05% Tween-20). Then, 100 μl of washing buffer containing 1 mg / ml BSA, 100 μg / ml competitor and MAT 6G1 in various concentrations were added to each well. The plates were incubated for 1 hour at 37 ° C with stirring. At the end of the incubation, antibodies that were not bound to the antigen were washed with washing buffer and a solution of horseradish peroxidase conjugated goat immunoglobulins to mouse immunoglobulins (Sigma, USA) was added to the wells according to the attached instructions. The plates were incubated for 1 hour at 37 ° C and stirring, after which they were thoroughly washed and stained with a solution of tetramethylbenzidine substrate (Khema, Russia). The results of a competitive assay with endogenous opioid peptides are shown in FIG. 2.

As can be seen from the data presented, 6G1 monoclonal antibodies do not compete with the selected opioid peptides even with a very large molar excess of competitor.

Example 3. Synthesis and sequencing of DNA encoding the variable parts of the light and heavy chains of monoclonal antibodies 6G1.

RNA was isolated from sG1 hybridoma cells, on the matrix of which DNA fragments encoding the variable regions of the heavy (VH) and light (VL) chains of the antibody were amplified using synthetic primer kits (Immunogenetics Information System http://www.imgt.org). The obtained DNA fragments were cloned into the pALTA vector (Eurogen, Russia) and sequenced from 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 of VH and VL are shown in SEQ ID NO: 3 and SEQ ID NO: 4. Analysis of amino acid sequences of the heavy and variable regions monoclonal antibody 6G1 light chains were produced by Cabot (Kabat E.A., Wu T.T., Perry N., Gottesman K. and Foeller C. (1991) SEQuences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91: 3242), which allowed us to identify sections of CDR that determine the complementarity of the antibody to the antigen.

Comparison of sequences of CDR regions with sequences of known antibodies to morphine components showed a lack of homology with similar regions of known antibodies.

The monoclonal antibody 6G1 of the present invention can serve as the basis for the construction of chimeric and humanized antibodies suitable for the creation of drugs that block the biological activity of morphine.

Claims (3)

1. Monoclonal antibody 6G1 to 6-hemisuccinyl ester of morphine, capable of blocking the biological activity of morphine, containing hypervariable regions of the heavy chain with sequences of SEQ ID NO: 5-7 and hypervariable regions of the light chain with sequences of SEQ ID NO: 8-10. 2. The monoclonal antibody according to claim 1, having the sequence of the variable region of the heavy chain according to SEQ ID NO: 3 and the sequence of the variable region of the light chain according to SEQ ID NO: 4. 3. The monoclonal antibody according to claim 2, encoded by a DNA containing the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

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