RU2741095C2 - Method of producing cell lines stably producing human monoclonal antibodies of class igg - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: disclosed is a method of producing a human IgG antibody with authentic human immunoglobulin sequences, produced by hybridoma (trioma): mouse myeloma—human lymphocyte—human lymphocyte.

EFFECT: present invention can find further application in producing human IgG antibodies of required specificity.

3 cl, 1 dwg, 3 tbl, 4 ex

Description

Technology area

The invention relates to the field of biotechnology, in particular to hybridoma technology, which makes it possible to obtain unmodified fully human IgG antibodies. The invention can be used in scientific organizations and pharmaceutical companies involved in the development of drugs based on therapeutic antibodies.

Prior art

Therapeutic monoclonal antibodies have become firmly established in clinical practice, the volume of the world market for drugs based on them is increasing every year. The experience of using chimeric, humanized and even fully human antibodies in the clinic shows that with prolonged administration, a significant number of patients develop an immune response to the drug, which greatly reduces its effectiveness. The point is that chimeric and humanized antibodies contain protein sequences of animal origin, which makes MAb "visible" to the immune system. Fully human are obtained either using phage display technology or using transgenic mice, and in both cases, the process involves mutagenesis steps to increase the affinity of antibodies, the so-called affinity maturation, which also leads to the appearance of "non-human" sequences.

The principal advantage of hybridoma technology for the production of human antibodies is that this technology preserves the authenticity of the DNA sequences of antibodies obtained from native B-lymphocytes, and does not involve genetic modifications that could lead to the appearance of antigenic determinants. However, so far not a single drug has been registered for which the antibody could be obtained using hybridoma technology without additional genetic modifications. The main reason for this is the ineffectiveness of hybridization of human B-lymphocytes with a tumor partner due to their incomplete genetic correspondence, which ultimately leads to a low yield of hybridoma clones and unstable antibody production.

In the 80s of the last century, many attempts were made to solve the problem using various methods. It was noted that the stability of antibody production is significantly increased if a human-mouse heterohybrid is used as a tumor partner. A number of such heterohybrid lines have been produced, and in all cases a murine myeloma lineage and a human tumor lineage were used. So lines were obtained by heterohybrid SPAZ-4 (Ostberg L, Pursch E. 1983. Human X (mouse X human) hybridomas stably producing human antibodies. Hybridoma 2: 361-367.), SHM-D33 (Teng NN, Lam KS, Calvo Riera F, Kaplan HS. 1983. Construction and testing of mouse-human heteromyelomas for human monoclonal antibody production. Proc Natl Acad Sci USA 80: 7308-7312), HMMA 2.5 (Posner MR, Elboim H, Santos D. 1987. The construction and use of a human-mouse myeloma analogue suitable for the routine production of hybridomas secreting human monoclonal antibodies. Hybridoma 6: 611-625), K6H6 / B5 (Carroll WL, Thielemans K, Dilley J, Levy R. 1986. Mouse x human heterohybridomas as fusion partners with human В cell tumors. J Immunol Methods 89: 61-72.), SPYMEG (Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, Saito H. 1985. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 66: 1384-1392.), B6B11 and MFP-2 (Kalantarov GF, Rudch enko SA, Lobel L, Trakht I. 2002. Development of a fusion partner cell line for efficient production of human monoclonal antibodies from peripheral blood lymphocytes. Hum Antibodies 11: 85-96.). Also, in all these cases, for effective hybridization of heteromyeloma with human lymphocytes, the latter were transformed with the Epstein-Barr virus (EBV), which determined the presence of this virus in the hybridoma producer. Contamination of the final product with this oncogenic virus can lead to iatrogenic infection and EBV-associated diseases, such as infectious mononucleosis and lymphogranulomatosis.

Closest to the claimed is a method for producing hybridoma monoclonal antibodies by somatic hybridization of immune lymphoblasts obtained from lymphoid organs (spleen, lymph nodes) and a myeloma cell line of a genotype similar to lymphoblasts. Genetic matching is achieved using linear animals, otherwise the hybridization efficiency and the stability of hybridoma antibody production are extremely low (reviewed by Microbiol Spectr. 2015 Feb; 3 (l): AID-0027-2014. Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies. Smith SA , Crowe JE Jr.). Such a method for obtaining human antibodies is not available due to the lack of linearity of the tumor partner and B-lymphocytes, as well as the limited availability of human lymphatic organs.

Disclosure of invention

The technical problem to be solved by the present invention is the development of a method for producing fully human antibodies with authentic immunoglobulin sequences and without any genetic modifications.

The technical result is obtained without the participation of human tumor lines and human viruses hybridoma lines, stably producing human IgG antibodies of the desired specificity.

The technical problem is solved by a method of obtaining a stable producer of a human IgG antibody, which is a hybridoma (trioma): mouse myeloma - human lymphocyte - human lymphocyte, while the producer is obtained by somatic hybridization of human-mouse heterohybridoma and human lymphoblasts obtained from mature human B-lymphocytes, with the subsequent selection and production of hybridoma clones secreting human immunoglobulins of the IgG class of the required specificity, the isolation of the obtained antibodies. In particular, a human-mouse heterohybrid is obtained by somatic hybridization of the mouse line P3 / NSI / 1-Ag4-1 [NS-1] (ATCC® THV-18 ™) and human peripheral blood B-lymphocytes using PEG4000. B-lymphocytes from human peripheral blood are isolated by any method known in the art, for example, using the commercial RosetteSep Human B cell Enrichment Cocktail (StemCell, USA), then the isolated B-lymphocytes are cultured in the presence of IL2, IL4, IL6, IL10, poke mitogen weed (Phytolacca americana). Hybrids (heterohybridomas) are selected on a selective medium with GAT (guanine-aminopterin-thymidine) and tested for the presence of human IgG antibodies in the culture fluid. Heterohybridomas are cultured by monitoring the production of antibodies by any suitable method, in particular, by enzyme immunoassay. The heterhybridoma clone, which retained the production of antibodies longer than the others, is produced in the presence of 8-azaguanine. 8-azaguanine is added according to the manufacturer's recommendations to restore GAT (guanine-aminopterin-thymidine) sensitivity.

To obtain mature human dendritic cells from human peripheral blood, monocytes are isolated by any method known in the art, for example, using the commercial RosetteSep Human Monocyte Enrichment Cocktail kit (StemCell, USA). Monocytes are cultured in the presence of GMCSF and IL4, while monocytes are converted into immature dendritic cells. To these cells is added the antigen (immunogen) to which the antibody is desired, for example, the glycoprotein G of the rabies virus or the rabies vaccine Rabipur. It is cultivated for the time required for maturation of dendritic cells under the action of antigen, usually within 1 day. During this time, immature dendritic cells are loaded with antigen and turn into mature dendritic cells.

Stimulated CD4 + T lymphocytes are required to produce mature human B lymphocytes. CD4 + T-lymphocytes are isolated from human peripheral blood by any method known in the art, for example, using the commercial RosetteSep Human CD4 + T cell Enrichment Cocktail kit (StemCell, USA). Selected CD4 + T-lymphocytes are stimulated with antigen-loaded dendritic cells. It is cultured for the time necessary to stimulate CD4 + T-lymphocytes with dendritic cells, usually for 4 hours. B-lymphocytes are isolated from human peripheral blood by any suitable method, for example, using the commercial RosetteSep Human B cell Enrichment Cocktail (StemCell, USA). The antigen is added, cultivated for the time required for the primary stimulation of B-lymphocytes, approximately for 8 hours. Stimulated CD4 + T-lymphocytes, IL2, IL4, IL6, IL10 are added to B-lymphocytes and cultured until maturation or for the time required for full maturation of B-lymphocytes, approximately 4-5 days). To the obtained mature B-lymphocytes, a mitogen is added and cultured for the time necessary for the transformation of mature B-lymphocytes into lymphoblasts (blast transformation, about 3 days).

To obtain hybridoma clones, the resulting lymphoblasts are subjected to somatic hybridization with a HAT-sensitive heterohybridoma using PEG4000. Seeded in culture 96-well plates in medium with HAT. After 7-10 days, the number of hybridoma clones and the hybridization efficiency (the ratio of grown clones to the initial number of B-lymphocytes) are assessed. After hybridization, the supernatants (culture liquids) of the grown hybridoma clones are tested for the content of antibodies of the required specificity. Testing can be performed by ELISA or other suitable method. The clones with the highest activity are selected for further work (cloning). Cloning is carried out in order to obtain a homogeneous population of hybridoma cells, that is, a cell line is obtained from a single cell. Cloning is carried out by the limiting dilution method or other suitable method. Hybridoma clone lines with stable antibody production are being developed. After each passage, the content of antibodies in the culture liquids is assessed. Cryopreservation of hybridoma cell lines is performed.

Isolation of antibodies from hybridoma culture liquids. The cell suspension is centrifuged to pellet the cells and cell fragments. The supernatant is filtered, the obtained antibodies are isolated by affinity chromatography for protein G-Sepharose or by another suitable method. The antibody solution is dialyzed against a phosphate buffer, pH 7.2-7.5, sterilized by filtration through a bacterial filter with a pore size of 0.22 μm, or a preservative is added or frozen.

The resulting antibodies are tested according to the following parameters:

protein concentration;

the relative content in the sample;

binding to antigen, determine the corresponding constants;

biological activity.

Brief Description of Drawings

The invention is illustrated by the following drawings.

FIG. 1 shows the results of electrophoresis of 12% SDS-PAGE of antibody samples under reducing conditions.

Implementation of the invention

Below is a more detailed description of the proposed method, which does not limit the scope of the claimed invention, but demonstrates the possibility of implementing the invention with the achievement of the claimed technical result.

To implement the proposed method were used:

1. heterohybridoma (trioma) human mouse, which significantly increased the efficiency of hybridization and the stability of the resulting hybridomas. This heterohybridoma was obtained by the method of hybridoma technology by somatic hybridization of the commercially available GAT (guanine-aminopterin-thymidine) sensitive mouse line P3 / NSI / 1-Ag4-1 [NS-1] (ATCC® TIB-18 ™) and peripheral B-lymphocytes human blood, pre-stimulated in vitro with cytokines IL-2, IL-4 and IL-6, mitogen and immunogen. A similar method is described in the work [Improved technology for obtaining monoclonal antibodies for the determination of transfusion-hazardous antigens of erythrocytes. Khlyabich G.N., Dubinkin I.V., Donskov S.I., Sukhanov Yu.S., Persanova L.V. Bulletin of the blood service of Russia. 2010. No. 1. S. 5-9; KOHLER G; MILSTEIN C: "Continuous cultures of fused cells secreting antibody of pre-defined specificity", NATURE, vol. 256, 1975, pages 495-7].

Lemieux R.). После потери антителопродукции гетерогибридому культивировали в присутствии 8-азагуанина (Sigma, США) для восстановления чувствительности к ГАТ. Через 2 месяца культивирования с 8-азагуанином чувствительность гетерогибридомы к ГАТ была полностью восстановлена. Теоретически таким способом может быть получена гетерогибридома мышиной миеломы NS1 с В-лимфоцитом любой видовой принадлежности.On selective medium with HAT, a hybridoma clone with the longest duration of human IgG1 production was selected (Hybrid Hybridomics. 2004 Dec; 23 (6): 362-7. Sensitive detection of human IgG in ELISA using a monoclonal anti-IgG-peroxidase conjugate. Chevrier MC ,

Lemieux R.). After loss of antibody production, the heterohybridoma was cultured in the presence of 8-azaguanine (Sigma, United States) to restore sensitivity to HAT. After 2 months of cultivation with 8-azaguanine, the sensitivity of the heterohybridoma to HAT was completely restored. Theoretically, this method can be used to obtain heterohybridoma of mouse NS1 myeloma with B-lymphocyte of any species.

2. human peripheral blood mononuclear cells. For effective hybridization, lymphoblasts are required, which are usually practically absent in the peripheral blood. Lymphoblasts specific to the desired antigen were obtained in 2 stages: obtaining mature B-lymphocytes and their blast transformation.

Mature B-lymphocytes were obtained as follows: a subpopulation of monocytes was isolated from human whole blood using a commercial kit, and immature dendritic cells were obtained from them by cultivation in the presence of granulocyte-macrophage-colony-stimulating factor (GMCSF) and IL4 (Patent WO 2011095592 A1 In vitro process for the preparation of antibodies of the igg type). After stimulation with antigen, immature dendritic cells turned into mature ones and could participate in the stimulation of B-lymphocytes as antigen-presenting cells. Then, CD4 + T-lymphocytes were obtained from the whole blood of the same donor using a commercial kit, and they were incubated with mature dendritic cells to obtain stimulated T-lymphocytes. B-lymphocytes were also isolated from the whole blood of an autologous donor and stimulated by the addition of antigen (Patent WO 2011095592 A1). For final maturation, B-lymphocytes were cultured in the presence of mature dendritic cells stimulated by CD4 + T-lymphocytes and cytokines IL2, IL4, IL6, and IL10 for 5 days. At the same time, cell proliferation and the formation of cell synapses were observed.

Mature B cells are unsuitable for obtaining hybridomas; therefore, they were transformed into lymphoblasts using the commercially available Lakonos American mitogen, cultivating them in its presence for another 3 days. Proliferation in the presence of mitogen continued, so lymphoblasts were obtained from 10 ml of donor blood without the participation of oncogenic viruses, the number of which was sufficient to obtain hybridomas.

Hybridization was performed according to a standard protocol using polyethylene glycol 4000. The cells were plated onto the feeder layer of mouse peritoneal cells in complete growth medium DMEM supplemented with 8% fetal calf serum and HAT. The supernatants were tested by enzyme immunoassay, clones secreting IgG antibodies to the required antigen were selected, cloned by the limiting dilution method and accumulated in culture flasks, monitoring antibody production. Antibodies were isolated from culture liquids by affinity chromatography for protein G-Sepharose, their concentration and activity were determined in ELISA. Additionally, the biological activity of antibodies was determined.

The claimed method can produce antibodies of any species, for example, horses, dogs, cats and other mammals. (But this is only theoretically, we have not tried it). This requires murine myeloma and the species-specific cytokines, if species-specific.

To implement the proposed method, any myeloma lines can be used (Improved technology for obtaining monoclonal antibodies to determine transfusion-hazardous antigens of erythrocytes. Khlyabich GN, Dubinkin IV, Donskov SI, Sukhanov Yu.S., Persanova L. V. Bulletin of the blood service of Russia. 2010. No. 1. S. 5-9).

Thus, the use of a heterohybridoma as a tumor partner ensures stable antibody production of the resulting hybridoma. The time separation of the processes of maturation of B-lymphocytes in vitro and their transformation into lymphoblasts made it possible to first obtain full-fledged mature B-lymphocytes, and then turn them into lymphoblasts suitable for hybridization, which led to the production of many primary IgG-producing hybridoma clones.

The following examples are provided for purposes of explanation and do not limit the scope of the present invention in any way.

Example 1. Obtaining a hybridoma stably producing a human monoclonal antibody against rabies virus glycoprotein G.

The procedure for in vitro stimulation of B-lymphocytes was carried out as follows: previously isolated from blood monocytes (300 thousand cells) were thawed, seeded in the wells of a 6-well culture plate in the presence of 800 IU / ml GMKSF and 500 IU / ml IL4, cultured for 5 days in a protein-free medium for dendritic cells CellGro DC manufactured by CellGenix GMP, Germany. As a result of culturing, monocytes acquired the morphology of immature dendritic cells. Then added Rabipur vaccine to a final dilution of 1: 200 and cultured for another 24 hours. By the end of the cultivation period, the cells acquired the morphology of mature dendritic cells. In this case, no proliferation or significant cell death was observed; accordingly, about 300 thousand mature dendritic cells loaded with antigens of the rabies virus were obtained.

B-lymphocytes were isolated from 10 ml of blood of an autologous donor and CD4 + T-lymphocytes were isolated from 5 ml of blood; for isolation, sets for the isolation of CD4 + T-lymphocytes, B-lymphocytes manufactured by RosetteSep (STEMCELL TECHNOLOGIES INC, USA) were used. As a result, 2 million CD4 + T-lymphocytes and 1.8 million B-lymphocytes were obtained. The isolated B-lymphocytes were cultured in CellGro DC medium for 8 hours with Rabipur antirabies vaccine at a final dilution of 1: 200. Mature dendritic cells removed from the surface of the flask with a cell scraper were added to the CD4 + T lymphocytes and cultured in CellGro DC medium for 4 hours. Then the B-lymphocytes stimulated with viral particles were transferred to the stimulated CD4 + T-lymphocytes and cytokines IL-2 were added to a final concentration of 20 U / ml, IL-4 to a final concentration of 500 U / ml, IL-6 to a final concentration of 1800 U / ml, IL-10 to a final concentration of 2 U / ml. Cultivated for 5 days. During this time, the cells formed large cellular synapses, visible to the naked eye, the total number of cells increased tenfold, which indicates their active proliferation and clonal expansion.

To transfer mature B-lymphocytes to the stage of lymphoblasts, after 5 days, the mitogen Phytolacca americana was added and incubated for another 3 days. In this case, further cell proliferation was observed. The morphology of the resulting cells was not studied, since almost all lymphocytes and lymphoblasts were located in dense cellular synapses. Half of all cells were frozen, the other half underwent hybridization with mouse-human heterohybridoma using polyethylene glycol 4000. The hybridized cells were plated on 96-well culture plates in complete growth medium DMEM in the presence of hypoxanthine-aminopterin-thymidine (HAT) with pre-seeded peritoneal mouse cells. The growth of hybridoma clones was recorded using a microscope; in total, about 1-2 thousand hybridoma clones were obtained. It is believed that with successful hybridization of lymphoblasts and myeloma cells from animals of the same line, the hybridization efficiency is 1:10 ⁴ cells, that is, one viable hybrid is obtained from 10,000 initial cells. Considering that a total of 1.8 million human B-lymphocytes were isolated, in the process of in vitro immunization their number increased 20-30 times, and half of the cells obtained were used, the total number of hybridomas should be about 2-3 thousand or 20 30 clones per well when plated per plate. Thus, the efficiency of hybridization was found to be comparable to the efficiency in obtaining murine hybridomas.

Example 2. Testing of hybridoma supernatants producing human antibodies against rabies virus

To select clones secreting antibodies of the IgG class against the rabies virus, the following scheme of enzyme-linked immunosorbent assay was used: a mouse antibody against G1 heavy chain of human immunoglobulins 2C11, 5 μg / ml was immobilized into the wells of an ELISA plate, then incubated with hybridoma supernatants diluted in half with PBS- AT, after washing, incubated with rabies vaccine Rabipur, diluted 1:25 in PBS-AT, then with murine antibody against rabies virus 1C5 conjugated with peroxidase. Thus, the immobilized 2C11 antibodies bind human IgG1 immunoglobulins from the supernatants; among them, antibodies specific to the rabies virus bind viral particles from the vaccine solution; these particles, in turn, bind mouse antibodies 1C5, which carry an enzymatic tag. With this scheme, the signal appears only if the supernatant contains an antibody against a viral particle of the IgG1 class. The test results showed that most of the wells of the plate contained clones producing antibodies against rabies virus. (Table 1).

Example 3. Production of hybridoma clones RabD4 and Rab5G6.

Hybridoma clones RabD4 and Rab5G6, secreting antibodies against rabies virus, were cultured for more than 3 months after at least 10 passages. Antibody production of each passage was monitored by ELISA using serial 2-fold dilutions of supernatants (Tables 2 and 3). The level of antibody production practically did not change from passage to passage for clone Rab5G6 and slightly increased for clone RabD4.

A total of 350 ml of RabD4 clone supernatant and 450 ml of Rab5G6 clone supernatant were produced. Antibodies were isolated from supernatants by protein G-Sepharose affinity chromatography (GE Healthcare, # 17-0618-05). The yield of antibodies from 1 ml of culture fluid was 0.47 mg for the RabD4 clone and 0.57 mg for the RabG6 clone. Analysis of antibody samples was performed by electrophoresis in 12% PAGE of FIG. one).

Example 4. Analysis of the biological activity of hybridoma human antibodies RabD4 and Rab5G6.

The antibodies to rabies virus, which have virus-neutralizing activity, are of the greatest value. The neutralizing activity was assessed by the FAVN method. Antibodies were incubated with rabies virus strain CVS11, then added to BHK-21 C13 cells. The neutralizing activity of antibodies was determined by the incorporation of viral particles into the cytoplasm of the cells in comparison with the international WHO International Laboratory For Biological Standards, Copengagen, Denmark: Second International Standard for Rabies Immunoglobulin, and the European Pharmacopoeia Reference Standard Human rabies immunoglobulin BPR, Strasburg. According to the results of the analysis, it was found that the RabD4 antibody has a high neutralizing activity of 305.7 IU / mg, while the Rab5G6 antibody did not show any neutralizing activity.

Thus, a method has been developed for producing fully human antibodies with authentic immunoglobulin sequences and without any genetic modifications, since does not imply manipulations with the genetic material of cells (isolation of DNA or RNA, amplification, construction of vectors, etc.), as well as the hybridoma lines obtained by the claimed method, stably produce human IgG antibodies of the desired specificity.

Claims (3)

1. A method of obtaining a human IgG antibody, with authentic human immunoglobulin sequences, produced by a hybridoma (trioma): mouse myeloma - human lymphocyte - human lymphocyte, characterized in that the producer is obtained by somatic hybridization of human-mouse heterohybridoma P3 / NSI / 1-Ag4- 1 [NS-1] (ATCC ^® TIB-18 ^™ ) and human lymphoblasts obtained from mature B-lymphocytes, pre-stimulated in vitro with mitogen, followed by selection and production of hybridoma clones secreting human IgG immunoglobulins of the required specificity, by isolating the obtained antibodies , while stimulation of human peripheral blood B-lymphocytes in vitro is carried out by adding stimulated CD4 + T-lymphocytes, using cytokines IL-2, IL-4, IL-6 and IL-10, and mature antigen-loaded human dendritic cells and CD4 + T - human lymphocytes, then culturing is carried out until the maturation of B-lymphocytes for 4-5 days, and mature dendri mate cells are obtained from human peripheral blood monocytes using GMCSF, IL-4 and immunogen, and the transformation of mature B-lymphocytes into lymphoblasts takes 3 days. 2. The method according to claim 1, characterized in that lectin from the American lakonos is used as the mitogen. 3. The method according to claim 1, characterized in that a protein or a protein complex against which an antibody is desired is used as an immunogen.

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