WO2021080541A1

WO2021080541A1 - Production of monoclonal antibody specific to cell receptor cd24

Info

Publication number: WO2021080541A1
Application number: PCT/TR2020/050972
Authority: WO
Inventors: Asuman SUNGUROĞLU; Hasan Çağlar UĞUR; Dilara AKÇORA YILDIZ
Original assignee: Ankara Ueniversitesi Rektoerluegue
Current assignee: Ankara Ueniversitesi Rektoerluegue
Priority date: 2019-10-23
Filing date: 2020-10-22
Publication date: 2021-04-29
Anticipated expiration: 2022-04-23
Also published as: EP3897723A1; EP3897723A4

Abstract

A monoclonal antibody that binds to the 27th-59th amino acids of CD24 surface receptor protein with high affinity is synthesized by means of the present invention. A therapeutic CD24 monoclonal antibody that can be used as a cancer stem cell marker to reduce the survival of cells of many types of cancer, especially breast cancer, was synthesized. In addition, it is understood that the CD24 12E9 antibody used has no toxic effect on normal blood cells.

Description

Production Of Monoclonal Antibody Specific To Cell Receptor CD 24

Technical Field of the Invention

The present invention relates to a novel monoclonal antibody that has therapeutic effects, and that is capable of binding to the CD24 surface marker with high affinity.

State of the Art

CD24 is found in bone marrow, colon, endocrine system, and chondrocyte cells. Moreover, scientific studies have indicated that it is responsible for tumor induction and resistance in many cancer types, particularly in breast cancer as a cancer stem cell marker. Therefore, a therapeutic CD24 monoclonal antibody was synthesized to be used in reducing the survival of cancer cells in the cancer types that belong to the abovementioned systems.

It was determined with the present invention that the 12E9 monoclonal antibody specific to CD24 reduces the viability of glioblastoma cells in vitro, is not toxic in vitro and in vivo. Anti- CD24 12E9 antibody was produced by hybridoma technology.

Technical Problems the Invention Seeks to Solve

It is possible to realize cheap, safe, and easy production of cells that produce monoclonal antibodies by means of the hybridoma technology with the present invention. Division of the cells that produce antibody is provided to continue with the fusion of spleen/lymph cells taken from mice which are immunized and whose antibody response is determined, with the mouse myeloma cells with this technology. The hybrid cells obtained with the invention are stored within liquid nitrogen. When it is needed, frozen cells can be thawed and used for many years by means of culturing the same. There is a potential for producing the same very economically. It can be used in Medical Biology, Molecular Biology, and Genetics, Oncology.

Description of the Invention

It is aimed to produce monoclonal antibody specific to CD24 protein and in this direction, immunization of the mice with CD24 peptide, studies in terms of monoclonal antibody production, purification, and characterization of the same were performed. It was realized to use the same in glioblastoma tumors in mice where death occurs in glioblastoma cells to which produced CD24 antibody was applied in vitro.

Detailed Description of the Invention

12E9 monoclonal antibody specific to CD24 was produced by using the hybridoma technology. The hybridoma technique is a method that is based on fusing the splenic lymphocytes of the animal that produce active antibody for the production of a monoclonal antibody with myeloma (cancer) cells. As a result of this process, immortal hybrid cells producing monoclonal antibody (Ab) that are devoted to a single determinant group (epitope) of an antigen are achieved.

Producing monoclonal antibody specific to CD24 protein is provided with the studies of immunization of the mice with CD24 peptide, producing the monoclonal antibody, purification, and characterization of the same.

The first step for the production of a monoclonal antibody is to achieve an immune response specific to the target molecule. For this reason, a peptide that covers the section between 27 ^th- 59^th amino acids of CD24 antigen that is produced recombinantly as an immunogen in the production studies of monoclonal antibody against CD24. CHO cells were preferred for the production of the peptide since there were many glycosylation sites in the CD24 antigen. The cloning strategy used to produce antigens is as follows:

In the invention, 4, 8-week-old female BALB/c mice species were used. The mice were immunized with antigens 4 times at 2-week intervals. For the first immunization, Complete Freund’s Adjuvant (CFA) equal to an amount of 50pg antigen was mixed and was injected into each mouse intraperitoneally (IP). The following immunizations were made by mixing 50 pg of antigen with an equal amount of Incomplete Freund’s Adjuvant (IF A). The measurement of the antibody responses formed in mice as a result of immunizations was provided by indirect EFISA. Determining the antibody level by EFISA method was performed as follows:

Each well of EFISA plates with 96-well was coated with 250-500ng/well CD24 antigen and kept for 1 night at 4°C with a closed cover.

The next day, the wells washed with 0.2% Tween-PBS were blocked for 1 hour with 200 pi of 1% milk powder (w/v in PBS). After the wells are washed with 0.2% Tween-PBS again, the diluted serum received from the mice in a ratio of 1/100, 1/1000, and 1/5000 was placed in the wells (IOOmI /well) and was kept at 37°C for 1 hour.

After washing the wells with 0.2% Tween-PBS, AP-conjugated secondary antibody developed against mouse antibodies was diluted in a ratio of 1/3000 and was added into the wells (100 mΐ /well). Plates are kept at 37°C for 1 hour.

After the wells were washed with 0.2% Tween-PBS 5 times, the substrate (4-nitrophenyl phosphate) was prepared such that it had a concentration of lmg/ml (substrate buffer was dissolved in ImM ZnC12, ImM MgC12, 0.1M glycine dH20 and pH was adjusted to 10.4, total volume was 200 ml) and 100 mΐ was added per each well.

After 30-60 minutes the absorbance (OD) was read at 405 nm.

ELISA measurements were made as of the second immunization. The base was coated with 250-500 ng / well CD24 antigen in these studies. Obtained mice serums were tested by diluting the same in a ratio of 1/100, 1/1000.

It was decided to fuse mice with OD⁴⁰⁵ nm value greater than 2, with high and specific antibody response as a result of the ELISA test and for this aim, 4 days before the fusion, a booster immunization was realized with 25 pg antigen without adjuvant from the tail in the same concentration. The stimulation of the cells producing antibodies against the antigen and their increase in numbers are aimed at the booster process.

Fusion and Hybridoma Selection

Hybridoma technology was used for monoclonal and in-vitro production of B cells that have anti-CD24 antibody production capacity. Division of the cells that produce antibody is provided to continue with the fusion of spleen/lymph cells taken from mice which are immunized and whose antibody response is determined, with the mouse myeloma cells with this technology.

After deciding to perform a fusion, the myeloma cells to be used in the fusion were removed from the liquid nitrogen and placed in the culture and were reproduced to 200-300 million by performing passage every other day, at least 10 days before the planned fusion. Moreover, 1 day before the fusion, macrophages that were not immunized and were taken from the abdominal cavity of another mouse were placed on the plates on which cells would be placed after the fusion, and the medium was enriched for hybridoma culture. After the spleen/lymph cells from the mice with positive immune response were isolated on the day of fusion were washed and mixed with the counted myeloma cells with determined ratios. The fusion of cells was realized by slowly adding PEG 4000 to this mixture. Then respectively;

Only DMEM (Dulbecco’ s Modified Eagle Medium),

DMEM with 20% added serum

DMEM with 20% added serum, 0.1% Gentamicin, 2% HAT (hypoxanthine- aminopterin-thymidine) was added.

HAT was used in the culture for the selection of hybridizing cells after fusion. HAT causes the death of myeloma cells with HGPRT mutation used in the fusion that do not fuse with the spleen cells. Spleen B lymphocytes and hybrid cells that can perform pyrimidine and purine synthesis will continue to survive in these selective conditions. However, spleen B lymphocytes that cannot make hybridization with the myeloma cells will die within 4-5 days under normal conditions. Therefore, only myeloma-spleen/lymph B lymphocyte hybrids will survive for a long time.

Screening the formation of hybrid clones on the plates is realized 10 days after the fusion and the plates on which clone formation was being observed were tested with ELISA for the presence of antibody on the plates. HT containing medium was added on the plates instead of the removed supernatant and after 10 days the wells of the clones with antibody response were marked with the ELISA test for the 2^nd time.

The fusion study was realized with the mice that were detected to give specific antibody responses. The fusion cells that were fed with a selective medium in the fusion study performed were distributed to the plates on which macrophages were placed previously and after 15 days they were examined under the microscope. As a result of the examinations made, an indirect Elisa test was applied to the clones by marking the ones that reached a determined size (with the supernatant in the well where there were -1056/156 clones) and it was observed that there were 8 responses in total against CD24 coated in the Elisa plate well. Clone screening was performed after 3 days and this time clones that were small in the previous screening but reached a certain density in the second screening were also included in the ELISA test. For CD24, which was positive as a result of ELISA, it was determined that it did not resulted in crossing in the test performed in the ELISA well that was not antigen-coated (only blocked with milk powder) for the 12e9 clone. As a result of this test, it was determined that the hybrid clone in the 12E9 well was CD24 specific, and following the increase in the number of cells, spares were prepared to the adjacent plate wells that are known to be empty. After fusion, dilution was conducted in 12E9 hybrid cells which were a clone giving response to CD24 so as to form a strain consisting of a single clone. On the 19^th day of the fusion, macrophages that were not immunized and were taken from the abdominal cavity of another mouse were placed on the plates on which subsequently cells of 12E9 clones would be placed after the fusion and the medium was enriched for hybridoma culture. On the 20^th day of the fusion, 12E9 hybrid cells were taken into tubes with the help of a pipette and cell count was carried out and distributed as a single cell into 4 pieces of 96-well culture plates. As a result of 1-week follow-up, ELISA studies were realized with the clone consisting of a single cell and it was seen that the clone gave a strong response and did not respond when ELISA was carried out in only the presence of PBS without antigen, and the studies of cell culture and accumulation of supernatant were initiated. In addition to these studies, some of the cells of the 12E9 clone that were dropped individually were frozen and backed up in liquid nitrogen.

Characterization of Anti-CD24 Antibody

It was characterized by using isotyping strips and ELISA so as to determine the type of antibody Immunoglobulin (Ig) produced by the clone named 12E9 determined to produce a reactive antibody with CD24. As a result of the characterization, the isotype of the antibody named 12E9 was determined as IgGl.

Purification of Antibody

Protein A was purified by an affinity chromatography method because the obtained antibody is IgG isotype. Immunoaffinity chromatography is one of the most commonly used methods on Protein A or Protein B bound solid phase so as to separate mAh’s with IgG isotype from serum proteins and IgGs. Protein A and Protein G are the bacterial proteins that can specifically bind to the Fc section of IgG class mouse immunoglobulins that do not bind to an antigen. While purification was made with Protein A; antibodies in the mixture were bind to the column and the other proteins were washed and removed. Then, bound antibodies are collected with the change of pH of the medium. The process of purification of antibodies was performed in two steps. The antibodies that are concentrated by being precipitated with AS in the first step were purified with Protein A affinity chromatography. Antibodies were loaded onto Protein A immobilized column within the binding buffer. After the loading process, the column was washed with binding buffer and unbound foreign molecules were removed, antibodies bound to the column were removed with the elution buffer so as to obtain pure antibody.

Table 1. Purification Scheme of Monoclonal antibody (12E9) specific to CD24.

50 pi Tris, pH 9 was added to each of the tubes in which elution fractions were collected so as to prevent activity loss of the eluted antibodies at low pH. The amount of protein taken from the column was evaluated by absorbance reading at 280 nm, the activity at the fractions was evaluated by indirect ELISA. Since purified antibodies subject to aggregation in water, antibodies were dialyzed against PBS and were frozen at -80 C and were lyophilized until they are completely dried. Lyophilized antibodies were stored at +4C.

Claims

1. A synthesis method of a monoclonal antibody that binds to the 27^lh-59^lh amino acids of CD24 surface receptor protein with high affinity, characterized in that; it comprises the following production steps:

Immunizing 4, 8-week-old female BALB/c mice species,

Performing fusion and Hybridoma Selection,

Performing characterization of Anti-CD24 Antibody,

Purifying antibodies,

Freezing purified antibodies at -80 C and lyophilizing the same until they are completely dried,

Keeping Lyophilized antibodies at +4C.

2. A peptide used for CD24 according to claim 1, characterized in that; it is EcoRI-Kozak Sequence-Artificial signal peptide-hlGlFc-Linkerl-FLAG / EK-CD24 (27-59) -Stop codon-Hindlll peptide