WO2021080542A1 - Production d'anticorps monoclonal spécifique à un marqueur de surface de cellule souche cd133 - Google Patents

Production d'anticorps monoclonal spécifique à un marqueur de surface de cellule souche cd133 Download PDF

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Publication number
WO2021080542A1
WO2021080542A1 PCT/TR2020/050973 TR2020050973W WO2021080542A1 WO 2021080542 A1 WO2021080542 A1 WO 2021080542A1 TR 2020050973 W TR2020050973 W TR 2020050973W WO 2021080542 A1 WO2021080542 A1 WO 2021080542A1
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WIPO (PCT)
Prior art keywords
antibody
fusion
antibodies
cells
elisa
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PCT/TR2020/050973
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English (en)
Inventor
Asuman SUNGUROĞLU
Hasan Çağlar UĞUR
Dilara AKÇORA YILDIZ
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Ankara Üniversitesi Rektörlüğü
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Publication of WO2021080542A1 publication Critical patent/WO2021080542A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype

Definitions

  • the present invention relates to a novel anti-CD133 7D7 antibody that is capable of binding to the CD 133 surface marker with high affinity.
  • a monoclonal antibody specific to CD133 protein is produced by hybridoma technology that can be used by western, flow cytometry, and immunofluorescence methods. Accordingly, studies concerning immunization of the mice with CD 133 peptide, monoclonal antibody production, purification, and characterization of the same were conducted.
  • 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.
  • myeloma cancer cells.
  • immortal hybrid cells producing a monoclonal antibody that are devoted to a single determinant group (epitope) of an antigen are achieved.
  • Hybridoma technology is used for monoclonal and in-vitro production of B cells that have anti- CD133 antibody production capacity. It is possible to realize cheap, safe, and easy production of cells that produce monoclonal anti-CD 133 antibodies by means of hybridoma technology. Division of the cells that produce antibodies is ensured to continue with the fusion of spleen/lymph cells taken from mice which are immunized and whose antibody response is determined, with the mice myeloma cells with this technology.
  • CD133 antibodies are already commercially available, most of these antibodies detect the glycosylated CD133 epitopes. However, more recently it is stated that cells have non-glycosylated CD133 epitopes during differentiation. For this reason, a high- affinity monoclonal antibody specific to the 2nd loop of CD 133 surface peptide was synthesized. It is evident that a 7D7 monoclonal antibody specific to CD133 can be used in a flow, western, immune fluorescent methods by making the same visible with any kind of secondary antibody.
  • a monoclonal antibody that can compete with commercial CD133’s in which CD 133 protein that is expressed in bone marrow, colon, endocrine system, liver, gall bladder, gastrointestinal system, male and female reproductive tissues, and that is accepted as one of the stem cell and cancer stem cell markers can be detected by a plurality of methods mentioned above.
  • a new monoclonal antibody that can be bind to the CD 133 surface marker with high affinity is produced with the present invention.
  • the inventive method can be used to detect CD133 protein in the fields of medical biology, molecular biology, genetics, and biotechnology.
  • 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 508th and 792nd amino acid so as to synthesize Ab specific to the 2nd loop for CD133 that is produced in a recombinant manner as an immunogen in the production studies of monoclonal antibody against CD133.
  • the cloning strategy used to produce antigen is as follows:
  • mice 48-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 ELISA. Determining the antibody level by ELISA method was performed as follows:
  • Each well of ELISA plates with 96-well was coated with 250-500ng/well CD133 antigen and kept for 1 night at 4°C with closed cover.
  • 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.
  • AP-conjugated secondary antibody developed against mouse antibodies was diluted in a ratio of 1/3000 and was added into the wells (100 m ⁇ /well). Plates are kept at 37°C for 1 hour.
  • 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 m ⁇ was added per each well.
  • mice serums were assessed by diluting the same in a ratio of 1/100, 1/1000. The responses of the mice increased after the 2 nd immunization.
  • 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;
  • DMEM fetal calf serum
  • 0.1% Gentamicin 0.1% Gentamicin
  • 2% HAT hypoxanthine- aminopterin-thymidine
  • 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 live 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.
  • the fusion study of the mice that were detected to give specific antibody responses was realized.
  • 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.
  • 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 32 responses in total against CD133 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.
  • 7D7 hybrid cells which were a clone giving response to CD133 so as to form a strain consisting of a single clone.
  • 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 7D7 clones would be placed after the fusion and the medium was enriched for hybridoma culture.
  • 7D7 cells were taken into tubes with the help of a pipette and cell counting was carried out and distributed as a single cell into 96-well culture plates.
  • Protein A was purified by an affinity chromatography method because the obtained antibody is of 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.
  • the purification Scheme of Monoclonal antibody (7D7) specific to CD133 is shown in Table 2.
  • Antibodies that were achieved in purified form were subject to Elisa after purification. Fluids on the cells were used as a positive control; Pbs was used as negative control.
  • Immunofluore scent staining was performed on the U87CD133 cell lines of the CD133 protein of the purely obtained CD133 (7D7) antibody and it was determined that the antibody also specifically worked for immunofluorescence staining under confocal microscopy.
  • 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. CD133 targeted treatments are appropriate for developing diagnostic methods.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un anticorps anti-CD133 7D7 qui est capable de se lier au marqueur de surface CD133 avec une affinité élevée.
PCT/TR2020/050973 2019-10-23 2020-10-22 Production d'anticorps monoclonal spécifique à un marqueur de surface de cellule souche cd133 WO2021080542A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TR2019/16361 2019-10-23
TR201916361 2019-10-23

Publications (1)

Publication Number Publication Date
WO2021080542A1 true WO2021080542A1 (fr) 2021-04-29

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011149493A1 (fr) * 2010-05-26 2011-12-01 Regents Of The Unviversity Of Minnesota Anticorps anti-cd133 à fragments monocaténaires variables et leurs utilisations
WO2016154623A2 (fr) * 2015-03-26 2016-09-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Anticorps monoclonaux anti-cd133 et compositions et méthodes associées

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011149493A1 (fr) * 2010-05-26 2011-12-01 Regents Of The Unviversity Of Minnesota Anticorps anti-cd133 à fragments monocaténaires variables et leurs utilisations
WO2016154623A2 (fr) * 2015-03-26 2016-09-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Anticorps monoclonaux anti-cd133 et compositions et méthodes associées

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HOLZLÖHNER PAMELA, HANACK KATJA: "Generation of Murine Monoclonal Antibodies by Hybridoma Technology", JOURNAL OF VISUALIZED EXPERIMENTS, vol. 119, 1 February 2017 (2017-02-01), pages e54832-1 - e54832-7, XP055819559, DOI: 10.3791/54832 *

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