Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies

Definitions

• the present invention relates to methods of producing human monoclonal antibodies and their use.
• RIA radioimmunoassay
• EIA enzyme immunoassay
• IFA immunofluorescence assay
• Antibodies against a few of the viral antigens It is also often only possible to obtain monoclonal antibodies against individual, but not against all, antigens of a virus from immunized animals.
• the object of the present invention is therefore to provide a method by which specific human monoclonal antibodies can be produced. According to the invention, this is achieved in a process which comprises the following process steps:
• B-cell precursors from tumor-infiltrated persons those infected with prokaryotic or eukaryotic pathogens, in particular viruses, or who have an inherited disease,
• antigen-presenting cells are preferably isolated from the pleural fluid of the persons specified.
• this liquid is diluted with a buffer, preferably Hanks, and centrifuged.
• the pelleted cells are then taken up in medium containing heat-inactivated fetal calf serum and incubated.
• the medium preferably also contains substances through which
• Dye molecule A is the condensation product of flavoprotein, namely that of riboflavin 5'-monophosphate sodium salt with alpha-lipoic acid, which remains after the addition of iodoacetamide and dialysis after distillation.
• Dye molecule B is understood to mean the rest
• Cells that have absorbed the fluorescent dyes mentioned are not only stimulated to grow, but are also fluorescence-labeled and thus recognizable under the microscope. According to the addressed
• Fluorescent dyes are preferably used in combination, for example with proteins or peptides. They are then called carrier-bound fluorescent dyes.
• serum-free, antigen-stimulating medium which promotes the proliferation of antigen-presenting cells
• IMDM Iscoves modified Dulbecco medium
• TCGF interleukin 2
• GM-CSF granulocyte-macrophage-colony-stimulating factor
• T "cell precursors and B cell precursors are preferably isolated from the venous blood of the persons specified.
• the cells are collected by several centrifugation steps and on with blood group sera and GM-CSF (specified above)
• T-H, cell precursors and B-cell precursors are characterized by
• IL-1 interleukin 1
• T n cell precursors According to the invention, T n cell precursors and
• serum-free, non-antigen stimulating medium As a medium, for example, the commercially available
• Base medium IMDM Iscoves modified Dulbecco medium
• Factors such as LAF (Interleukin 1), TRF (T-cell replacing factor) and GM-CSF (given above) are added to this medium.
• LAF Interleukin 1
• TRF T-cell replacing factor
• GM-CSF GM-CSF
• Mature B cells then mature into antibody-producing (secreting) cells (plasma cells).
• Mature B cells can be distinguished from immature B cells by incubation with an antibody directed against their surface immunoglobulin. Mature B cells re-synthesize their surface immunoglobulin within 24 hours, while immature B cells are unable to do so.
• B cells are preferably isolated from the venous blood of the persons specified.
• the cells are washed, incubated in a serum-free, non-antigen-stimulating medium, to which the fluorescent dye indicated above is preferably added to stimulate the proliferation of the cells, then centrifuged and washed.
• the pelleted cells are again taken up in the same medium and carefully mixed with antigen-activated T ⁇ cell precursors and B cell precursors and then incubated.
• the action of UV light and the drop in CO 2 pressure release the antigen-activated precursor cells. They are centrifuged off, while the stuck activated B cells are washed several times in medium, centrifuged and resuspended in medium.
• the activated B cells are then placed in wells of microtiter plates and incubated. The plates are centrifuged and after macrophages and remaining cell precursors have been removed, the activated B cells are again labeled with the fluorescent dye indicated above and incubated for 8 days. Antibody production is checked every two days. To do this, the protrusions of the spots are tested. B cells, the supernatants of which are positive, are added to eating cells or macrophages, whereby the B cells are stimulated to proliferate. After differentiation to antibody-secreting -7-
• Plasma cells are cloned. For this purpose, the cells present in the positive supernatants are separated and cultivated.
• the method according to the invention makes it possible to produce human monoclonal antibodies without having to use recombination or cell fusion methods. In this way, the chromosome losses that often occur with these methods can be avoided, which also eliminates the associated risk of obtaining non-specifically binding antibodies.
• the method according to the invention also has the great advantage that antibodies are not only obtained from a limited number of different, mature human B cells (plasma cells), as is customary, but that, through the use of Tn cell precursors and B cell precursors, the overall Potential of all possible antibody variations in a person can be exploited. This gives you the opportunity to obtain a wide variety of antibodies. For example, antibodies against the HIV surface protein gp 120 and its precursor gp 160 were also isolated.
• Human monoclonal antibodies produced according to the invention have, as already mentioned, the ability to bind specifically, so they are suitable for the detection of very specific antigens and thus for the diagnosis of tumors, hereditary or caused by prokaryotic or eukaryotic pathogens, in particular viruses.
• AIDS, multiple sclerosis and Alzheimer's disease are particularly meant as viral diseases, while small cell lung cancer is particularly addressed as a tumor disease.
• Human monoclonal antibodies produced according to the invention are also suitable for therapeutic measures, since due to the lack of non-host determinants in treated patients there is no antibody production directed against the antibodies produced according to the invention.
• the crystalline dye molecules A and B according to DE-OS 38 11 692 (page 2 and claim 1) are treated separately in PBS buffer in an ultrasonic bath until a clear solution. 4 mmol each of the dye molecules A and B are in
• reaction mixture is checked with starch-iodide test strips. If there is free ENT “, the test strip shows a blue-black color.
• the dye conjugate is dialyzed at 4 ° C. for 1 h and sterile filtered.
• the ready-to-use fluorescent dye is adjusted to pH -7.5 and stored in the refrigerator. -9-
• a carrier-bound fluorescent dye is obtained by coupling the ready-to-use fluorescent dye from Example 1 with proteins, peptides or other substances.
• the coupling reaction is carried out at 4 ° C and a pH of 9.0.
• a protein with a molecular weight of 155,000 (IgG) requires 0.08 mmol for coupling. This amount of protein is in 100 ml borate buffer, or as
• reaction solution is stirred for 2 hours at 4 ° C. with frequent pH control, the reaction solution is left to stand in the refrigerator overnight and then dialysed against 5-7 L 0.15 M NaCl for 12 hours. After dialysis is complete, the pH is adjusted to 7.5.
• the coupling can alternatively also be carried out by means of an equilibrium dialysis, two dialysis chambers with different membranes are put together with an intermediate membrane filter and
• the membrane In the chamber, the membrane, the diffusion of the ready-to-use fluorescent dye, but not that of
• the protein solution is introduced into the other chamber of the ready-to-use fluorescent dye off.
• the equilibrium is reached when the concentration of the free protein is the same on both sides. Free and bound protein is measured in a fluorometer. The absorption and emission spectra show free and bound amounts of protein.
• Hyaluronidase (MW 89,000) is an important diffusion factor for the incorporation of proteins, peptides and other substances into living cells.
• 1 ml of dissolved hyaluronidase (in 0.9% NaCl) is pipetted into 100 ml of the ready-to-use fluorescent dye from Example 1.
• the reaction mixture is then stirred vigorously at 37 ° C. for 10 min, cooled to 4 ° C., dialyzed and adjusted to pH -7.5.
• certain proteins, peptides or other substances are coupled with the ready-to-use fluorescent dye from Example 1.
• Pleural fluids from tumor or HIV patients are used to obtain antigen-presenting cells.
• the pleural fluid is centrifuged and the cell precipitate obtained is washed with modified Hanks solution, centrifuged and filled into 3 ml aliquots.
• the prepared cells (aliquots) are resuspended in M 199 medium supplemented with 2% heat-inactivated fetal calf serum and 100 ⁇ l fluorescent dye (from Example 1) and then transferred to culture bottles and cultured for 24 hours. Thereafter, non-adherent cells are washed out and fixed labeled cells are treated with 0.25 M EDTA, pH 7.5 to remove contaminating fibroblasts, macrophages and mesothelial cells.
• Specific cells are then transferred to serum-free, antigen stimulating medium.
• It is the basic medium - IMDM (Iscoves modified Dulbecco medium), without L-glutamine, which with factors such as 1 U / ml TCGF, 0.1, ul / ml GM-CSF and 10, ul / ml fluorescent dye (from
• Example 1 The obtained fluorooorreesszzeennzzmmaarrkkiieerrtteenn KKlloonnee wweerrddeen in 100, ul aliquots stored in liquid nitrogen,
• T "cell precursors and B cell precursors are made from heparinized according to the known" buffy coat B method
• the petri dishes were additionally GM - CSF (100 / ul 1:10 and 100, ul 1: 500) -12-
• Non-adherent cells are washed out and the stuck cells are resuspended in serum-free, antigen-stimulating medium (see Example 3) and stored in the refrigerator until reused.
• Ficoll-Hypaque gradient centrifugation method is isolated, washed and incubated for 1 h at 37 ° C. with the addition of 100 ⁇ l fluorescent dye from example 1 in serum-free, non-antigen stimulating medium. It is the basic medium IMDM (Iscoves modified Dulbecco medium), without L-glutamine, the factors such as 1 U / ml LAF, l.ul / ml TRF, 0, l, ul / ml GM-CSF and lO.ul / ml. Fluorescent dye (from Example 1) are added. The mixture is then centrifuged at 500 g for 5 min.
• IMDM Iscoves modified Dulbecco medium
• the cell precipitate is resuspended in the medium and the cell suspension is then adjusted to 2x10 cells / ml and stored. With 9-20% fluorescent dye in the medium, cell numbers of 4.85 x 10 to 5.36 x 10 cells are achieved. Dye concentrations below 50% have no negative effect on cell viability.
• 100 / ul fluorescent-labeled antigen presenting cells are made with 100, ul T ⁇ cell precursor and B cell precursor
• the antigen-activated cell precursors are resuspended in serum-free, antigen-stimulating medium (see Example 3) and stored until they are used.
• B cells are pretreated as described in Example 5. 50 / ul fluorescence-labeled, antigen-activated
• T ⁇ cell precursors and B cell precursors are mixed with 50, ul
• Non-adherent cells are washed out with serum-free, non-antigen stimulating medium (see Example 5).
• Adherent cells are 90 min in serum-free, - non-antigen stimulating medium -14-
• the B cells are again treated with 100, ul of
• Fluorescent dye (from Example 1) labeled, then aliquoted and stored at -20 ° C until use q or in wells of microtiter plates (1x10 cells / ml and
• the B cells After stimulation by eating cells or markophages, the B cells proliferate and differentiate as clones into plasma cells that secrete antibodies.
• 10 feed cells / ml or 4x10 macrophages / ml are placed in the wells of the microtiter plates. The cells present in the positive supernatant are counted. 10 cells are sufficient for cloning. -15-
• Sera from 5 seropositive / virus-positive patients, 5 seropositive / virus-negative patients and 5 normal persons are isolated and processed. -16-
• regression method simply linear and directed linear regression, split interpolation and polygonal interpolation.
• Absorption ratio less than 2.5 negative between 2.5-5 acceptance of HIV-AK to low-level greater than 5-11 high level
• Cells are reactivated with an unlabeled antibody, washed, transferred into chamber slides and placed on thinly applied medium (from Example 3) and fixed.
• the cells to be examined are pretreated with 50 .mu.g / ml L-lysine or with carrier-bound fluorescent dye (from Example 2).
• Binding capacity is prevented after 10 minutes with 1 to 2 drops (25, ul to 50, ul) blocking solution (sterile whey).
• the cells are then washed with medium, incubated for one hour in a moist chamber with an antibody fluorescently labeled according to the invention or with an antibody labeled according to customary methods at RT or 37 ° C. (the incubation temperature depends on the type of antibody), then washed again with medium and examined under the microscope in the chamber slides.
• biopsy material especially lymph nodes, can be examined immediately.
• human-human lymph nodes are charged with a drop of a human monoclonal antibody which is fluorescence-labeled and additionally complement-amplified according to the invention, incubated for 10 minutes and washed with physiological, sterile saline solution. -18-

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• 1989
  • 1989-06-28 DE DE19893921211 patent/DE3921211C1/de not_active Expired - Lifetime
• 1990
  • 1990-06-20 CA CA 2033984 patent/CA2033984A1/en not_active Abandoned
  • 1990-06-20 EP EP19900909725 patent/EP0431125A1/de not_active Withdrawn
  • 1990-06-20 WO PCT/EP1990/000976 patent/WO1991000342A1/de not_active Ceased

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