WO2002046233A1 - Method for preparing a human monoclonal antibody, fragments thereof or antibodies comprising such fragments, resulting antibodies and use thereof - Google Patents

Method for preparing a human monoclonal antibody, fragments thereof or antibodies comprising such fragments, resulting antibodies and use thereof Download PDF

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WO2002046233A1
WO2002046233A1 PCT/FR2001/003881 FR0103881W WO0246233A1 WO 2002046233 A1 WO2002046233 A1 WO 2002046233A1 FR 0103881 W FR0103881 W FR 0103881W WO 0246233 A1 WO0246233 A1 WO 0246233A1
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lymphocytes
fragments
antibodies
antibody
fragment
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PCT/FR2001/003881
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French (fr)
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Jacques Noury
Hervé Bazin
Dominique Latinne
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Technopharm
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the present invention relates to a method for preparing a human monoclonal antibody, fragments thereof or antibodies comprising such fragments.
  • the subject of the invention is also the hybridomas and the antibodies thus obtained and their uses in diagnostic and therapeutic compositions.
  • Monoclonal antibodies directed against an antigenic determinant have been produced by a large number of laboratories since the introduction of the technique of cell fusion between a myeloma cell and a lymphoid cell of an immunized animal.
  • the first model of ybridomas secreting monoclonal antibodies developed by K ⁇ hler and Milstein (1975, Nature, vol. 256, page 495) was a murine model.
  • the present invention relates to a method for preparing monoclonal antibodies having the advantage not to require the prior immortalization of the B lymphocytes such as for example by the EBV virus as proposed in the prior art.
  • the process of the invention is remarkable in that it is based on the successive and combined use of various cytokines at defined times and of a non-specific activating system.
  • the method of the invention is also remarkable in that the immortalization of differentiated human lymphocytes is carried out by means of a rat myeloma line.
  • the process for obtaining human monoclonal antibodies comprising a fusion of differentiated human lymphocytes with cells of a rat fusion line, leads to the production of particularly stable hybridomas and suitable for the production of human monoclonal antibodies.
  • the process for preparing human monoclonal antibodies or fragments thereof or of antibodies comprising such a fragment, from B lymphocytes comprises the following steps: a) isolating B lymphocytes from a sample originating from an individual of the human species, b) differentiating said B lymphocytes into plasma cells with at least one non-specific activating system and at least one cytokine, c ) the immortalization of said plasma cells by cell fusion with a rat fusion line, d) the in vitro culture of the fused cells until hybridization is obtained, and advantageously the screening of specific clones, e) the purification of the monoclonal antibodies produced by the hybridomas, f) optionally the cleavage of said antibodies into fragments, and the purification of one or more of said fragments, g) optionally the association of one or more of the fragments obtained with step (f) with fragments of other antibodies.
  • the invention relates both to the antibodies obtained in step (e) and to fragments of these obtained in step (f) free or associated with other molecules or substances, of any what isotype, humans or seeking to approach it.
  • monoclonal antibodies from mammals close to humans For example, monoclonal antibodies from mammals close to humans
  • the process of the invention may optionally include an additional step (h) characterized by the transfer of the genetic material of the hybridomas encoding all or part of the antibodies into any other expression system described in the specialized literature.
  • the B lymphocytes are isolated from a sample from an individual of the human species or from any other origin having the same characteristics.
  • the B lymphocytes are isolated in step (a) of the method according to the invention from human blood.
  • the term “plasma cells” in step (b) means modified B lymphocytes producing immunoglobulins and capable of being fused with another cell to give a hybridoma secreting immunoglobulins. This type of modified B cell can sometimes be called "activated B cell".
  • step (b) of differentiating B lymphocytes comprises the following steps: i) mixing a suspension of B lymphocytes with 0.01 to 1% of a non-specific activating system and from 1 to 8, 5 ng / ml of IL2, then incubation of the mixture for 24 hours to 11 days at a temperature between 36.5 and 37.8 ° C, ii) centrifugation of the suspension obtained in (i) from 600 to 3500 rpm for 2 to 12 minutes, then removal of the supernatant and resuspension of the cell pellet, iii) mixing the suspension obtained in (ii) with from 1 to 8.5 ng / ml of IL2 and 10 at 350 ng / ml of IL10, then incubation of the mixture for 48 hours to 15 days at a temperature between 36.5 and 37.8 ° C.
  • step (b) of differentiating B lymphocytes comprises the following steps: i) mixing a suspension of B lymphocytes with 0.04% of a non-specific activating system and 2 , 5 ng / ml of IL2, then incubation of the mixture for 3 days at a temperature of 37 ° C under 5% of C0 2 , ii) centrifugation of the suspension obtained in (i) at 1300 rpm for ⁇ minutes , then removing the supernatant and resuspending the cell pellet, iii) mixing the suspension obtained in (ii) with 2.5 ng / ml of IL2 and 100 ng / ml of IL10, then incubation of the mixture for 5 days at a temperature of 37 ° C.
  • the non-specific activating system is chosen from a lipopolysaccharide or any substance capable of having an identical action in whole or in part, pansorbine, phytohemagglutinin, muramyldipeptide used alone or in combination.
  • the fusion line used for the immortalization of the plasma cells in step (c) is a myeloma rat line and preferably a myeloma rat line of the LOU strain.
  • the fusion line used for the immortalization of the plasma cells in step (c) is the myeloma rat line LOU IR 983 F / TEC deposited in the National Collection of Cultures of Microorganisms at the Institut Pasteur (Paris) under No. 1-2584, November 29, 2000, this deposit having been made within the framework of the Budapest Treaty.
  • step (b) of differentiating B lymphocytes into plasma cells is carried out in the presence of a specific antigen of an infectious agent.
  • the B lymphocytes are isolated from blood from an organism vaccinated against an infectious agent or recovering from an infection by said infectious agent .
  • the B lymphocytes are isolated from the blood of a subject vaccinated against influenza.
  • the process of the invention makes it possible to produce monoclonal antibodies or fragments thereof or antibodies comprising such a fragment from B lymphocytes from mammals or from any animal species capable of showing a specific humoral immune response, but preferably these are isolated from human blood or mammals close to humans.
  • the invention obviously relates very particularly to the preparation of human monoclonal antibodies from human B lymphocytes.
  • the method of the invention allows the preparation of human monoclonal antibodies or of fragments thereof or of antibodies comprising such a fragment directed against any type of infectious agent, such as for example in a non-exhaustive manner the influenza virus.
  • infectious agent such as for example in a non-exhaustive manner the influenza virus.
  • agents classically objectified in biological warfare such as anthrax, smallpox, tularemia, the Ebola virus etc. and any pathogenic agent of man, animals and in fact of the animal and vegetable kingdom.
  • the invention also relates to a hybridoma capable of being obtained after steps (a) to (d) of a method described above, as well as the monoclonal antibodies capable of being produced by these hybridomas, or a fragment thereof. , and more particularly the Fab fragments including the parts of the antibody site which can bind to the antigen. These antibodies and their fragments can be identified by an ELISA test. The specificity of the monoclonal antibodies vis-à-vis the infectious agent can be evaluated:
  • monoclonal antibodies belong to the class of IgG, IgM, IgE, IgD or IgA, but they can also be modified, whatever their class, into antibodies of another class of immunoglobulins for example by techniques described in molecular biology (Lewis et al., 1992, Hum. Antibodies Hybridomas, vol. 3, pages 146-52 Lewis et al., 1993, J. Immunol., Vol. 151, pages 2829-38 Hall et al., 1994, Cancer Res ., vol. 54, pages 5178-85 Shepherd and Dean, "Monoclonal antibodies", Oxford university press, 2000, 479 pages).
  • the invention relates to a monoclonal antibody capable of being obtained by steps (a) to (e) of the method previously, or a fragment thereof obtained by steps (a) to (f) of said method.
  • Such a monoclonal antibody or fragment thereof can belong to the class of IgG, IgM, IgE, IgD or IgA.
  • the invention also relates to an antibody or antibody fragment comprising at least one of the above antibody fragments.
  • Such antibodies can be prepared by techniques known to those skilled in the art to combine different antibody fragments.
  • the subject of the invention is also a nucleic acid comprising a nucleotide sequence coding for an antibody obtained according to the method of the invention or for a fragment thereof.
  • the invention also relates to pharmaceutical compositions comprising, as active agent, an effective amount of at least one antibody or antibody fragment comprising at least one antibody fragment obtained according to the method of the invention.
  • compositions are useful for the diagnosis and for the curative or preventive treatment of infections by the infectious agents against which the antibodies and antibody fragments have been prepared according to the method described above.
  • the subject of the invention is a kit for detecting an infection with an infectious agent, comprising at least one antibody or at least one antibody fragment against said infectious agent obtained as described above.
  • DMEM culture medium Biowhittaker, ref: M12914F
  • RPMI 1640 culture medium Biowhittaker, ref: M12115F
  • IL2 Recombinant human interleukin 2 (R and D, ref:
  • IL6 Recombinant human interleukin 6 (R and D, ref .:
  • IL10 Recombinant human interleukin 10 (R & D, ref .: 217 IL025)
  • HEPES 1M Life Technology, ref .: 15630-056
  • PEG Polyethylene glycol 4000 (Fisher Sci., Ref:
  • Aminopterin 100X (Life Technology, ref .: 11362-019)
  • Thymidine 50X Hypoxantine Supplement (Life Technology, Ref: 41065-012)
  • the blood was taken from volunteers vaccinated with an influenza vaccine or recovering from an influenza infection. Samples are taken 0, 8, 15 or 21 days after vaccination.
  • Human blood from EDTA tubes is diluted in the same volume of PBS.
  • the diluted blood is placed in a 50 ml centrifuge tube containing the lymphocyte separation medium (1/3 of medium for 2/3 of blood). After centrifugation at 3000 rpm without brake for 20 min, the ring of lymphocytes is recovered at the interface using a 10 ml pipette.
  • the pipette is emptied into a 15 ml centrifuge tube and complete RPMI culture medium, RPMI culture medium containing 1% L-glutamine, 0.1% gentamicin, 50 ⁇ M of 2-mercaptoethanol and 10% serum.
  • fetal calf is added qs 15 ml.
  • the cell pellet is resuspended in 10 ml of complete RPMI culture medium. After centrifugation at 1300 rpm with brake for 6 min, the supernatant is removed. The cell pellet is resuspended in complete RPMI medium so as to have from 1 to 2.10 6 cells per ml.
  • the cell suspension is placed in culture flasks at a rate of 10 ml per 50 ml flask. The monocytes are removed by adhesion by incubating the bottles horizontally in an oven at 37 ° C. and 5% of CO 2 overnight.
  • a solution of 0.4 g of AET, pH 9 adjusted with NaOH, is prepared for 10 ml of distilled water.
  • the solution is filtered on a 0.22 ⁇ m filter in a 10 ml tube.
  • the cell suspension of the vials is recovered.
  • An equal volume of cell suspension and 4% solution of sheep red blood cells-AET are placed in a 50 ml tube.
  • Half a volume of fetal calf serum is added.
  • the whole is mixed gently and centrifuged 5 min at 800 rpmi / min.
  • the mixture with the supernatant is incubated for 1 hour in ice.
  • the supernatant is removed and the pellet is resuspended and then placed in a 15 ml centrifuge tube containing the lymphocyte separation medium, 1/3 of medium for 2/3 of pellet. After centrifugation at 3000 rpm without brake for 20 min, the ring of lymphocytes is recovered at the interface using a pipette.
  • the pipette is emptied into a 15 ml centrifuge tube and complete RPMI culture medium is added to 15 ml. After centrifugation at 1300 rpm with brake for 6 min, the supernatant is removed. The cell pellet is resuspended in 10 ml of complete RPMI culture medium. After centrifugation at 1300 rpm with brake for 6 min, the supernatant is removed. The cell pellet is resuspended in complete RPMI medium and the cell suspension is placed in culture flasks at a rate of 10 ml per 50 ml flask.
  • the cell suspension is put in a 15 ml centrifuge tube and the IR 983 F / TEC cells in another tube.
  • the tubes are centrifuged for 6 min at 1200 rpm.
  • the supernatants are removed and the cells are resuspended in 10 ml of complete MEM REGA 3 medium, MEM REGA 3 medium containing 1% of HEPES and 0.1% of gentamicin, at 37 ° C.
  • a count of the two cell types is carried out.
  • the tubes are centrifuged for 6 min at 1200 rpm.
  • the supernatants are removed and the cells are resuspended in 10 ml of complete MEM REGA 3 medium at 37 ° C.
  • the cells are mixed in a 50 ml centrifuge tube in a ratio of 1 IR 983 F / TEC for a plasma cell. After centrifugation at 1200 rpm for 6 min, the supernatants are removed and the pellet is resuspended in the last drop.
  • a PEG solution is obtained by heating 5 g of PEG 4000 in 7 ml of PBS and 1 ml of DMSO to 50 ° C. and then filtering the solution on a 0.2 ⁇ M filter. A ml of PEG solution is added to the pellet over 1 min 30 sec by shaking the tube.
  • MEM REGA 3 Leave to stand for 30 sec. Are added 1 ml of MEM REGA 3 at 37 ° C in 1 min 30 sec by shaking the tube then 20 ml of MEM REGA 3 at 37 ° C in 4 min by shaking the tube. After one centrifugation at 1200 rpm for 6 min, the supernatants are removed and the pellet is resuspended in complete DMEM medium with 1% aminopterin and 2% hypoxantine-thymidine so as to have 1.10 6 cells per ml.
  • the complete DMEM medium contains 1% L-glutamine, 1% NEAA, 1% sodium pyruvate, 0.1% gentamicin, 50 ⁇ M 2-mercaptoethanol, 5% fetal calf serum and 5% serum of horse.
  • the cell suspension is distributed on 96-well culture plates containing 100 ⁇ l of peritoneal cells, the suspension is distributed at the rate of 100 ⁇ l per well.
  • the plates are incubated for 7 days in an oven at 37 ° C and 5% C0 2 .
  • the culture medium of the plates is removed and are then placed, per well, 200 ⁇ l of complete DMEM medium containing 2% of hypoxanthine-thymidine and 2.5 ng / ml of IL6.
  • the culture medium of the plates is removed and are then placed, per well, 200 ⁇ l of complete DMEM medium containing 2.5 ng / ml of IL6.
  • the plates are incubated in an oven at 37 ° C and 5% C0 2 , the culture medium is changed in the same way until the appearance of the clones. V- Results of the various mergers carried out.
  • the hybridomas obtained are tested for the production of human immunoglobulins (Ig) by the ELISA technique using antibodies to human anti-IgG and anti-IgM rats. Table 1 summarizes the results obtained (number of clones and production of Ig).
  • the immunoglobulins are recovered from the culture supernatants of the 9 clones FAL 11 and of the clone FAL 8. They are purified by affinity chromatography on a Sepharose column coupled with anti-human Ig antibodies which are the LO-hK antibody -3 (anti-human Ig Kappa, ref: TEC 146, Technopharm-France) and the antibody LO-hL-2 (anti-human Ig lambda, ref: TEC 147, Technopharm-France). The immunoglobulin concentration is evaluated by an ELISA test in comparison with a standard (human IgM at 1 mg / ml, ref: 18260, Sigma).
  • HHA 06-5 Hemagglutinin Influenza
  • the antigens diluted to a concentration of 5 to 10 ⁇ g / ml in carbonate bicarbonate buffer at pH 9.5 are distributed at the rate of 100 ⁇ l per well in 96-well plates. These plates are incubated overnight at 4 ° C.
  • the plates are then washed 3 times with PBS containing 2% of Tween 20.
  • the plates are saturated with PBS containing 2% of Tween 20 and 5% of skimmed milk at a rate of 200 ⁇ l per well and incubated one hour at 37 ° vs.
  • the plates are then washed 3 times with PBS containing 2% Tween 20.
  • the immunoglobulin samples at different dilutions are distributed in the plates at a rate of 10 ⁇ l per well. These plates are incubated for 2 hours at 37 ° C. The plates are then washed 6 times with PBS containing 2% Tween 20.
  • the antibodies coupled to the peroxidase are diluted in PBS containing 2% of Tween 20 (0.5 ⁇ l / ml) and deposited in the plates at a rate of 100 ⁇ l per well. The plates are incubated for one hour at 37 ° C. The plates are washed 6 times with PBS containing 2% Tween 20.
  • OPD pellet O Phenylenediamine, ref: P8787, Sigma
  • development buffer citrate phosphate buffer at pH 5.5
  • oxygen peroxide oxygen peroxide
  • the solution is deposited on the plates at the rate of 100 ⁇ l per well.
  • the plates are incubated for 10 to 20 minutes in the dark.
  • the enzymatic reaction is stopped by adding 50 ⁇ l per well of 2 M H 2 S0 4 .
  • the results are read at 490 nm in the plate reader.
  • ELISA tests are therefore carried out in order to test the specificity of the immunoglobulins on the viral antigens A (H3N2), A (H1N1) and B of the strains of winter 1998/99 (Table 2).
  • the tests were positive for 2 FAL 11 clones (3D2 and 2C3) and for the FAL 8 clone (1G4).
  • Table 2 Result of the ELISA test for specificity on the different clones.
  • mice 5-week-old female BALB / c mice are grouped in batches of 6. They are housed in an autonomous enclosure in cages covered with a filtering cap.
  • Antibodies Human total immunoglobulins (Ivlg) stored at -80 ° C and then diluted to the desired concentration immediately before use (500 ⁇ g / 50 ⁇ l) are used as control.
  • the purified antibodies AB1, AB2, AB9 and AB10 stored at 4 ° C are adjusted to an equivalent concentration for each antibody (1 ⁇ g / 50 ⁇ l) • ii) Protocol.
  • mice are lightly anesthetized according to a protocol described in the specialized books and receive 50 ⁇ l of suspension of antibody (AB1, AB2, AB9, AB10 or total human Ig: Ivlg) or physiological serum intra-nasally (controls ). The next day, after anesthesia, they receive 50 ⁇ l of the lethal viral suspension in 10 days.
  • mice are monitored daily.
  • Protection effectiveness is measured in survival / lethality.
  • mice receiving antibodies of the Ig M lambda type from batch AB2 are all alive on D15;
  • mice receiving antibodies of the Ig M lambda type from batch AB9 all died at Jll; - that 5 of the 6 mice receiving antibodies of the Ig M Kappa type from batch AB1 died on D15;
  • mice receiving the total immunoglobulins at a dose of 500 ⁇ g are all alive on D15.
  • the results show that the lambda IgM monoclonal antibodies secreted by the clone FAL 11 2C3 protect the mice from the fatal infection by the A / Scotland / 74 (H3N2) virus at a dose of 1 ⁇ g in 50 ⁇ l of PBS compared to total human immunoglobulins at a dose of 500 ⁇ g in 50 ⁇ l of PBS which also protect mice from fatal respiratory infection by the same influenza virus
  • Table 3 Test for protection of a viral infection in mice using human antibodies

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Abstract

The invention concerns a method for preparing human monoclonal antibodies or fragments thereof or antibodies comprising such a fragment, from B lymphocytes. The invention is characterised in that it comprises the following steps: a) isolating B lymphocytes from a sample derived from a human subject; b) differentiating said B lymphocytes in plasma cells with at least a non-specific activating system and at least a cytokine; c) immortalising said plasma cells by cell fusion with a fusion line; d) culturing in vitro the fused cells until hybridomas are obtained; e) purifying the monoclonal antibodies produced by the hybridomas; f) optionally cleaving said antibodies into fragments and purifying one or several said fragments; g) optionally combining one or several fragments obtained at step f) with fragments of other antibodies.

Description

PROCEDE DE PREPARATION D'UN ANTICORPS MONOCLONAL HUMAIN, DE FRAGMENTS DE CELUI-CI OU D'ANTICORPS COMPRENANT DE TELS FRAGMENTS, LES ANTICORPS AINSI OBTENUS ET LEUR UTILISATION. PROCESS FOR THE PREPARATION OF A HUMAN MONOCLONAL ANTIBODY, FRAGMENTS THEREOF OR ANTIBODIES COMPRISING SUCH FRAGMENTS, THE ANTIBODIES THUS OBTAINED AND THEIR USE
La présente invention concerne un procédé de préparation d'un anticorps monoclonal humain, de fragments de celui-ci ou d'anticorps comprenant de tels fragments. L'invention a également pour objet les hybridomes et les anticorps ainsi obtenus et leurs utilisations dans des compositions de diagnostic et thérapeutiques .The present invention relates to a method for preparing a human monoclonal antibody, fragments thereof or antibodies comprising such fragments. The subject of the invention is also the hybridomas and the antibodies thus obtained and their uses in diagnostic and therapeutic compositions.
Les anticorps monoclonaux dirigés contre un déterminant antigénique sont produits par un grand nombre de laboratoires depuis l'introduction de la technique de fusion cellulaire entre une cellule myélomateuse et une cellule lymphoïde d'un animal immunisé. Le premier modèle d' ybridomes sécrétant des anticorps monoclonaux développé par Kδhler et Milstein (1975, Nature, vol. 256, page 495) fut un modèle murin.Monoclonal antibodies directed against an antigenic determinant have been produced by a large number of laboratories since the introduction of the technique of cell fusion between a myeloma cell and a lymphoid cell of an immunized animal. The first model of ybridomas secreting monoclonal antibodies developed by Kδhler and Milstein (1975, Nature, vol. 256, page 495) was a murine model.
Les anticorps monoclonaux produits par des hybridomes présentent des intérêts multiples qui ont été décrits de manière exhaustive dans plusieurs ouvragesMonoclonal antibodies produced by hybridomas have multiple interests which have been fully described in several books
(Bazin, « Rat hybridomas and rat monoclonal antibodies », CRC Press, 1990, 515 pages ; Goding, « Monoclonal antibodies : principles and practise », 3leme édition, Académie Press, 1996, 492 pages ; Shepherd et Dean, « Monoclonal antibodies », Oxford university press, 2000, 479 pages) . Ces anticorps sont utiles à des emplois aussi bien de diagnostic que de thérapeutiques préventives et/ou curatives .(Bazin, "Rat hybridomas and rat monoclonal antibodies", CRC Press, 1990, 515 pages; Goding, "Monoclonal antibodies: principles and practice", 3 Leme edition, Academic Press, 1996, 492 pages; Shepherd and Dean, "Monoclonal antibodies ", Oxford university press, 2000, 479 pages). These antibodies are useful for diagnostic as well as preventive and / or curative therapeutic uses.
La présente invention concerne un procédé de préparation d'anticorps monoclonaux présentant l'avantage de ne pas nécessiter l' immortalisation préalable des lymphocytes B comme par exemple par le virus E. B. V. tel que proposé dans l'art antérieur. Le procédé de l'invention est remarquable en ce qu'il est basé sur l'utilisation successive et combinée de diverses cytokines à des temps définis et d'un système activateur non spécifique.The present invention relates to a method for preparing monoclonal antibodies having the advantage not to require the prior immortalization of the B lymphocytes such as for example by the EBV virus as proposed in the prior art. The process of the invention is remarkable in that it is based on the successive and combined use of various cytokines at defined times and of a non-specific activating system.
Le procédé de l'invention est également remarquable en ce que l' immortalisation des lymphocytes humains différenciés est effectuée au moyen d'une lignée myélomateuse de rat.The method of the invention is also remarkable in that the immortalization of differentiated human lymphocytes is carried out by means of a rat myeloma line.
Il est en effet connu que la manipulation et le maintien en culture de lignées de fusion de rat sont aléatoires, et de ce fait, des nombreuses équipes ont toujours refusé l'utilisation de telles lignées de fusion, préférant les lignées de fusion murines. (Goding, « Monoclonal antibodies : principles and practice », 3ιeme édition, Académie Press, 1996, pages 62 et pages 147-148) .It is indeed known that the manipulation and maintenance in culture of rat fusion lines are random, and therefore many teams have always refused the use of such fusion lines, preferring murine fusion lines. (Goding, "Monoclonal antibodies: principles and practice" 3 ιeme edition, Academic Press, 1996, pages 62 and 147-148 pages).
De manière surprenante, le procédé d'obtention d'anticorps monoclonaux humains mis en œuvre par la demanderesse, comprenant une fusion de lymphocytes humains différenciés avec des cellules d'une lignée de fusion de rat, conduit à l'obtention d' hybridomes particulièrement stables et adaptés à la production d'anticorps monoclonaux humains .Surprisingly, the process for obtaining human monoclonal antibodies implemented by the applicant, comprising a fusion of differentiated human lymphocytes with cells of a rat fusion line, leads to the production of particularly stable hybridomas and suitable for the production of human monoclonal antibodies.
Plus particulièrement, le procédé de préparation d'anticorps monoclonaux humains ou de fragments de ceux-ci ou d'anticorps comprenant un tel fragment, à partir de lymphocytes B, comprend les étapes suivantes : a) l'isolement de lymphocytes B à partir d'un prélèvement provenant d'un individu de l'espèce humaine, b) la différenciation desdits lymphocytes B en cellules plasmocytaires avec au moins un système activateur non spécifique et au moins une cytokine, c) l' immortalisation desdites cellules plasmocytaires par fusion cellulaire avec une lignée de fusion de rat, d) la culture in vi tro des cellules fusionnées jusqu'à l'obtention d'hybrido es, et avantageusement le criblage des clones spécifiques, e) la purification des anticorps monoclonaux produits par les hybridomes, f) éventuellement le clivage desdits anticorps en fragments, et la purification d'un ou plusieurs desdits fragments, g) éventuellement l'association d'un ou plusieurs des fragments obtenu (s) à l'étape (f) avec des fragments d'autres anticorps.More particularly, the process for preparing human monoclonal antibodies or fragments thereof or of antibodies comprising such a fragment, from B lymphocytes, comprises the following steps: a) isolating B lymphocytes from a sample originating from an individual of the human species, b) differentiating said B lymphocytes into plasma cells with at least one non-specific activating system and at least one cytokine, c ) the immortalization of said plasma cells by cell fusion with a rat fusion line, d) the in vitro culture of the fused cells until hybridization is obtained, and advantageously the screening of specific clones, e) the purification of the monoclonal antibodies produced by the hybridomas, f) optionally the cleavage of said antibodies into fragments, and the purification of one or more of said fragments, g) optionally the association of one or more of the fragments obtained with step (f) with fragments of other antibodies.
Ainsi, l'invention se rapporte tant aux anticorps obtenus à l'étape (e) qu'à des fragments de ceux- ci obtenus à l'étape (f) libres ou associés à d'autres molécules ou substances, de n'importe quel isotype, humains ou cherchant à s'en approcher. Comme par exemple, des anticorps monoclonaux de mammifères proches de l'hommeThus, the invention relates both to the antibodies obtained in step (e) and to fragments of these obtained in step (f) free or associated with other molecules or substances, of any what isotype, humans or seeking to approach it. For example, monoclonal antibodies from mammals close to humans
(singes supérieurs) , soit chimérisés avec des parties constantes d' immunoglobulines humaines, soit humanisés, modifiés artificiellement ou non dans le but d'obtenir des propriétés similaires à ceux de l'espèce humaine dans leur forme membranaire, circulante ou sécrétée.(superior monkeys), either chimerized with constant parts of human immunoglobulins, or humanized, modified artificially or not in order to obtain properties similar to those of the human species in their membrane, circulating or secreted form.
Le procédé de l'invention peut éventuellement comporter une étape supplémentaire (h) caractérisée par le transfert du matériel génétique des hybridomes codant pour tout ou partie des anticorps dans tout autre système d'expression décrit dans la littérature spécialisée.The process of the invention may optionally include an additional step (h) characterized by the transfer of the genetic material of the hybridomas encoding all or part of the antibodies into any other expression system described in the specialized literature.
A l'étape (a) du procédé selon l'invention, les lymphocytes B sont isolés à partir d'un prélèvement d'un individu de l'espèce humaine ou de toute autre origine ayant les mêmes caractéristiques. Avantageusement, les lymphocytes B sont isolés à l'étape (a) du procédé selon l'invention à partir de sang humain. On entend par « cellules plasmocytaires » à l'étape (b) des lymphocytes B modifiés produisant des immunoglobulines et aptes à être fusionnés avec une autre cellule pour donner un hybridome sécrétant des immunoglobulines. Ce type de lymphocytes B modifiés peut être parfois appelé « lymphocytes B activés » . Avantageusement, l'étape (b) de différenciation des lymphocytes B comprend les étapes suivantes : i) le mélange d'une suspension de lymphocytes B avec de 0,01 à 1 % d'un système activateur non spécifique et de 1 à 8,5 ng/ml d'IL2, puis l'incubation du mélange pendant 24 heures à 11 jours à une température comprise entre 36,5 et 37,8°C, ii) la centrifugation de la suspension obtenue en (i) de 600 à 3500 rpm pendant 2 à 12 minutes, puis le retrait du surnageant et la remise en suspension du culot cellulaire, iii) le mélange de la suspension obtenue en (ii) avec de 1 à 8,5 ng/ml d' IL2 et de 10 à 350 ng/ml d'ILlO, puis l'incubation du mélange pendant 48 heures à 15 jours à une température comprise entre 36,5 et 37,8°C. Ainsi, à titre d'exemple préféré, l'étape (b) de différenciation des lymphocytes B comprend les étapes suivantes : i) le mélange d'une suspension de lymphocytes B avec 0,04 % d'un système activateur non spécifique et 2,5 ng/ml d'IL2, puis l'incubation du mélange pendant 3 jours à une température de 37°C sous 5% de C02, ii) la centrifugation de la suspension obtenue en (i) à 1300 rpm pendant β minutes, puis le retrait du surnageant et la remise en suspension du culot cellulaire, iii) le mélange de la suspension obtenu en (ii) avec 2,5 ng/ml d'IL2 et 100 ng/ml d'ILlO, puis l'incubation du mélange pendant 5 jours à une température de 37 °C sous 5% de C02. Le système activateur non spécifique est choisi parmi un lipopolysaccharide ou toute substance susceptible d'avoir une action identique en tout ou partie, la pansorbine, la phytohémagglutinine, le muramyldipeptide utilisés seuls ou en association.In step (a) of the method according to the invention, the B lymphocytes are isolated from a sample from an individual of the human species or from any other origin having the same characteristics. Advantageously, the B lymphocytes are isolated in step (a) of the method according to the invention from human blood. The term “plasma cells” in step (b) means modified B lymphocytes producing immunoglobulins and capable of being fused with another cell to give a hybridoma secreting immunoglobulins. This type of modified B cell can sometimes be called "activated B cell". Advantageously, step (b) of differentiating B lymphocytes comprises the following steps: i) mixing a suspension of B lymphocytes with 0.01 to 1% of a non-specific activating system and from 1 to 8, 5 ng / ml of IL2, then incubation of the mixture for 24 hours to 11 days at a temperature between 36.5 and 37.8 ° C, ii) centrifugation of the suspension obtained in (i) from 600 to 3500 rpm for 2 to 12 minutes, then removal of the supernatant and resuspension of the cell pellet, iii) mixing the suspension obtained in (ii) with from 1 to 8.5 ng / ml of IL2 and 10 at 350 ng / ml of IL10, then incubation of the mixture for 48 hours to 15 days at a temperature between 36.5 and 37.8 ° C. Thus, as a preferred example, step (b) of differentiating B lymphocytes comprises the following steps: i) mixing a suspension of B lymphocytes with 0.04% of a non-specific activating system and 2 , 5 ng / ml of IL2, then incubation of the mixture for 3 days at a temperature of 37 ° C under 5% of C0 2 , ii) centrifugation of the suspension obtained in (i) at 1300 rpm for β minutes , then removing the supernatant and resuspending the cell pellet, iii) mixing the suspension obtained in (ii) with 2.5 ng / ml of IL2 and 100 ng / ml of IL10, then incubation of the mixture for 5 days at a temperature of 37 ° C. under 5% of CO 2 . The non-specific activating system is chosen from a lipopolysaccharide or any substance capable of having an identical action in whole or in part, pansorbine, phytohemagglutinin, muramyldipeptide used alone or in combination.
La lignée de fusion utilisée pour 1' immortalisation des cellules plasmocytaires à l'étape (c) est une lignée myélomateuse de rat et de préférence une lignée myélomateuse de rat de souche LOU.The fusion line used for the immortalization of the plasma cells in step (c) is a myeloma rat line and preferably a myeloma rat line of the LOU strain.
Selon une forme de réalisation toute particulière du procédé de l'invention, la lignée de fusion utilisée pour l' immortalisation des cellules plasmocytaires à l'étape (c) est la lignée myélomateuse de rat LOU IR 983 F/ TEC déposée à la Collection Nationale de Cultures de Micro-organismes à l'Institut Pasteur (Paris) sous le No. 1-2584, le 29 novembre 2000, ce dépôt ayant été réalisé dans le cadre du Traité de Budapest. Selon une première forme de mise en œuvre préférée du procédé de l'invention, l'étape (b) de différenciation des lymphocytes B en cellules plasmocytaires est effectuée en présence d'un antigêne spécifique d'un agent infectieux.According to a very particular embodiment of the process of the invention, the fusion line used for the immortalization of the plasma cells in step (c) is the myeloma rat line LOU IR 983 F / TEC deposited in the National Collection of Cultures of Microorganisms at the Institut Pasteur (Paris) under No. 1-2584, November 29, 2000, this deposit having been made within the framework of the Budapest Treaty. According to a first preferred form of implementation of the method of the invention, step (b) of differentiating B lymphocytes into plasma cells is carried out in the presence of a specific antigen of an infectious agent.
Selon une seconde forme de mise en œuvre préférée du procédé de l'invention, à l'étape (a) les lymphocytes B sont isolés de sang d'un organisme vacciné contre un agent infectieux ou en convalescence d'une infection par ledit agent infectieux. Avantageusement, à l'étape (a), les lymphocytes B sont isolés de sang d'un sujet vacciné contre la grippe.According to a second preferred embodiment of the method of the invention, in step (a) the B lymphocytes are isolated from blood from an organism vaccinated against an infectious agent or recovering from an infection by said infectious agent . Advantageously, in step (a), the B lymphocytes are isolated from the blood of a subject vaccinated against influenza.
Le procédé de l'invention permet de produire des anticorps monoclonaux ou des fragments de ceux-ci ou des anticorps comprenant un tel fragment à partir de lymphocytes B de mammifères ou de toute espèce animale susceptible de montrer une réponse immune humorale spécifique, mais de préférence ceux-ci sont isolés à partir de sang humain ou de mammifères proches de l'homme. L'invention concerne bien entendu tout particulièrement la préparation d'anticorps monoclonaux humains à partir de lymphocytes B humains .The process of the invention makes it possible to produce monoclonal antibodies or fragments thereof or antibodies comprising such a fragment from B lymphocytes from mammals or from any animal species capable of showing a specific humoral immune response, but preferably these are isolated from human blood or mammals close to humans. The invention obviously relates very particularly to the preparation of human monoclonal antibodies from human B lymphocytes.
Ainsi, le procédé de l'invention permet la préparation d'anticorps monoclonaux humains ou de fragments de ceux-ci ou d'anticorps comprenant un tel fragment dirigés contre tout type d'agent infectieux, comme par exemple de manière non exhaustive le virus grippal, les agents classiquement objectivés dans la guerre biologique comme le charbon, la variole, la tularémie, le virus Ebola etc.. et tout agent pathogène de l'homme, des animaux et en fait du règne animal et végétal .Thus, the method of the invention allows the preparation of human monoclonal antibodies or of fragments thereof or of antibodies comprising such a fragment directed against any type of infectious agent, such as for example in a non-exhaustive manner the influenza virus. , agents classically objectified in biological warfare such as anthrax, smallpox, tularemia, the Ebola virus etc. and any pathogenic agent of man, animals and in fact of the animal and vegetable kingdom.
L'invention concerne aussi un hybridome susceptible d'être obtenu après les étapes (a) à (d) d'un procédé décrit précédemment, ainsi que les anticorps monoclonaux susceptibles d'être produits par ces hybridomes, ou un fragment de celui-ci, et plus particulièrement les fragments Fab incluant les parties du site anticorps qui peut se fixer à l'antigène. Ces anticorps et leurs fragments peuvent être identifiés par un test ELISA. La spécificité des anticorps monoclonaux vis-à-vis de l'agent infectieux peut être évaluée :The invention also relates to a hybridoma capable of being obtained after steps (a) to (d) of a method described above, as well as the monoclonal antibodies capable of being produced by these hybridomas, or a fragment thereof. , and more particularly the Fab fragments including the parts of the antibody site which can bind to the antigen. These antibodies and their fragments can be identified by an ELISA test. The specificity of the monoclonal antibodies vis-à-vis the infectious agent can be evaluated:
- par un test ELISA de spécificité vis-à-vis de l'agent infectieux ;- by an ELISA test of specificity vis-à-vis the infectious agent;
- par un test d'inhibition d'hémagglutination avec les antigènes de l'agent infectieux ;- by a hemagglutination inhibition test with the antigens of the infectious agent;
- ou par un test in vivo sur un modèle animal de protection vis-à-vis de l'agent infectieux. Ces anticorps monoclonaux appartiennent à la classe des IgG, IgM, IgE, IgD ou IgA, mais ils peuvent être aussi modifiés, quelle que soit leur classe, en anticorps d'une autre classe d' immunoglobulines par exemple par des techniques décrites de biologie moléculaire (Lewis et al . , 1992, Hum. Antibodies Hybridomas , vol. 3, pages 146-52 Lewis et al . , 1993, J. Immunol . , vol. 151, pages 2829-38 Hall et al . , 1994, Cancer Res . , vol. 54, pages 5178-85 Shepherd et Dean, « Monoclonal antibodies », Oxford university press, 2000, 479 pages) .- or by an in vivo test on an animal model of protection against the infectious agent. These monoclonal antibodies belong to the class of IgG, IgM, IgE, IgD or IgA, but they can also be modified, whatever their class, into antibodies of another class of immunoglobulins for example by techniques described in molecular biology (Lewis et al., 1992, Hum. Antibodies Hybridomas, vol. 3, pages 146-52 Lewis et al., 1993, J. Immunol., Vol. 151, pages 2829-38 Hall et al., 1994, Cancer Res ., vol. 54, pages 5178-85 Shepherd and Dean, "Monoclonal antibodies", Oxford university press, 2000, 479 pages).
Ainsi, l'invention se rapporte à un anticorps monoclonal susceptible d'être obtenu par les étapes (a) à (e) du procédé précédemment, ou un fragment de celui-ci obtenu par les étapes (a) à (f) dudit procédé.Thus, the invention relates to a monoclonal antibody capable of being obtained by steps (a) to (e) of the method previously, or a fragment thereof obtained by steps (a) to (f) of said method.
Un tel anticorps monoclonal ou fragment de celui-ci peut appartenir à la classe des IgG, IgM, IgE, IgD ou IgA.Such a monoclonal antibody or fragment thereof can belong to the class of IgG, IgM, IgE, IgD or IgA.
L'invention concerne aussi un anticorps ou fragment d'anticorps comprenant au moins un fragment d'anticorps ci-dessus. De tels anticorps peuvent être préparés par des techniques connues de l'homme du métier pour associer des fragments d'anticorps différents.The invention also relates to an antibody or antibody fragment comprising at least one of the above antibody fragments. Such antibodies can be prepared by techniques known to those skilled in the art to combine different antibody fragments.
L'invention a également pour objet un acide nucléique comprenant une séquence de nucléotides codant pour un anticorps obtenu selon le procédé de l'invention ou pour un fragment de celui-ci.The subject of the invention is also a nucleic acid comprising a nucleotide sequence coding for an antibody obtained according to the method of the invention or for a fragment thereof.
L'invention se rapporte encore à des compositions pharmaceutiques comprenant à titre d'agent actif une quantité efficace d'au moins un anticorps ou fragment d'anticorps comprenant au moins un fragment d'anticorps obtenu selon le procédé de l'invention. Ces compositions sont utiles pour le diagnostic et pour le traitement curatif ou préventif d'infections par les agents infectieux contre lesquels les anticorps et fragments d'anticorps ont été préparés selon le procédé décrit précédemment. Ainsi, l'invention a pour objet un kit de détection d'une infection par un agent infectieux, comprenant au moins un anticorps ou au moins un fragment d'anticorps contre ledit agent infectieux obtenus comme décrit précédemment.The invention also relates to pharmaceutical compositions comprising, as active agent, an effective amount of at least one antibody or antibody fragment comprising at least one antibody fragment obtained according to the method of the invention. These compositions are useful for the diagnosis and for the curative or preventive treatment of infections by the infectious agents against which the antibodies and antibody fragments have been prepared according to the method described above. Thus, the subject of the invention is a kit for detecting an infection with an infectious agent, comprising at least one antibody or at least one antibody fragment against said infectious agent obtained as described above.
D'autres avantages et caractéristiques de l'invention apparaîtront de la description qui suit rapportant des exemples de préparation d'anticorps monoclonaux et leur propriété .Other advantages and characteristics of the invention will emerge from the description which follows reporting examples of preparation of monoclonal antibodies and their properties.
I- Isolement des lymphocytes B humains. 1) Matériel.I- Isolation of human B lymphocytes. 1) Material.
Tubes EDTA (CML, réf : T200QS)EDTA tubes (CML, ref: T200QS)
Seringues 20 ml (Fisher Sci., réf : A14669625)20 ml syringes (Fisher Sci., Ref: A14669625)
Filtres Minisart 0,22 μM (CML, réf : 17597K)0.22 μM Minisart filters (CML, ref: 17597K)
Plaques de culture 96 puits (Life Technologies, réf : 167008A)96-well culture plates (Life Technologies, ref: 167008A)
Flacons de culture à bouchons ventilés (Fisher Sci., réf :Culture flasks with ventilated caps (Fisher Sci., Ref:
A12078050)A12078050)
Pipettes stériles 10 ml (CML, réf : P1051 B)10 ml sterile pipettes (CML, ref: P1051 B)
Tubes à centrifugation 50 ml (Fisher Sci., réf : A12832506) Tubes centrifugeurs 15 ml (Fisher Sci., réf : A12832163)Centrifuge tubes 50 ml (Fisher Sci., Ref: A12832506) Centrifuge tubes 15 ml (Fisher Sci., Ref: A12832163)
Tubes 12 ml stériles ml (Fisher Sci., réf : A12926183)12 ml sterile ml tubes (Fisher Sci., Ref: A12926183)
Cellule à numération (CML, réf :KOVAS)Counting cell (CML, ref: KOVAS)
Bleu de trypan (Sigma, réf : T-8154)Trypan blue (Sigma, ref: T-8154)
Milieu de culture DMEM (Biowhittaker, réf : M12914F) Milieu de culture RPMI 1640 (Biowhittaker, réf : M12115F)DMEM culture medium (Biowhittaker, ref: M12914F) RPMI 1640 culture medium (Biowhittaker, ref: M12115F)
Sérum de veau fœtal (Labconsul, réf : P30-0702)Fetal calf serum (Labconsul, ref: P30-0702)
Sérum de cheval (Labconsul, réf : 17-905C)Horse serum (Labconsul, ref: 17-905C)
L-glutamine 200 mM (Biowhittaker, réf : 17-905C)L-glutamine 200 mM (Biowhittaker, ref: 17-905C)
Gentamicine 50 mg/ml (Life Technologies, réf : 15750-045) NEAA 100X (Biowhittaker, réf : BE13-114E)Gentamicin 50 mg / ml (Life Technologies, ref: 15750-045) NEAA 100X (Biowhittaker, ref: BE13-114E)
Pyruvate de sodium 100 mM (Biowhittaker, réf : 13-115E)100 mM sodium pyruvate (Biowhittaker, ref: 13-115E)
2-mercaptoéthanol (Sigma, réf. : M7522)2-mercaptoethanol (Sigma, ref .: M7522)
Milieu de séparation des lymphocytes (Biowhittaker, réf. :Lymphocyte separation medium (Biowhittaker, ref.:
MI 7829E) Sang de mouton défibriné (Eurobio, réf. : 905705)MI 7829E) Defibrinated sheep blood (Eurobio, ref .: 905705)
HBSS (Life Technology, réf. : 14025-050)HBSS (Life Technology, ref .: 14025-050)
PBS (Biowhittaker, réf. : MI 7512F) AET : 2-Aminoethylisothiouroniumbromide (Sigma, réf. :PBS (Biowhittaker, ref .: MI 7512F) AET: 2-Aminoethylisothiouroniumbromide (Sigma, ref.:
A5879)A5879)
Eau distilléeDistilled water
NaOH (Fisher Sci., réf : A43211977) IL2 : Interleukine 2 humaine recombinante (R et D, réf. :NaOH (Fisher Sci., Ref: A43211977) IL2: Recombinant human interleukin 2 (R and D, ref:
202 IL050)202 IL050)
IL6 : Interleukine 6 humaine recombinante (R et D, réf. :IL6: Recombinant human interleukin 6 (R and D, ref .:
206 IL050)206 IL050)
IL10 : Interleukine 10 humaine recombinante (R et D, réf. : 217 IL025)IL10: Recombinant human interleukin 10 (R & D, ref .: 217 IL025)
Pansorbine (France Biochem SA, réf. : 507851)Pansorbine (France Biochem SA, ref .: 507851)
Milieu de fusion MEM REGA 3 (Life Technology, réf. : 19993-MEM REGA 3 fusion medium (Life Technology, ref .: 19993-
013)013)
HEPES 1M (Life Technology, réf. : 15630-056) PEG : Polyéthylène glycol 4000 (Fisher Sci., réf :HEPES 1M (Life Technology, ref .: 15630-056) PEG: Polyethylene glycol 4000 (Fisher Sci., Ref:
A48691512)A48691512)
DMSO : Diméthylsuifoxyde (Fisher Sci., réf : A43320281)DMSO: Dimethylsuifoxide (Fisher Sci., Ref: A43320281)
Aminoptérine 100X (Life Technology, réf. : 11362-019)Aminopterin 100X (Life Technology, ref .: 11362-019)
Supplément d' hypoxantine thymidine 50X (Life Technology, réf. : 41065-012)Thymidine 50X Hypoxantine Supplement (Life Technology, Ref: 41065-012)
2) Méthode. a) Première étape.2) Method. a) First step.
Le sang a été prélevé sur des volontaires vaccinés par un vaccin antigrippal ou en convalescence d'une infection grippale. Les prélèvements sont réalisés à 0, 8, 15 ou 21 jours après la vaccination.The blood was taken from volunteers vaccinated with an influenza vaccine or recovering from an influenza infection. Samples are taken 0, 8, 15 or 21 days after vaccination.
Le sang humain provenant de tubes EDTA est dilué dans le même volume de PBS . Le sang dilué est déposé dans un tube à centrifuger de 50 ml contenant le milieu de séparation des lymphocytes (1/3 de milieu pour 2/3 de sang) . Après une centrifugation à 3000 rpm sans frein pendant 20 min, l'anneau de lymphocytes est récupéré à l'interface à l'aide d'une pipette de 10 ml. La pipette est vidée dans un tube à centrifuger de 15 ml et du milieu de culture complet RPMI, milieu de culture RPMI contenant 1% de L-glutamine, 0,1% de gentamicine, 50μM de 2- mercaptoéthanol et 10% de sérum de veau fœtal, est ajouté qsp 15 ml. Après une centrifugation à 1300 rpm avec frein pendant 6 min, le surnageant est enlevé. Le culot cellulaire est remis en suspension dans 10 ml de milieu de culture complet RPMI. Après une centrifugation à 1300 rpm avec frein pendant 6 min, le surnageant est enlevé. Le culot cellulaire est remis en suspension dans du milieu complet RPMI de façon à avoir de 1 à 2.106 cellules par ml. La suspension cellulaire est mise dans des flacons de culture à raison de 10 ml par flacon de 50 ml. Les monocytes sont enlevés par adhérence en incubant les flacons à l'horizontale dans une étuve à 37°C et 5% de C02 pendant une nuit .Human blood from EDTA tubes is diluted in the same volume of PBS. The diluted blood is placed in a 50 ml centrifuge tube containing the lymphocyte separation medium (1/3 of medium for 2/3 of blood). After centrifugation at 3000 rpm without brake for 20 min, the ring of lymphocytes is recovered at the interface using a 10 ml pipette. The pipette is emptied into a 15 ml centrifuge tube and complete RPMI culture medium, RPMI culture medium containing 1% L-glutamine, 0.1% gentamicin, 50 μM of 2-mercaptoethanol and 10% serum. fetal calf, is added qs 15 ml. After centrifugation at 1300 rpm with brake for 6 min, the supernatant is removed. The cell pellet is resuspended in 10 ml of complete RPMI culture medium. After centrifugation at 1300 rpm with brake for 6 min, the supernatant is removed. The cell pellet is resuspended in complete RPMI medium so as to have from 1 to 2.10 6 cells per ml. The cell suspension is placed in culture flasks at a rate of 10 ml per 50 ml flask. The monocytes are removed by adhesion by incubating the bottles horizontally in an oven at 37 ° C. and 5% of CO 2 overnight.
Une solution à 0,4 g de AET, pH 9 ajusté avec NaOH, est préparée pour 10 ml d'eau distillée. La solution est filtrée sur filtre 0,22 μm dans un tube de 10 ml.A solution of 0.4 g of AET, pH 9 adjusted with NaOH, is prepared for 10 ml of distilled water. The solution is filtered on a 0.22 μm filter in a 10 ml tube.
Douze ml de sang de mouton défibriné sont mis dans un tube à centrifuger de 15 ml . Après une centrifugation à 2100 rpm pendant 10 min, le surnageant est enlevé et le culot de globules rouges est remis en suspension dans 10 ml de HBSS . Après une centrifugation à 2100 rpm pendant 10 min, le surnageant est enlevé. Deux ml de globules rouges et 8 ml de solution d'AET sont mélangés dans un tube à centrifuger de 15 ml. Après une incubation d'une heure à 37°C, 5 ml de PBS sont ajoutés. Le surnageant est enlevé après une centrifugation de 10 min à 1300 rpm. Deux ml du culot constitué de globules rouges et d'AET sont mis dans un tube à centrifuger de 50 ml contenant 48 ml de milieu complet RPMI. Une incubation est réalisée toute une nuit à 4°C. b) Deuxième étape .Twelve ml of defibrinated sheep blood is placed in a 15 ml centrifuge tube. After centrifugation at 2100 rpm for 10 min, the supernatant is removed and the pellet of red blood cells is resuspended in 10 ml of HBSS. After centrifugation at 2100 rpm for 10 min, the supernatant is removed. Two ml of red blood cells and 8 ml of AET solution are mixed in a 15 ml centrifuge tube. After a one hour incubation at 37 ° C, 5 ml of PBS is added. The supernatant is removed after a 10 min centrifugation at 1300 rpm. Two ml of the pellet consisting of red blood cells and AET are placed in a 50 ml centrifuge tube containing 48 ml of complete RPMI medium. An incubation is carried out overnight at 4 ° C. b) Second step.
La suspension cellulaire des flacons est récupérée. Un volume égal de suspension cellulaire et de solution à 4% de globules rouges de mouton-AET sont mis dans un tube de 50 ml. Un demi volume de sérum de veau fœtal est ajouté. Le tout est mélangé doucement et centrifugé 5 min à 800 rpmi/min. Le mélange avec le surnageant est incubé 1 heure dans la glace. Le surnageant est enlevé et le culot est remis en suspension puis déposé dans un tube à centrifuger de 15 ml contenant le milieu de séparation des lymphocytes, 1/3 de milieu pour 2/3 de culot. Après une centrifugation à 3000 rpm sans frein pendant 20 min, l'anneau de lymphocytes est récupéré à l'interface à l'aide d'une pipette. La pipette est vidée dans un tube à centrifuger de 15 ml et du milieu de culture complet RPMI est ajouté qsp 15 ml. Après une centrifugation à 1300 rpm avec frein pendant 6 min, le surnageant est enlevé. Le culot cellulaire est remis en suspension dans 10 ml de milieu de culture complet RPMI. Après une centrifugation à 1300 rpm avec frein pendant 6 min, le surnageant est enlevé. Le culot cellulaire est remis en suspension dans du milieu complet RPMI et la suspension cellulaire est mise dans des flacons de culture à raison de 10 ml par flacon de 50 ml.The cell suspension of the vials is recovered. An equal volume of cell suspension and 4% solution of sheep red blood cells-AET are placed in a 50 ml tube. Half a volume of fetal calf serum is added. The whole is mixed gently and centrifuged 5 min at 800 rpmi / min. The mixture with the supernatant is incubated for 1 hour in ice. The supernatant is removed and the pellet is resuspended and then placed in a 15 ml centrifuge tube containing the lymphocyte separation medium, 1/3 of medium for 2/3 of pellet. After centrifugation at 3000 rpm without brake for 20 min, the ring of lymphocytes is recovered at the interface using a pipette. The pipette is emptied into a 15 ml centrifuge tube and complete RPMI culture medium is added to 15 ml. After centrifugation at 1300 rpm with brake for 6 min, the supernatant is removed. The cell pellet is resuspended in 10 ml of complete RPMI culture medium. After centrifugation at 1300 rpm with brake for 6 min, the supernatant is removed. The cell pellet is resuspended in complete RPMI medium and the cell suspension is placed in culture flasks at a rate of 10 ml per 50 ml flask.
II- Différenciation en cellules plasmocytaires.II- Differentiation into plasma cells.
Sont ajoutés à la suspension cellulaire 0,04% de pansorbine et 2,5 ng/ml d' IL2. La suspension cellulaire est incubée pendant 3 jours dans une étuve à 37°C et 5% de0.04% pansorbine and 2.5 ng / ml IL2 are added to the cell suspension. The cell suspension is incubated for 3 days in an oven at 37 ° C and 5% of
C02 avant d'être centrifugée 6 min à 1300 rpm. Le surnageant est enlevé et le culot cellulaire est remis en suspension dans du milieu complet RPMI. La suspension cellulaire est mise dans des flacons de culture à raison de 10 ml par flacon de 50 ml. Sont ajoutés à la suspension cellulaire 2,5 ng/ml d'IL2 et 100 ng/ml d'ILlO. La suspension cellulaire est incubée pendant 5 jours dans une étuve à 37°C et 5% de C02.C0 2 before being centrifuged 6 min at 1300 rpm. The supernatant is removed and the cell pellet is resuspended. suspension in complete RPMI medium. The cell suspension is placed in culture flasks at a rate of 10 ml per 50 ml flask. 2.5 ng / ml IL2 and 100 ng / ml IL10 are added to the cell suspension. The cell suspension is incubated for 5 days in an oven at 37 ° C and 5% C0 2 .
III- Fusion cellulaire.III- Cell fusion.
La suspension cellulaire est mise dans un tube à centrifuger de 15 ml et les cellules IR 983 F/ TEC dans un autre tube. Les tubes sont centrifugés pendant 6 min à 1200 rpm. Les surnageants sont enlevés et les cellules sont remises en suspension dans 10 ml de milieu MEM REGA 3 complet, milieu MEM REGA 3 contenant 1% d'HEPES et 0,1% de gentamicine, à 37°C. Après une centrifugation à 1200 rpm pendant 6 min, les surnageants sont enlevés et les cellules sont remises en suspension dans 10 ml de milieu MEM REGA 3 complet à 37°C. Un comptage des deux types cellulaires est effectué. Les tubes sont centrifugés pendant 6 min à 1200 rpm. Les surnageants sont enlevés et les cellules sont remises en suspension dans 10 ml de milieu MEM REGA 3 complet à 37°C. Les cellules sont mélangées dans un tube à centrifuger de 50 ml suivant un rapport de 1 IR 983 F/ TEC pour une cellule plasmocytaire . Après une centrifugation à 1200 rpm pendant 6 min, les surnageants sont enlevés et le culot est remis en suspension dans la dernière goutte. Une solution de PEG est obtenue en chauffant à 50°C 5 g de PEG 4000 dans 7 ml de PBS et 1 ml de DMSO puis en filtrant la solution sur filtre 0,2 μM. Un ml de solution de PEG est ajouté au culot en 1 min 30 sec en agitant le tube. On laisse reposer 30 sec. Sont ajoutés 1 ml de MEM REGA 3 à 37°C en 1 min 30 sec en agitant le tube puis 20 ml de MEM REGA 3 à 37°C en 4 min en agitant le tube. Après une centrifugation à 1200 rpm pendant 6 min, les surnageants sont enlevés et le culot est remis en suspension dans du milieu complet DMEM avec 1% d' aminoptérine et 2% d'hypoxantine-thymidine de façon à avoir 1.106 cellules par ml . Le milieu complet DMEM contient 1% de L-glutamine, 1% de NEAA, 1% de pyruvate de sodium, 0,1% de gentamicine, 50 μM de 2-mercaptoethanol, 5% de sérum de veau fœtal et 5% de sérum de cheval. La suspension cellulaire est répartie sur des plaques de culture 96 puits contenant 100 μl de cellules péritonéales, la suspension est répartie à raison de 100 μl par puits. Les plaques sont incubées 7 jours dans une étuve à 37°C et 5% de C02.The cell suspension is put in a 15 ml centrifuge tube and the IR 983 F / TEC cells in another tube. The tubes are centrifuged for 6 min at 1200 rpm. The supernatants are removed and the cells are resuspended in 10 ml of complete MEM REGA 3 medium, MEM REGA 3 medium containing 1% of HEPES and 0.1% of gentamicin, at 37 ° C. After centrifugation at 1200 rpm for 6 min, the supernatants are removed and the cells are resuspended in 10 ml of complete MEM REGA 3 medium at 37 ° C. A count of the two cell types is carried out. The tubes are centrifuged for 6 min at 1200 rpm. The supernatants are removed and the cells are resuspended in 10 ml of complete MEM REGA 3 medium at 37 ° C. The cells are mixed in a 50 ml centrifuge tube in a ratio of 1 IR 983 F / TEC for a plasma cell. After centrifugation at 1200 rpm for 6 min, the supernatants are removed and the pellet is resuspended in the last drop. A PEG solution is obtained by heating 5 g of PEG 4000 in 7 ml of PBS and 1 ml of DMSO to 50 ° C. and then filtering the solution on a 0.2 μM filter. A ml of PEG solution is added to the pellet over 1 min 30 sec by shaking the tube. Leave to stand for 30 sec. Are added 1 ml of MEM REGA 3 at 37 ° C in 1 min 30 sec by shaking the tube then 20 ml of MEM REGA 3 at 37 ° C in 4 min by shaking the tube. After one centrifugation at 1200 rpm for 6 min, the supernatants are removed and the pellet is resuspended in complete DMEM medium with 1% aminopterin and 2% hypoxantine-thymidine so as to have 1.10 6 cells per ml. The complete DMEM medium contains 1% L-glutamine, 1% NEAA, 1% sodium pyruvate, 0.1% gentamicin, 50 μM 2-mercaptoethanol, 5% fetal calf serum and 5% serum of horse. The cell suspension is distributed on 96-well culture plates containing 100 μl of peritoneal cells, the suspension is distributed at the rate of 100 μl per well. The plates are incubated for 7 days in an oven at 37 ° C and 5% C0 2 .
IV- Entretien des plaques de fusion. Sept jours après la fusion, le milieu de culture des plaques est enlevé. Par puits, sont ajoutés 200 μl de milieu complet DMEM contenant 1% d' aminoptérine et 1% d'hypoxanthine-thymidine . Les plaques sont incubées pendant 5 jours dans une étuve à 37°C et 5% de C02. Le milieu de culture des plaques est enlevé puis sont mis, par puits, 200 μl de milieu complet DMEM contenant 2% d'hypoxanthine- thymidine. Après une incubation de 4 jours dans une étuve à 37°C et 5% de C02, le milieu de culture des plaques est enlevé puis sont mis, par puits, 200 μl de milieu complet DMEM contenant 2% d'hypoxanthine-thymidine et 2,5 ng/ml d'IL6. Après une incubation de 4 jours dans une étuve à 37°C et 5% de C02, le milieu de culture des plaques est enlevé puis sont mis, par puits, 200 μl de milieu complet DMEM contenant 2,5 ng/ml d'IL6. les plaques sont mises à incuber dans une étuve à 37°C et 5% de C02, le milieu de culture est changé de la même manière jusqu'à l'apparition des clones . V- Résultats des différentes fusions réalisées.IV- Maintenance of the melting plates. Seven days after fusion, the plate culture medium is removed. Per well, 200 μl of complete DMEM medium containing 1% of aminopterin and 1% of hypoxanthine-thymidine are added. The plates are incubated for 5 days in an oven at 37 ° C and 5% C0 2 . The culture medium for the plates is removed and then 200 μl of complete DMEM medium containing 2% of hypoxanthine-thymidine are placed per well. After an incubation of 4 days in an oven at 37 ° C and 5% of C0 2 , the culture medium of the plates is removed and are then placed, per well, 200 μl of complete DMEM medium containing 2% of hypoxanthine-thymidine and 2.5 ng / ml of IL6. After an incubation of 4 days in an oven at 37 ° C and 5% of C0 2 , the culture medium of the plates is removed and are then placed, per well, 200 μl of complete DMEM medium containing 2.5 ng / ml of IL6. the plates are incubated in an oven at 37 ° C and 5% C0 2 , the culture medium is changed in the same way until the appearance of the clones. V- Results of the various mergers carried out.
Les lymphocytes B (LcB) provenant de volontaires vaccinés par un vaccin anti-grippal et prélevés 0, 8, 15 ou 21 jours après la vaccination ont été fusionnés avec la lignée IR 983 F/ TEC suivant le protocole décrit ci-dessus. Les lymphocytes B d'un donneur X ont servi de contrôle. Les hybridomes obtenus sont testés pour la production d' immunoglobulines (Ig) humaines par la technique ELISA en utilisant des anticorps de rats anti-Ig G et anti-Ig M humaines. Le tableau 1 récapitule les résultats obtenus (nombre de clones et production d'Ig) .The B lymphocytes (LcB) originating from volunteers vaccinated with an influenza vaccine and collected 0, 8, 15 or 21 days after the vaccination were fused with the line IR 983 F / TEC according to the protocol described above. B cells from donor X were used as controls. The hybridomas obtained are tested for the production of human immunoglobulins (Ig) by the ELISA technique using antibodies to human anti-IgG and anti-IgM rats. Table 1 summarizes the results obtained (number of clones and production of Ig).
Tableau 1 : Résultats obtenus pour chaque fusion.Table 1: Results obtained for each merger.
Figure imgf000016_0001
Figure imgf000016_0001
VI- Purification des immunoglobulines.VI- Purification of immunoglobulins.
Les immunoglobulines sont récupérées à partir des surnageants de culture des 9 clones FAL 11 et du clone FAL 8. Elles sont purifiées par chromatographie d'affinité sur une colonne de Sépharose couplée à des anticorps anti- Ig humaines qui sont l'anticorps LO-hK-3 (anti-Ig humaines Kappa, réf : TEC 146, Technopharm-France) et l'anticorps LO-hL-2 (anti-Ig humaines lambda, réf : TEC 147, Technopharm-France) . La concentration en immunoglobulines est évaluée par un test ELISA en comparaison avec un étalon (IgM humaine à 1 mg/ml, réf : 18260, Sigma) .The immunoglobulins are recovered from the culture supernatants of the 9 clones FAL 11 and of the clone FAL 8. They are purified by affinity chromatography on a Sepharose column coupled with anti-human Ig antibodies which are the LO-hK antibody -3 (anti-human Ig Kappa, ref: TEC 146, Technopharm-France) and the antibody LO-hL-2 (anti-human Ig lambda, ref: TEC 147, Technopharm-France). The immunoglobulin concentration is evaluated by an ELISA test in comparison with a standard (human IgM at 1 mg / ml, ref: 18260, Sigma).
VII- Activité des anticorps.VII- Activity of antibodies.
1) Test ELISA de spécificité, i) Matériel .1) ELISA specificity test, i) Material.
- Antigênes viraux du virus Influenza : A/Sidney/5/97 (H3N2)-like lot : HVR 13-1, A/Beijing/262/95 (H1N1) -like lot : HXH 19-4,- Influenza virus viral antigens: A / Sidney / 5/97 (H3N2) -like lot: HVR 13-1, A / Beijing / 262/95 (H1N1) -like lot: HXH 19-4,
B/Beijing/184/93 (H3N2) -like lot : HHA 06-5, Hémagglutinine Influenza (réf : 12149, Sigma)B / Beijing / 184/93 (H3N2) -like lot: HHA 06-5, Hemagglutinin Influenza (ref: 12149, Sigma)
- Anticorps monoclonaux LO-HK-3 lot : 5330 et LO-HL-2 lot : 4451 marqués à la péroxydase.- LO-HK-3 lot monoclonal antibodies: 5330 and LO-HL-2 lot: 4451 labeled with peroxidase.
ii) Protocole .ii) Protocol.
Les antigènes dilués à une concentration de 5 à 10 μg/ml dans du tampon carbonate bicarbonate au pH de 9,5 sont répartis à raison de 100 μl par puits dans des plaques 96 puits. Ces plaques sont incubées une nuit à 4°C.The antigens diluted to a concentration of 5 to 10 μg / ml in carbonate bicarbonate buffer at pH 9.5 are distributed at the rate of 100 μl per well in 96-well plates. These plates are incubated overnight at 4 ° C.
Les plaques sont ensuite lavées 3 fois avec du PBS contenant 2% de Tween 20. Les plaques sont saturées avec du PBS contenant 2% de Tween 20 et 5% de lait écrémé à raison de 200 μl par puits et incubées une heure à 37°C. Les plaques sont ensuite lavées 3 fois avec du PBS contenant 2% de Tween 20.The plates are then washed 3 times with PBS containing 2% of Tween 20. The plates are saturated with PBS containing 2% of Tween 20 and 5% of skimmed milk at a rate of 200 μl per well and incubated one hour at 37 ° vs. The plates are then washed 3 times with PBS containing 2% Tween 20.
Les échantillons d' immunoglobulines à différentes dilutions sont répartis dans les plaques à raison de 10 μl par puits. Ces plaques sont incubées 2 heures à 37°C. Les plaques sont ensuite lavées 6 fois avec du PBS contenant 2% de Tween 20.The immunoglobulin samples at different dilutions are distributed in the plates at a rate of 10 μl per well. These plates are incubated for 2 hours at 37 ° C. The plates are then washed 6 times with PBS containing 2% Tween 20.
Les anticorps couplés à la péroxydase sont dilués dans du PBS contenant 2% de Tween 20 (0,5 μl/ml) et déposés dans les plaques à raison de 100 μl par puits. Les plaques sont incubées une heure à 37°C. Les plaques sont lavées 6 fois avec du PBS contenant 2% de Tween 20.The antibodies coupled to the peroxidase are diluted in PBS containing 2% of Tween 20 (0.5 μl / ml) and deposited in the plates at a rate of 100 μl per well. The plates are incubated for one hour at 37 ° C. The plates are washed 6 times with PBS containing 2% Tween 20.
Une pastille d'OPD (O Phénylenediamine, réf : P8787, Sigma) est dissoute dans le tampon de révélation (tampon citrate phosphate au pH de 5,5) avec du peroxyde d'oxygène. La solution est déposée sur les plaques à raison de 100 μl par puits. Les plaques sont incubées de 10 à 20 minutes à l'obscurité. La réaction enzymatique est arrêtée par l'ajout de 50 μl par puits de H2S04 à 2M. Les résultats sont lus à 490 nm au lecteur de plaques.An OPD pellet (O Phenylenediamine, ref: P8787, Sigma) is dissolved in the development buffer (citrate phosphate buffer at pH 5.5) with oxygen peroxide. The solution is deposited on the plates at the rate of 100 μl per well. The plates are incubated for 10 to 20 minutes in the dark. The enzymatic reaction is stopped by adding 50 μl per well of 2 M H 2 S0 4 . The results are read at 490 nm in the plate reader.
iii) Résultats .iii) Results.
Des tests ELISA sont donc réalisés afin de tester la spécificité des immunoglobulines sur les antigènes viraux A(H3N2) , A(H1N1) et B des souches de l'hiver 1998/99 (Tableau 2) . Les tests se sont révélés positifs pour 2 clones FAL 11 (3D2 et 2C3) et pour le clone FAL 8 (1G4) .ELISA tests are therefore carried out in order to test the specificity of the immunoglobulins on the viral antigens A (H3N2), A (H1N1) and B of the strains of winter 1998/99 (Table 2). The tests were positive for 2 FAL 11 clones (3D2 and 2C3) and for the FAL 8 clone (1G4).
L'absence de spécificité étroite pour une des 3 souches de virus est particulièrement intéressante sur le plan thérapeutique dans le sens où les anticorps seraient capables d'être actifs sur différentes souches de virus.The absence of narrow specificity for one of the 3 strains of virus is particularly interesting from a therapeutic point of view in the sense that the antibodies would be capable of being active on different strains of virus.
Tableau 2 : Résultat du Test ELISA de spécificité sur les différents clones.Table 2: Result of the ELISA test for specificity on the different clones.
Figure imgf000018_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000019_0001
2) Test de protection d'une infection virale chez la souris à l'aide d'anticorps humains. i) Matériel .2) Test for protection of a viral infection in mice using human antibodies. i) Material.
Souris : Des souris BALB/c femelles de 5 semaines sont groupées par lot de 6. Elles sont hébergées dans une enceinte autonome dans des cages recouvertes d'une cape filtrante.Mice: 5-week-old female BALB / c mice are grouped in batches of 6. They are housed in an autonomous enclosure in cages covered with a filtering cap.
- Virus : Le virus Influenza A souche Scotland- Virus: The Scotland Influenza A strain
[A/Scotland/74 (H3N2)] est adapté à la souris par passages pulmonaires successifs. Le stock viral est conservé en broyâts pulmonaires à -80°C. La dilution en tampon PBS pour l'infection expérimentale est effectuée exte poranément .[A / Scotland / 74 (H3N2)] is adapted to the mouse by successive pulmonary passages. The viral stock is kept in crushed lungs at -80 ° C. The dilution in PBS buffer for the experimental infection is carried out externally.
Anticorps : Des immunoglobulines totales humaines (Ivlg) conservées à -80°C puis diluées à la concentration souhaitée extemporanément (500 μg/ 50 μl) sont utilisées comme contrôle. Les anticorps purifiés AB1, AB2, AB9 et AB10 conservés à 4°C sont ajustés à une concentration équivalente pour chaque anticorps (1 μg/ 50 μl) • ii) Protocole.Antibodies: Human total immunoglobulins (Ivlg) stored at -80 ° C and then diluted to the desired concentration immediately before use (500 μg / 50 μl) are used as control. The purified antibodies AB1, AB2, AB9 and AB10 stored at 4 ° C are adjusted to an equivalent concentration for each antibody (1 μg / 50 μl) • ii) Protocol.
Les souris sont anesthésiées légèrement suivant un protocole décrit dans les ouvrages spécialisés et reçoivent par voie intra-nasale 50 μl de suspension d'anticorps (AB1, AB2, AB9, AB10 ou d'Ig totales humaines : Ivlg) ou de sérum physiologique (Témoins) . Le lendemain, après anesthesie, elles reçoivent 50 μl de la suspension virale létale en 10 jours.The mice are lightly anesthetized according to a protocol described in the specialized books and receive 50 μl of suspension of antibody (AB1, AB2, AB9, AB10 or total human Ig: Ivlg) or physiological serum intra-nasally (controls ). The next day, after anesthesia, they receive 50 μl of the lethal viral suspension in 10 days.
Les souris sont surveillées chaque jour.The mice are monitored daily.
L'efficacité de la protection est mesurée en survie/ létalité.Protection effectiveness is measured in survival / lethality.
iii) Résultats .iii) Results.
Les résultats (Tableau 3) montrent : que les souris recevant le sérum physiologique sont mortes dès Jll ;The results (Table 3) show: that the mice receiving the physiological saline died from Jll;
- que les souris recevant des anticorps de type Ig M lambda du lot AB2 sont toutes vivantes à J15 ;- that the mice receiving antibodies of the Ig M lambda type from batch AB2 are all alive on D15;
- que les souris recevant des anticorps de type Ig M lambda du lot AB9 sont toutes mortes à Jll ; - que 5 des 6 souris recevant des anticorps de type Ig M Kappa du lot AB1 sont mortes à J15 ;- that the mice receiving antibodies of the Ig M lambda type from batch AB9 all died at Jll; - that 5 of the 6 mice receiving antibodies of the Ig M Kappa type from batch AB1 died on D15;
- que les souris recevant les immunoglobulines totales à la dose de 500 μg sont toutes vivantes à J15. Les résultats montrent que les anticorps monoclonaux IgM lambda sécrétés par le clone FAL 11 2C3 protègent les souris de l'infection mortelle par le virus A/Scotland/74 (H3N2) de la grippe à la dose de 1 μg dans 50 μl de PBS comparativement à des immunoglobulines humaines totales à la dose de 500 μg dans 50 μl de PBS qui protègent également les souris de l'infection respiratoire mortelle par le même virus de la grippe- that the mice receiving the total immunoglobulins at a dose of 500 μg are all alive on D15. The results show that the lambda IgM monoclonal antibodies secreted by the clone FAL 11 2C3 protect the mice from the fatal infection by the A / Scotland / 74 (H3N2) virus at a dose of 1 μg in 50 μl of PBS compared to total human immunoglobulins at a dose of 500 μg in 50 μl of PBS which also protect mice from fatal respiratory infection by the same influenza virus
Tableau 3 : Test de protection d'une infection virale chez la souris à l'aide d'anticorps humainsTable 3: Test for protection of a viral infection in mice using human antibodies
(exprimé en nombre de survivants par groupe de 6) .(expressed in number of survivors per group of 6).
Figure imgf000021_0001
Figure imgf000021_0001

Claims

REVENDICATIONS
1) Procédé de préparation d'anticorps monoclonaux humains ou de fragments de ceux-ci ou d'anticorps comprenant un tel fragment, à partir de lymphocytes B, caractérisé en ce qu'il comprend les étapes suivantes : a) l'isolement de lymphocytes B à partir d'un prélèvement provenant d'un individu de l'espèce humaine, b) la différenciation desdits lymphocytes B en cellules plasmocytaires avec au moins un système activateur non spécifique et au moins une cytokine, c) l' immortalisation desdites cellules plasmocytaires par fusion cellulaire avec une lignée de fusion de rat, d) la culture in vi tro des cellules fusionnées jusqu'à l'obtention d' hybridomes, e) la purification des anticorps monoclonaux produits par les hybridomes, f) éventuellement le clivage desdits anticorps en fragments, et la purification d'un ou plusieurs desdits fragments, g) éventuellement l'association d'un ou plusieurs des fragments obtenu (s) à l'étape (f) avec des fragments d'autres anticorps.1) Process for the preparation of human monoclonal antibodies or of fragments thereof or of antibodies comprising such a fragment, from B lymphocytes, characterized in that it comprises the following steps: a) isolation of lymphocytes B from a sample from an individual of the human species, b) the differentiation of said B lymphocytes into plasma cells with at least one non-specific activating system and at least one cytokine, c) the immortalization of said plasma cells by cell fusion with a rat fusion line, d) in vitro culture of the fused cells until hybridomas are obtained, e) purification of the monoclonal antibodies produced by the hybridomas, f) optionally the cleavage of said antibodies into fragments, and the purification of one or more of said fragments, g) optionally the association of one or more of the fragments obtained in step (f) with fragments of other antibodies.
2) Procédé selon la revendication 1, caractérisé en ce que l'étape (b) de différenciation des lymphocytes B comprend les étapes suivantes : i) le mélange d'une suspension de lymphocytes B avec de 0,01 à 1 % d'un système activateur non spécifique et de 1 à 8,5 ng/ml d'IL2, puis l'incubation du mélange pendant 24 heures à 11 jours à une température comprise entre 36,5 et 37,8°C, ii) la centrifugation de la suspension obtenue en (i) de 600 à 3500 rpm pendant 2 à 12 minutes, puis le retrait du surnageant et la remise en suspension du culot cellulaire, iii) le mélange de la suspension obtenue en (ii) avec de 1 à 8,5 ng/ml d'IL2 et de 10 à 350 ng/ml d'ILlO, puis l'incubation du mélange pendant 48 heures à 15 jours à une température comprise entre 36,5 et 37,8°C.2) Method according to claim 1, characterized in that step (b) of differentiating B lymphocytes comprises the following steps: i) mixing a suspension of B lymphocytes with from 0.01 to 1% of a non-specific activator system and from 1 to 8.5 ng / ml of IL2, then incubation of the mixture for 24 hours to 11 days at a temperature between 36.5 and 37.8 ° C, ii) centrifugation of the suspension obtained in (i) from 600 to 3500 rpm for 2 to 12 minutes, then removal of the supernatant and resuspending the cell pellet, iii) mixing the suspension obtained in (ii) with 1 to 8.5 ng / ml IL2 and 10 to 350 ng / ml IL10, then incubating the mixing for 48 hours to 15 days at a temperature between 36.5 and 37.8 ° C.
3) Procédé selon l'une des revendications 1 ou 2, caractérisé en ce que l'étape (b) de différenciation des lymphocytes B comprend les étapes suivantes : i) le mélange d'une suspension de lymphocytes B avec 0,04 % de système activateur non spécifique et 2,5 ng/ml d'IL2, puis l'incubation du mélange pendant 3 jours à une température de 37°C sous 5% de C02, ii) la centrifugation de la suspension obtenue en (i) à 1300 rpm pendant 6 minutes, puis le retrait du surnageant et la remise en suspension du culot cellulaire, iii) le mélange de la suspension obtenue en (ii) avec 2,5 ng/ml d' IL2 et 100 ng/ml d'ILlO, puis l'incubation du mélange pendant 5 jours à une température de 37°C sous 5% de C02.3) Method according to one of claims 1 or 2, characterized in that step (b) of differentiating B lymphocytes comprises the following steps: i) mixing a suspension of B lymphocytes with 0.04% of non-specific activator system and 2.5 ng / ml of IL2, then incubation of the mixture for 3 days at a temperature of 37 ° C under 5% of CO 2 , ii) centrifugation of the suspension obtained in (i) at 1300 rpm for 6 minutes, then removal of the supernatant and resuspension of the cell pellet, iii) mixing the suspension obtained in (ii) with 2.5 ng / ml of IL2 and 100 ng / ml of IL10, then incubation of the mixture for 5 days at a temperature of 37 ° C under 5% CO 2 .
4) Procédé selon l'une quelconque des revendications 1 à 3, caractérisé en ce que le système activateur non spécifique est choisi parmi un lipopolysaccharide ou toute substance susceptible d'avoir une action identique en tout ou partie, la pansorbine, la phytohémagglutinine, le muramyldipeptide utilisés seuls ou en association. 5) Procédé selon l'une quelconque des revendications 1 à 4, caractérisé en ce que la lignée de fusion de rat utilisée pour l' immortalisation des plasmocytes à l'étape (c) est une lignée myélomateuse de rat de souche LOU.4) Method according to any one of claims 1 to 3, characterized in that the non-specific activating system is chosen from a lipopolysaccharide or any substance capable of having an identical action in whole or in part, pansorbine, phytohemagglutinin, muramyldipeptide used alone or in combination. 5) Method according to any one of claims 1 to 4, characterized in that the rat fusion line used for the immortalization of plasma cells in step (c) is a myeloma rat line of LOU strain.
6) Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que la lignée de fusion utilisée pour l' immortalisation des plasmocytes à l'étape (c) est la lignée myélomateuse de rat LOU IR 983 F/ TEC déposée à la Collection Nationale de Cultures de Microorganismes à l'Institut Pasteur (Paris) sous le No. 1-2584.6) Method according to any one of the preceding claims, characterized in that the fusion line used for the immortalization of the plasma cells in step (c) is the rat myeloma line LOU IR 983 F / TEC deposited in the Collection Nationale de Cultures de Microorganismes at the Institut Pasteur (Paris) under No. 1-2584.
7) Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce qu'à l'étape7) Method according to any one of the preceding claims, characterized in that in step
(b) la différenciation des lymphocytes B en cellules plasmocytaires est effectuée en présence d'un antigène spécifique d'un agent infectieux.(b) the differentiation of B lymphocytes into plasma cells is carried out in the presence of a specific antigen of an infectious agent.
8) Procédé selon l'une quelconque des revendications 1 à 6, caractérisé en ce qu'à l'étape (a) les lymphocytes B sont isolés de sang d'un individu vacciné contre un agent infectieux ou en convalescence d'une infection par ledit agent infectieux.8) Method according to any one of claims 1 to 6, characterized in that in step (a) the B lymphocytes are isolated from the blood of an individual vaccinated against an infectious agent or recovering from an infection by said infectious agent.
9) Procédé selon la revendication 8, caractérisé en ce qu'à l'étape (a) les lymphocytes B sont isolés de sang d'un sujet vacciné contre la grippe.9) Method according to claim 8, characterized in that in step (a) the B lymphocytes are isolated from the blood of a subject vaccinated against influenza.
10) Un hybridome susceptible d'être obtenu après les étapes (a) à (d) d'un procédé selon l'une quelconque des revendications 1 à 9. 11) Un anticorps monoclonal susceptible d'être obtenu par les étapes (a) à (e) d'un procédé selon l'une quelconque des revendications 1 à 9, ou un fragment de celui-ci obtenu par les étapes (a) à (f) d'un procédé selon l'une quelconque des revendications 1 à 9.10) A hybridoma capable of being obtained after steps (a) to (d) of a method according to any one of claims 1 to 9. 11) A monoclonal antibody capable of being obtained by steps (a) to (e) of a method according to any one of claims 1 to 9, or a fragment thereof obtained by steps (a) to (f) a method according to any one of claims 1 to 9.
12) Un anticorps monoclonal ou un fragment de celui-ci selon la revendication 11, appartenant à la classe des IgG, IgM, IgE, IgD ou IgA.12) A monoclonal antibody or a fragment thereof according to claim 11, belonging to the class of IgG, IgM, IgE, IgD or IgA.
13) Un anticorps ou fragment d'anticorps comprenant au moins un fragment d'anticorps selon l'une des revendications 11 ou 12.13) An antibody or antibody fragment comprising at least one antibody fragment according to one of claims 11 or 12.
14) Un acide nucléique comprenant une séquence codant pour un anticorps ou pour un fragment de celui-ci selon l'une quelconque des revendications 11 à 13.14) A nucleic acid comprising a sequence coding for an antibody or for a fragment thereof according to any one of claims 11 to 13.
15) Composition pharmaceutique comprenant à titre d'agent actif une quantité efficace d'au moins un anticorps monoclonal ou d'un fragment d'anticorps selon l'une des revendications 11 à 13.15) Pharmaceutical composition comprising, as active agent, an effective amount of at least one monoclonal antibody or of an antibody fragment according to one of claims 11 to 13.
16) Un kit de détection d'une infection par un agent infectieux, caractérisé en ce qu'il comprend au moins un anticorps ou un fragment d'anticorps selon l'une des revendications 11 à 13. 16) A kit for detecting an infection by an infectious agent, characterized in that it comprises at least one antibody or an antibody fragment according to one of claims 11 to 13.
PCT/FR2001/003881 2000-12-07 2001-12-07 Method for preparing a human monoclonal antibody, fragments thereof or antibodies comprising such fragments, resulting antibodies and use thereof WO2002046233A1 (en)

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