EP1545614A2 - Treatment of pathologies which escape the immune response, using optimised antibodies - Google Patents

Treatment of pathologies which escape the immune response, using optimised antibodies

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Publication number
EP1545614A2
EP1545614A2 EP03775438A EP03775438A EP1545614A2 EP 1545614 A2 EP1545614 A2 EP 1545614A2 EP 03775438 A EP03775438 A EP 03775438A EP 03775438 A EP03775438 A EP 03775438A EP 1545614 A2 EP1545614 A2 EP 1545614A2
Authority
EP
European Patent Office
Prior art keywords
anticoφs
cells
cell
use according
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03775438A
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German (de)
French (fr)
Inventor
Christophe De Romeuf
Christine Gaucher
Sylvie Jorieux
Dominique Bourel
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LFB SA
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LFB SA
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Priority claimed from FR0211416A external-priority patent/FR2844521B1/en
Priority claimed from FR0211415A external-priority patent/FR2844520B1/en
Application filed by LFB SA filed Critical LFB SA
Priority to EP15182965.2A priority Critical patent/EP3001198A1/en
Publication of EP1545614A2 publication Critical patent/EP1545614A2/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • 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
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A61P33/12—Schistosomicides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to the use of optimized chimeric, humanized or human monoclonal antibodies which are produced in selected cell lines, said antibodies having a high affinity for the CD 16 receptor of effector cells of the immune system but also the property of inducing the secretion of cytokines and interleukins, in particular IFN ⁇ or 1TL2, for the treatment of pathologies for which the target cells express only a low antigenic density and in which the effector cells can only be recruited at low amount.
  • HLA class 1 or class 2 molecules for example in the case of carcinomas, melanomas, ovarian cancers, prostate cancers are generally poorly expressed on the surface of target cells. tumor.
  • HLA class 1 or class 2 molecules express only few viral molecules on their membrane.
  • the objective is to obtain new antibodies having a better efficacy compared to current antibodies, which would make it possible to envisage their use in therapy for pathologies having few expressed molecular targets or a low antigenic density as well as a number limited effector cells capable of being activated.
  • CD 16 can be further reinforced by additional conditions aimed at producing antibodies which also induce the production of cytokines, in particular the production of IFN ⁇ or IL2 by cells of the immune system.
  • the two characteristics mentioned above complement each other. Indeed, the production of IFN ⁇ or IL2 induced by the antibodies selected by the process of the invention can reinforce the cytotoxic activity. The mechanism of action of such activation is probably due to an autocrine regulation of effector cells. It can be postulated that the antibodies bind to CD 16 causing cytotoxic activity but also induce the production of Y IFN ⁇ or IL2 which ultimately leads to an even greater increase in cytotoxic activity.
  • the invention relates to the use of an optimized chimeric, humanized or human monoclonal antibody characterized in that: a) it is produced in a cell line selected for its glycosylation properties of the Fc fragment of an antibody, or b ) the glycan structure of Fcgamma has been modified ex vivo, and / or c) its primary sequence has been modified so as to increase its reactivity towards Fc receptors; said antibody having i) an ADCC level via Fc ⁇ RIII (CD 16) greater than 50%, preferably greater than 100% for an E / T ratio (effector cells / target cells) less than 5/1, preferably less than 2 / 1, compared to the same antibody produced in a CHO line; and ii) a production rate of at least one cytokine by an effector cell of the immune system expressing the CD 16 receptor greater than 50%, 100% or preferably greater than 200% compared to the same antibody produced in a CHO line; for the preparation of a medicament intended for the treatment of pathologies
  • cytokines released by the optimized antibodies are chosen from interleukins, interferons and tissue necrosis factors (TNF).
  • the antibody is selected for its ability to induce the secretion of at least one cytokine chosen from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 , IL-8, IL-9, IL-10, ... TNFa, TGF ⁇ , IP10 and IFN ⁇ by effector cells of the immune system expressing the CD 16 receptor.
  • cytokine chosen from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 , IL-8, IL-9, IL-10, ... TNFa, TGF ⁇ , IP10 and IFN ⁇ by effector cells of the immune system expressing the CD 16 receptor.
  • the antibody selected has the capacity to induce the secretion of IFN ⁇ or IL2 by effector cells of the immune system expressing the CD 16 receptor or of 1TL2 by the Jurkat CD 16 cell for a small number of antigenic sites. present on the surface of target cells or for a small number of antigens accessible to antibodies.
  • the secreted IFN ⁇ or IL2 level reflects the quality of the antibody fixed by the CD 16 receptor as regards its integrity (Fc function) and its efficiency (antigenic site) of binding to the antigen.
  • the secretion of IFN ⁇ or IL2 by cells of the immune system can activate the cytotoxic activity of effector cells.
  • the antibodies of the invention are also useful for the treatment of pathologies for which the number of activated or recruited effector cells is low.
  • Effector cells can express endogenous CD 16 or be transformed.
  • the term transformed cell means a cell genetically modified so as to express a receptor, in particular the CD 16 receptor.
  • the antibody of the invention is capable of inducing the secretion of at least one cytokine by a leukocyte cell, in particular of the family of NK (natural killer) or by cells of the monocyte group -macrophages.
  • a Jurkat line transfected with an expression vector coding for the CD 16 receptor as effector cell is used for the selection of the antibodies. This line is particularly advantageous because it is immortalized and develops indefinitely in culture media.
  • the level of interleukin IL2 secreted reflects the quality of the antibody fixed by the CD 16 receptor as regards its integrity (Fc function) and its efficiency (antigenic site) of binding to the antigen.
  • the optimized antibody can be prepared after being purified and / or modified ex vivo by modification of the glycan structure of the Fc fragment.
  • any suitable chemical, chromatographic or enzymatic means can be used to modify the glycan structure of the antibodies.
  • the antibody can be produced by cells of rat myeloma lines, in particular YB2 / 0 and its derivatives.
  • Other lines can be selected for their properties of producing the antibodies defined above.
  • the production in CHO serves as a reference (CHO being used for the production of drug antibodies) to compare and select the production systems leading to the antibodies according to the invention.
  • the general glycan structure of the antibody corresponds to a biantennary type, with short chains, low sialylation, mannoses and GlcNAc at the non-intermediate terminal attachment point, and low fucosylation.
  • the level of intermediate GlcNac is not zero.
  • the invention relates to the use of an antibody described above for the preparation of a medicament intended for the treatment of a pathology escaping the immune response notably chosen from hemolytic disease of the newborn, Sézary Syndrome , chronic myeloid leukemia, cancers for which the antigenic targets are weakly expressed, in particular breast cancer, pathologies linked to the environment targeting in particular people exposed to polychlorinated biphenyls, infectious diseases, in particular tuberculosis, chronic fatigue (CFS), parasitic infections such as schistosomules.
  • a pathology escaping the immune response notably chosen from hemolytic disease of the newborn, Sézary Syndrome , chronic myeloid leukemia, cancers for which the antigenic targets are weakly expressed, in particular breast cancer, pathologies linked to the environment targeting in particular people exposed to polychlorinated biphenyls, infectious diseases, in particular tuberculosis, chronic fatigue (CFS), parasitic infections such as schistosomules.
  • FIG. 1 ADCC on red cells: comparison of normal red cells (N) versus red cells over-expressing the Rhesus antigen (GR6) (Teg 500 ⁇ g / well, ADCC 375 03 017)
  • Figure 2 ADCC activity induced by chimeric anti-HLA-DR antibodies expressed in CHO or YB2 / 0 as a function of the E / T ratio.
  • Lym-1 on the ADCC activity induced by the chimeric anti-HLA-DR antibodies expressed in CHO (square) or YB2 / 0 (triangle).
  • Figure 4 Influence of the number of HLA-DR antigens expressed on Raji (blocking by Lym-1) on the activation of Jurkat CD16 (IL2) induced by chimeric anti-HLA-DR antibodies expressed in CHO (square) or YB2 / 0 (triangle).
  • Figure 6 Correlation between the ADCC test and the secretion of IL2 by Jurkat CD 16.
  • Figure 7 IL8 secreted by CMN in the presence or absence of target
  • FIG 10 Secretion of cytokines by NK induced by anti-Rhesus antibodies
  • Figure 11 Secretion of TNF alpha by NK cells, induced by anti-CD20 and anti-HLA-DR antibodies expressed in CHO and YB2 / 0 (324 03 082).
  • Figure 12 Secretion of gamma IFN by NK cells, induced by anti-CD20 and anti-HLA-DR antibodies expressed in CHO and YB2 / 0 (324 03 082).
  • Example 1 ADCC induced by anti-Rhesus antibodies as a function of the number of antigenic sites.
  • the same sequence coding for an IgG1 specific for the Rhesus D antigen is transfected into CHO and YB2 / 0.
  • the cytotoxic activity of the antico ⁇ s is compared with respect to Rhesus positive red cells expressing on their surface different quantities of Rhesus antigen, ie: normal O + red cells (10-20,000 sites) and red cells over-expressing the antigen rhesus (> 60,000 sites).
  • ADCC activity of the antico ⁇ s expressed in CHO (triangle) or YB2 / 0 (square) on normal red cells (N, empty) or overexpressing the Rhesus antigen (GR6, full) are compared.
  • the same sequence coding for an IgG1 specific for the HLA-DR antigen is transfected into CHO and YB2 / 0.
  • the cytotoxic activity of the antico ⁇ s is compared with respect to the Raji cell in the presence of different effector / target ratios (see Figure 2).
  • the relative percentage of lysis induced by the antico ⁇ s expressed in CHO (100% being the value of the antico ⁇ s expressed in YB2 / 0 for each ratio) is 61%, 52%, 48% and 36% respectively.
  • the antico ⁇ s expressed in YB2 / 0 proves to be more cytotoxic than when it is produced by
  • Example 3 ADCC Induced by Anti-HLA DR Antibodies as a Function of the Quantity of Antigens Accessible
  • the same sequence coding for an IgG1 specific for the HLA-DR antigen is transfected into CHO and YB2 / 0.
  • the cytotoxic activity of the antico ⁇ s is compared with respect to the Raji cell in the presence of different effector / target ratios (E / T ratio).
  • the cytotoxic activity of the antico ⁇ s is compared with respect to Raji cells whose antigenic sites have been previously blocked with increasing amounts of an inactive (non-cytotoxic) murine antico DRs anti-HLA-DR, so as to have a decreasing number of 'HLA-DR antigens available with regard to the antico ⁇ s to be evaluated (see Figure 3).
  • the same sequence coding for an IgG1 specific for the HLA-DR antigen is transfected into CHO and YB2 / 0.
  • the activation of the effector cell (secretion of IL2 by Jurkat CD 16) induced by antico ⁇ s is compared with respect to Raji cells whose antigenic sites have been previously blocked with increasing amounts of a murine anti-HLA antico ⁇ s DR, so as to have a decreasing number of HLA-DR antigen available vis-à-vis the antico ⁇ s to be evaluated (see Figure 4).
  • Example 5 ADCC induced by anti-CD20 antibodies as a function of the quantity of antigens.
  • the same sequence coding for an IgG1 specific for the CD20 antigen is transfected into CHO and YB2 / 0.
  • the activation of the effector cell (secretion of IL2 by Jurkat CD 16) induced by antico ⁇ s is compared with respect to Raji cells whose antigenic sites have been previously blocked with increasing amounts of an inactive murine antico ⁇ s anti-CD20 , so as to have a decreasing number of CD20 antigens available with respect to the antico ⁇ s to be evaluated (see Figure 5).
  • Pantico ⁇ s optimized that is to say produced in YB2 / 0 can thus relate to target cells expressing on their surface a poorly expressed antigen.
  • the optimized antico ⁇ s prove to be particularly useful for therapeutic applications when the target cells express on their surface few antigens, and this whatever the antigen.
  • Example 7 Correlation in vitro between ADCC and release of IL-2 by the Jurkat CD16 cell.
  • the monoclonal antibody (Mab) DF5-EBV was produced by human B Lymphocytes obtained from an immunized D-negative donor and immortalized by transformation with EBV. This antibody was used as a negative control since in a clinical trial it was unable to remove Rhesus positive red blood cells from the circulation.
  • the monoclonal antico Ms (Mab) DF5-YB2 / 0 was obtained by expressing the primary sequence of DF5-EBV in the line YB2 / 0.
  • the monoclonal antibody R297 and other recombinant antibodies were also expressed in YB2 / 0.
  • Antico ⁇ s are tested in vitro for their ability to induce lysis of red blood cells treated with papain using mononuclear cells (PBL) as an effector.
  • PBL mononuclear cells
  • IVIg is thought to bind with high affinity to FcgammaRI (CD64).
  • the two Mab DF5-YB2 / 0 and R297 induce lysis of red blood cells at a level comparable to that of WinRho polyclonal antibodies.
  • the Mab DF5-YB2 / 0 and R297 induce lysis of red blood cells at a level comparable to that of WinRho polyclonal antibodies.
  • the Mab DF5-YB2 / 0 and R297 induce lysis of red blood cells at a level comparable to that of WinRho polyclonal antibodies.
  • IL2 produced by effector cells activated by complex antigen-antico ⁇ s immune cells, induces activation of T lymphocytes and NK cells, which can go as far as stimulating cell proliferation.
  • IFN ⁇ stimulates the activity of
  • CTLs and can enhance the activity of macrophages.
  • TNF produced by effector cells activated by immune systems antigen-antico ⁇ s complexes, stimulates the proliferation of macrophages and lymphocytes infiltrating tumors.
  • LTL-IRa is a cytokine that competes with IL1 at its receptor and thus exerts an anti-inflammatory effect.
  • LTL10 is a molecule that inhibits the activation of different effector cells and the production of cytokines.
  • the IL10 produced by the effector cells activated by antigen-antico ⁇ s complex immune systems can have a regulatory role in the cytotoxic activity of the antico ⁇ s with respect to normal cells, but expressing antigens common with the target cells targeted, and also modulate the effects of TNF alpha.
  • Example 11 Induction of the secretion of cytokines by different effector cells.
  • cytokine synthesis is dependent on the presence of the target. There are few differences in the ability of anti-D R297 and polyclonal antibodies to induce the production of different cytokines. On the other hand, AD1 is very often not inducing secretion of cytokines.
  • the monoclonal antibody R297 and the anti-D polyclonal antibody anti-D WinRho induce an important secretion of TNF alpha, less strong but greater than those induced by AD1 of IL6, of IFN gamma, IP10, TNF alpha and TGF Beta.
  • the secretion dIL6, of IFN gamma, of IP10 increases, but that of TNF alpha and the TGF Beta decreases (FIG. 8).
  • the monoclonal antico ⁇ s R297 and the polyclonal anti-D anti-D WinRho induce a very weak secretion, but superior to AD1, of IL2, of IFN gamma, of IPlO and of TNF by the polymorphonuclear cells. This secretion is dependent on the concentration of antico ⁇ s ( Figure 9).
  • the monoclonal antibody R297 and the anti-D polyclonal antibody anti-D WinRho induce significant secretion of IFN gamma, IP10 and TNF by the NK cells. This secretion is dependent on the concentration of antico ⁇ s ( Figure 10).
  • Example 11 Optimized chimeric anti-CD 20 and anti-HLA DR antibodies produced in YB2 / 0
  • Anti-CD20 the chimeric anti-CD20 antico ⁇ s transfected in YB2 / 0 is compared to a commercial anti-CD20 antico (s produced in CHO (Rituxan).
  • Anti-HLA DR the same sequence encoding the chimeric anti-HLA DR antibody is transfected into CHO (Bl 1) or YB2 / 0 (4B7).
  • Target cells Raji cells expressing on their surface the CD20 antigen and HLA-DR
  • Effector cells human NK cells purified by negative selection from a human blood bag.
  • TNF alpha The results are expressed in pg / ml of TNF alpha assayed in the supernatants. On the abscissa are the different concentrations of antico ⁇ s added to the reaction mixture ( Figure 11).
  • the chimeric anti-CD 20 and anti-HLA DR antico ⁇ s produced in YB2 / 0 induce higher levels of TNF in the presence of their target (Raji) compared to the same antico ⁇ s produced in CHO.
  • the amount of TNF alpha is dose dependent on the concentration of added antico ⁇ s.
  • 10 ng / ml of antico ⁇ s 5 times more TNF alpha is induced with the antico ⁇ s produced in YB2 / 0 compared to the antico ⁇ s produced in CHO.
  • IFN gamma The results are expressed in pg / ml of IFN gamma assayed in the supernatants. On the abscissa are the different concentrations of antico ⁇ s added to the reaction mixture ( Figure 12).
  • the chimeric anti-CD 20 and anti-HLA DR antibodies produced in YB2 / 0 induce higher levels of gamma IFN in the presence of their target (Raji) compared to the same antibodies produced in CHO.
  • the amount of IFN gamma is dose dependent on the concentration of added antico ⁇ s. At all the concentrations used (10 to 200 ng / ml) the anti-HLA DR antico ⁇ s produced in CHO does not induce IFN gamma secretion, while 40 ng / ml of the antico ⁇ s produced in YB2 / 0 induces approximately lOOOpg / ml of IFN gamma.

Abstract

The invention relates to the use of optimised human or humanised chimeric monoclonal antibodies which are produced in selected cell lines, said antibodies having a strong affinity for receptor CD16 of the effector cells of the immune system and being able to induce the secretion of cytokines and interleukins, in particular 1' IFN? or 1'IL2, for the treatment of pathologies for which the target cells only express a low antigenic density and in which the effector cells can only be recruited in small quantities.

Description

1 1
Traitement des pathologies échappant à la réponse immune par des anticorps optimisésTreatment of pathologies that escape the immune response with optimized antibodies
La présente invention concerne l'utilisation d'anticorps monoclonaux chimériques, humanisés ou humains optimisés qui sont produits dans des lignées cellulaires sélectionnées, lesdits anticorps présentant une forte affinité pour le récepteur CD 16 des cellules effectrices du système immunitaire mais également la propriété d'induire la sécrétion de cytokines et d'interleukines, en particulier l'IFNγ ou 1TL2, pour le traitement de pathologies pour lesquelles les cellules cibles n'expriment qu'une faible densité antigénique et dans lesquelles les cellules effectrices ne peuvent être recrutées qu'en faible quantité.The present invention relates to the use of optimized chimeric, humanized or human monoclonal antibodies which are produced in selected cell lines, said antibodies having a high affinity for the CD 16 receptor of effector cells of the immune system but also the property of inducing the secretion of cytokines and interleukins, in particular IFNγ or 1TL2, for the treatment of pathologies for which the target cells express only a low antigenic density and in which the effector cells can only be recruited at low amount.
L'immunothérapie au moyen d'anticorps monoclonaux est en passe de devenir un des aspects les plus importants de la médecine. En revanche, les résultats obtenus lors d'essais cliniques apparaissent contrastés. En effet, il peut s'avérer que l'anticorps monoclonal ne soit pas suffisamment efficace. De nombreux essais cliniques sont arrêtés pour diverses causes telles que le manque d'efficacité et des effets secondaires incompatibles avec une utilisation en thérapie clinique. Ces deux aspects sont étroitement liés sachant que des anticorps peu actifs sont administrés à forte dose pour compenser et obtenir une réponse thérapeutique. L'administration de fortes doses induit non seulement des effets secondaires mais est économiquement peu viable.Immunotherapy with monoclonal antibodies is fast becoming one of the most important aspects of medicine. On the other hand, the results obtained in clinical trials appear to be mixed. Indeed, it may turn out that the monoclonal antibody is not sufficiently effective. Many clinical trials have been stopped for various causes such as ineffectiveness and side effects incompatible with use in clinical therapy. These two aspects are closely linked, given that weakly active antibodies are administered in high doses to compensate for and obtain a therapeutic response. The administration of large doses not only induces side effects but is economically unsustainable.
Ces problèmes sont majeurs dans l'industrie des anticorps monoclonaux chimériques humanisés ou humains.These problems are major in the humanized or human chimeric monoclonal antibody industry.
Or, ce problème est exacerbé pour un certain nombre de pathologies pour lesquelles la densité antigénique exprimée par les cellules cibles est faible et/ou le faible nombre de cellules effectrices disponibles et activées est limité, rendant ainsi techniquement impossible l'utilisation d'anticorps à visée thérapeutique avec les anticorps actuellement disponibles. Par exemple, dans le Syndrome de Sézary, l'antigène spécifique, KIR3DL2, est faiblement exprimé (seulement environ 10 000 molécules). L'expression des antigènes tumoraux peut également être régulée négativement, comme HER2-neu dans le cancer du sein. Par ailleurs, lorsque l'on cherche à inhiber l'angiogénèse via le ciblage du NEGFR2, peu de cibles moléculaires sont effectivement accessibles car le récepteur est internalisé. De même, les peptides spécifiques d'antigènes de tumeurs présentés par les molécules HLA de classe 1 ou classe 2, par exemple dans le cas de carcinomes, mélanomes, cancers ovarien, cancers de la prostate sont généralement peu exprimés à la surface des cellules cibles tumorales. Enfin, une autre situation peut se trouver lors d'infections virales dans lesquelles les cellules infectées par certains virus (VHB, NHC, NIH) n'expriment que peu de molécules virales sur leur membrane.However, this problem is exacerbated for a certain number of pathologies for which the antigenic density expressed by the target cells is low and / or the small number of effector cells available and activated is limited, thus making technically impossible the use of antibodies to therapeutic aim with currently available antibodies. For example, in Sézary Syndrome, the antigen specific, KIR3DL2, is weakly expressed (only around 10,000 molecules). The expression of tumor antigens can also be downregulated, like HER2-neu in breast cancer. Furthermore, when one seeks to inhibit angiogenesis via the targeting of NEGFR2, few molecular targets are effectively accessible because the receptor is internalized. Similarly, the peptides specific for tumor antigens presented by HLA class 1 or class 2 molecules, for example in the case of carcinomas, melanomas, ovarian cancers, prostate cancers are generally poorly expressed on the surface of target cells. tumor. Finally, another situation can be found during viral infections in which cells infected with certain viruses (HBV, NHC, NIH) express only few viral molecules on their membrane.
Ce problème se pose aussi pour toutes les pathologies qui présentent une diminution du nombre de cellules ΝK, ou de leur activité ou de leur nombre de CD 16 (Cavalcanti M et al, Irréversible cancer cell-induced functional anergy and apoptosis in resting and activated ΝK cells, Int J Oncol 1999 Feb;14(2):361-6). On peut citer à titre d'exemple, les leucémies myéloides chroniques (Parrado A. et al., Νatural killer cytotoxicity and lymphocyte subpopulations in patients with acute leukaemia, Leuk Res 1994 Mar;18(3):191-7), les pathologies liées à l'environnement visant notamment les personnes exposées aux biphényles polychlorinés (Svensson BG. et al., Parameters of immunological compétence in subjects with high consumption of fïsh contaminated with persistent organochlorine compounds, Int Arch Occup Environ Health 1994;65(6):351-8), les maladies infectieuses, notamment la tuberculose (Restrepo LM. et al, Νatural killer cell activity in patients with pulmonary tuberculosis and in healthy controls, Tubercle 1990 Jun;71(2):95-102), le syndrome de la fatigue chronique (CFS) (Whiteside TL, Friberg D, Νatural killer cells and natural killer cell activity in chronic fatigue syndrome, Am J Med 1998 Sep 28;105(3A):27S-34S), et toutes les infections parasitaires comme par exemple les schistosomules (Feldmeier H, et al, Relationship between intensity of infection and immunomodulation in human This problem also arises for all pathologies which present a decrease in the number of ΝK cells, or in their activity or in their number of CD 16 (Cavalcanti M et al, Irreversible cancer cell-induced functional anergy and apoptosis in resting and activated ΝK cells, Int J Oncol 1999 Feb; 14 (2): 361-6). By way of example, mention may be made of chronic myeloid leukemia (Parrado A. et al., Νatural killer cytotoxicity and lymphocyte subpopulations in patients with acute leukaemia, Leuk Res 1994 Mar; 18 (3): 191-7), pathologies related to the environment, targeting in particular people exposed to polychlorinated biphenyls (Svensson BG. et al., Parameters of immunological competence in subjects with high consumption of fïsh contaminated with persistent organochlorine compounds, Int Arch Occup Environ Health 1994; 65 (6): 351-8), infectious diseases, in particular tuberculosis (Restrepo LM. Et al, Νatural killer cell activity in patients with pulmonary tuberculosis and in healthy controls, Tubercle 1990 Jun; 71 (2): 95-102), chronic fatigue (CFS) (Whiteside TL, Friberg D, Νatural killer cells and natural killer cell activity in chronic fatigue syndrome, Am J Med 1998 Sep 28; 105 (3A): 27S-34S), and all parasitic infections such as example the schistoso mules (Feldmeier H, et al, Relationship between intensity of infection and immunomodulation in human
schistosomiasis. IL NK cell activity and in vitro lymphocyte prolifération, Clin Exp Immunol 1985 May; 60(2):234-40).schistosomiasis. IL NK cell activity and in vitro lymphocyte proliferation, Clin Exp Immunol 1985 May; 60 (2): 234-40).
Ainsi, l'objectif est d'obtenir de nouveaux anticorps présentant une meilleure efficacité comparée aux anticorps actuels, ce qui permettrait d'envisager leur utilisation en thérapie pour les pathologies présentant peu de cibles moléculaires exprimées ou une faible densité antigénique ainsi qu'un nombre limité de cellules effectrices capables d'être activées.Thus, the objective is to obtain new antibodies having a better efficacy compared to current antibodies, which would make it possible to envisage their use in therapy for pathologies having few expressed molecular targets or a low antigenic density as well as a number limited effector cells capable of being activated.
Nous avions montré dans notre demande WO 01/77181 (LFB) l'importance de sélectionner des lignées cellulaires permettant de produire des anticorps présentant une forte activité ADCC via le FcγRIII (CD 16). Nous avions trouvé que la modification de la glycosylation du fragment constant des anticorps produits dans des lignées de myélomes de rat telle que YB2/0 conduisait à améliorer l'activité ADCC. Les structures glycanniques desdits anticorps sont de type biantennées, avec des chaînes courtes, une faible sialylation, des mannoses et GlcNAc du point d'attache terminaux non intercalaires, et une faible fucosylation.We had shown in our application WO 01/77181 (LFB) the importance of selecting cell lines making it possible to produce antibodies having a high ADCC activity via FcγRIII (CD 16). We had found that modifying the glycosylation of the constant fragment of the antibodies produced in rat myeloma lines such as YB2 / 0 led to improving the ADCC activity. The glycan structures of said antibodies are of the biantennary type, with short chains, low sialylation, mannoses and GlcNAc at the non-intermediate terminal attachment point, and low fucosylation.
Or, dans le cadre de la présente invention, nous avons découvert que l'avantage de présenter une forte affinité pour le CD 16 peut encore être renforcé par des conditions supplémentaires visant à produire des anticorps qui induisent également la production de cytokines, notamment la production d' IFNγ ou l'IL2 par les cellules du système immunitaire.However, in the context of the present invention, we have discovered that the advantage of having a strong affinity for CD 16 can be further reinforced by additional conditions aimed at producing antibodies which also induce the production of cytokines, in particular the production of IFNγ or IL2 by cells of the immune system.
Les deux caractéristiques précitées se complémentent. En effet, la production d' IFNγ ou l'IL2 induite par les anticorps sélectionnés par le procédé de l'invention peut renforcer l'activité cytotoxique. Le mécanisme d'action d'une telle activation tient probablement à une régulation positive autocrine des cellules effectrices. On peut postuler que les anticorps se lient au CD 16 provoquant une activité cytotoxique mais également induisent la production de Y IFNγ ou l'IL2 qui au final conduit à augmenter encore davantage l'activité cytotoxique.The two characteristics mentioned above complement each other. Indeed, the production of IFNγ or IL2 induced by the antibodies selected by the process of the invention can reinforce the cytotoxic activity. The mechanism of action of such activation is probably due to an autocrine regulation of effector cells. It can be postulated that the antibodies bind to CD 16 causing cytotoxic activity but also induce the production of Y IFNγ or IL2 which ultimately leads to an even greater increase in cytotoxic activity.
Nous montrons ici que les anticorps optimisés de l'invention conservent une bonne efficacité même lorsque la densité antigénique est faible ou le nombre de cellules effectrices limité. Ainsi, à des doses compatibles avec une utilisation en thérapie clinique, il est désormais possible de traiter des pathologies pour lesquelles un traitement par anticorps n'était pas envisageable à ce jour.We show here that the optimized antibodies of the invention retain good efficacy even when the antigen density is low or the number of effector cells limited. Thus, at doses compatible with use in clinical therapy, it is now possible to treat pathologies for which antibody treatment has not been possible to date.
DescriptionDescription
Ainsi, l'invention concerne l'utilisation d'un anticorps monoclonal chimérique, humanisé ou humain optimisé caractérisé en ce que : a) il est produit dans une lignée cellulaire sélectionnée pour ses propriétés de glycosylation du fragment Fc d'un anticorps, ou b) la structure glycannique du Fcgamma a été modifiée ex vivo, et/ou c) sa séquence primaire a été modifiée de façon à augmenter sa réactivité vis à vis des récepteurs Fc ; ledit anticorps présentant i) un taux ADCC via le FcγRIII (CD 16) supérieur à 50 %, de préférence supérieur à 100 % pour un ratio E/T (cellules effectrices/cellules cibles) inférieur à 5/1, de préférence inférieur à 2/1, comparé au même anticorps produit dans une lignée CHO ; et ii) un taux de production d'au moins une cytokine par une cellule effectrice du système immunitaire exprimant le récepteur CD 16 supérieur à 50 %, 100 % ou de préférence supérieur à 200 % comparé au même anticorps produit dans une lignée CHO; pour la préparation d'un médicament destiné au traitement de pathologies pour lesquelles le nombre de sites antigéniques ou la densité antigénique sont faibles, les antigènes sont peu accessibles aux anticorps, ou encore pour lesquelles le nombre de cellules effectrices activées ou recrutées est faible. Avantageusement, le nombre de sites antigéniques est inférieur à 250 000, de préférence inférieur à 100 000 ou 50 000 par cellule cible.Thus, the invention relates to the use of an optimized chimeric, humanized or human monoclonal antibody characterized in that: a) it is produced in a cell line selected for its glycosylation properties of the Fc fragment of an antibody, or b ) the glycan structure of Fcgamma has been modified ex vivo, and / or c) its primary sequence has been modified so as to increase its reactivity towards Fc receptors; said antibody having i) an ADCC level via FcγRIII (CD 16) greater than 50%, preferably greater than 100% for an E / T ratio (effector cells / target cells) less than 5/1, preferably less than 2 / 1, compared to the same antibody produced in a CHO line; and ii) a production rate of at least one cytokine by an effector cell of the immune system expressing the CD 16 receptor greater than 50%, 100% or preferably greater than 200% compared to the same antibody produced in a CHO line; for the preparation of a medicament intended for the treatment of pathologies for which the number of antigenic sites or the antigenic density are low, the antigens are not very accessible to antibodies, or even for which the number of effector cells activated or recruited is low. Advantageously, the number of antigenic sites is less than 250,000, preferably less than 100,000 or 50,000 per target cell.
Lesdites cytokines libérées par les anticorps optimisés sont choisies parmi des interleukines, des interférons et des facteurs de nécrose tissulaire (TNF).Said cytokines released by the optimized antibodies are chosen from interleukins, interferons and tissue necrosis factors (TNF).
Ainsi, l'anticorps est sélectionné pour sa capacité d'induire la sécrétion d'au moins une cytokine choisie parmi IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,... TNFa, TGFβ, IP10 et IFNγ par les cellules effectrices du système immunitaire exprimant le récepteur CD 16.Thus, the antibody is selected for its ability to induce the secretion of at least one cytokine chosen from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 , IL-8, IL-9, IL-10, ... TNFa, TGFβ, IP10 and IFNγ by effector cells of the immune system expressing the CD 16 receptor.
De préférence, l'anticorps sélectionné présente la capacité d'induire la sécrétion d' IFNγ ou d'IL2 par les cellules effectrices du système immunitaire exprimant le récepteur CD 16 ou de 1TL2 par la cellule Jurkat CD 16 pour un faible nombre de sites antigéniques présents à la surface des cellules cibles ou pour un faible nombre d'antigènes accessibles aux anticorps. Le taux IFNγ ou d'IL2 sécrété reflète la qualité de l'anticorps fixé par le récepteur CD 16 quant à son intégrité (fonction Fc) et à son efficacité (site antigénique) de liaison à l'antigène. En outre, la sécrétion d' IFNγ ou d'IL2 par les cellules du système immunitaire peut activer l'activité cytotoxique des cellules effectrices. Ainsi, les anticorps de l'invention sont également utiles pour le traitement de pathologies pour lesquelles le nombre de cellules effectrices activées ou recrutées est faible.Preferably, the antibody selected has the capacity to induce the secretion of IFNγ or IL2 by effector cells of the immune system expressing the CD 16 receptor or of 1TL2 by the Jurkat CD 16 cell for a small number of antigenic sites. present on the surface of target cells or for a small number of antigens accessible to antibodies. The secreted IFNγ or IL2 level reflects the quality of the antibody fixed by the CD 16 receptor as regards its integrity (Fc function) and its efficiency (antigenic site) of binding to the antigen. In addition, the secretion of IFNγ or IL2 by cells of the immune system can activate the cytotoxic activity of effector cells. Thus, the antibodies of the invention are also useful for the treatment of pathologies for which the number of activated or recruited effector cells is low.
Les cellules effectrices peuvent exprimer un CD 16 endogène ou être transformées. On entend par cellule transformée, une cellule modifiée génétiquement de sorte à exprimer un récepteur, en particulier le récepteur CD 16. Dans un mode de réalisation particulier, l'anticorps de l'invention est capable d'induire la sécrétion d'au moins une cytokine par une cellule leucocytaire, en particulier de la famille des NK (natural killer) ou par des cellules du groupe monocytes-macrophages. De préférence, on utilise pour la sélection des anticorps une lignée Jurkat transfectée avec un vecteur d'expression codant pour le récepteur CD 16 comme cellule effectrice. Cette lignée est particulièrement avantageuse car elle est immortalisée et se développe indéfiniment dans des milieux cultures. Le taux d'interleukine IL2 sécrétée reflète la qualité de l'anticorps fixé par le récepteur CD 16 quant à son intégrité (fonction Fc) et à son efficacité (site antigénique) de liaison à l'antigène.Effector cells can express endogenous CD 16 or be transformed. The term transformed cell means a cell genetically modified so as to express a receptor, in particular the CD 16 receptor. In a particular embodiment, the antibody of the invention is capable of inducing the secretion of at least one cytokine by a leukocyte cell, in particular of the family of NK (natural killer) or by cells of the monocyte group -macrophages. Preferably, a Jurkat line transfected with an expression vector coding for the CD 16 receptor as effector cell is used for the selection of the antibodies. This line is particularly advantageous because it is immortalized and develops indefinitely in culture media. The level of interleukin IL2 secreted reflects the quality of the antibody fixed by the CD 16 receptor as regards its integrity (Fc function) and its efficiency (antigenic site) of binding to the antigen.
Dans un autre mode de réalisation, l'anticorps optimisé peut être préparé après avoir été purifié et/ou modifié ex vivo par modification de la structure glycannique du fragment Fc. A cet effet, on peut utiliser tout moyen chimique, chromatographique ou enzymatique approprié pour modifier la structure glycannique des anticorps.In another embodiment, the optimized antibody can be prepared after being purified and / or modified ex vivo by modification of the glycan structure of the Fc fragment. To this end, any suitable chemical, chromatographic or enzymatic means can be used to modify the glycan structure of the antibodies.
Dans un autre mode de réalisation, l'anticorps peut être produit par des cellules de lignées de myélomes de rat, en particulier YB2/0 et ses dérivées. D'autres lignées peuvent être sélectionnées pour leurs propriétés de produire les anticorps définis ci- dessus. On pourra tester par exemple les cellules lymphoblastoïdes humaines, les cellules d'insectes et les cellules de myélomes murines. La sélection peut également être appliquée à l'évaluation des anticorps produits par des plantes transgéniques ou de mammifères transgéniques. A cet effet, la production dans CHO sert de référence (CHO étant employée pour la production d'anticorps médicament) pour comparer et sélectionner les systèmes de production conduisant aux anticorps selon l'invention.In another embodiment, the antibody can be produced by cells of rat myeloma lines, in particular YB2 / 0 and its derivatives. Other lines can be selected for their properties of producing the antibodies defined above. We can test for example human lymphoblastoid cells, insect cells and murine myeloma cells. Selection can also be applied to the evaluation of antibodies produced by transgenic plants or transgenic mammals. To this end, the production in CHO serves as a reference (CHO being used for the production of drug antibodies) to compare and select the production systems leading to the antibodies according to the invention.
La structure glycannique générale de l'anticorps correspond à un type biantenné, avec des chaînes courtes, une faible sialylation, des mannoses et GlcNAc du point d'attache terminaux non intercalaires, et une faible fucosylation. Dans ces anticorps, le taux de GlcNac intermédiaire est non nul. Ainsi, l'invention vise l'utilisation d'un anticorps décrit ci-dessus pour la préparation d'un médicament destiné au traitement d'une pathologie échappant à la réponse immune notamment choisie parmi la maladie hémolytique du nouveau né, le Syndrome de Sézary, les leucémies myéloides chroniques, les cancers dont les cibles antigéniques sont faiblement exprimées, notamment le cancer du sein, les pathologies liées à l'environnement visant notamment les personnes exposées aux biphényles polychlorinés, les maladies infectieuses, notamment la tuberculose, le syndrome de la fatigue chronique (CFS), les infections parasitaires comme par exemple les schistosomules.The general glycan structure of the antibody corresponds to a biantennary type, with short chains, low sialylation, mannoses and GlcNAc at the non-intermediate terminal attachment point, and low fucosylation. In these antibodies, the level of intermediate GlcNac is not zero. Thus, the invention relates to the use of an antibody described above for the preparation of a medicament intended for the treatment of a pathology escaping the immune response notably chosen from hemolytic disease of the newborn, Sézary Syndrome , chronic myeloid leukemia, cancers for which the antigenic targets are weakly expressed, in particular breast cancer, pathologies linked to the environment targeting in particular people exposed to polychlorinated biphenyls, infectious diseases, in particular tuberculosis, chronic fatigue (CFS), parasitic infections such as schistosomules.
Légendes et titres des figures:Legends and titles of figures:
Figure 1 : ADCC sur hématies : comparaison hématies normales (N) versus hématies sur-exprimant l'antigène Rhésus (GR6) (Teg 500 μg /puits, ADCC 375 03 017)Figure 1: ADCC on red cells: comparison of normal red cells (N) versus red cells over-expressing the Rhesus antigen (GR6) (Teg 500 μg / well, ADCC 375 03 017)
Figure 2: Activité ADCC induite par les anticorps chimériques anti-HLA-DR exprimés dans CHO ou YB2/0 en fonction du ratio E/T.Figure 2: ADCC activity induced by chimeric anti-HLA-DR antibodies expressed in CHO or YB2 / 0 as a function of the E / T ratio.
Figure 3: Influence du nombre d'antigènes HLA-DR exprimés sur Raji (blocage parFigure 3: Influence of the number of HLA-DR antigens expressed on Raji (blocking by
Lym-1) sur l'activité ADCC induite par les anticorps chimériques anti-HLA-DR exprimés dans CHO(carré) ou YB2/0 (triangle).Lym-1) on the ADCC activity induced by the chimeric anti-HLA-DR antibodies expressed in CHO (square) or YB2 / 0 (triangle).
Figure 4: Influence du nombre d'antigènes HLA-DR exprimés sur Raji (blocage par Lym-1) sur l'activation de Jurkat CD16 (IL2) induite par les anticorps chimériques anti-HLA-DR exprimés dans CHO(carré) ou YB2/0 (triangle).Figure 4: Influence of the number of HLA-DR antigens expressed on Raji (blocking by Lym-1) on the activation of Jurkat CD16 (IL2) induced by chimeric anti-HLA-DR antibodies expressed in CHO (square) or YB2 / 0 (triangle).
Figure 5: Influence du nombre d'antigènes CD20 exprimés sur Raji (blocage par CATFigure 5: Influence of the number of CD20 antigens expressed on Raji (blocking by CAT
13) sur l'activation de Jurkat CD16.13) on the activation of Jurkat CD16.
Figure 6: Corrélation entre le test ADCC et la sécrétion d'IL2 par Jurkat CD 16. Figure 7: IL8 sécrétée par les CMN en présence ou absence de cibleFigure 6: Correlation between the ADCC test and the secretion of IL2 by Jurkat CD 16. Figure 7: IL8 secreted by CMN in the presence or absence of target
Figure 8: Sécrétion de cytokines par les CMN induite par les anticorps anti-RhésusFigure 8: Secretion of cytokines by CMN induced by anti-Rhesus antibodies
(valeur sans cible déduite) Tox 324 03 062(value without target deducted) Tox 324 03 062
Figure 9: Sécrétion de cytokines par les polynucléaires induite par les anticorps antiFigure 9: Polynuclear secretion of cytokines induced by anti antibodies
Rhésus. Figure 10: Sécrétion de cytokines par les NK induite par les anticorps anti-Rhésus Figure 11 : Sécrétion de TNF alpha par les cellules NK, induite par les anticoφs anti- CD20 et anti-HLA-DR exprimés dans CHO et YB2/0 (324 03 082). Figure 12: Sécrétion d'IFN gamma par les cellules NK, induite par les anticoφs anti- CD20 et anti-HLA-DR exprimés dans CHO et YB2/0 (324 03 082).Rhesus. Figure 10: Secretion of cytokines by NK induced by anti-Rhesus antibodies Figure 11: Secretion of TNF alpha by NK cells, induced by anti-CD20 and anti-HLA-DR antibodies expressed in CHO and YB2 / 0 (324 03 082). Figure 12: Secretion of gamma IFN by NK cells, induced by anti-CD20 and anti-HLA-DR antibodies expressed in CHO and YB2 / 0 (324 03 082).
Exemple 1 : ADCC induit par des anticorps anti-Rhésus en fonction du nombre de sites antigéniques.Example 1: ADCC induced by anti-Rhesus antibodies as a function of the number of antigenic sites.
La même séquence codant pour une IgGl spécifique de l'antigène Rhésus D est transfectée dans CHO et YB2/0. L'activité cytotoxique des anticoφs est comparée vis à vis des hématies Rhésus positif exprimant à leur surface différentes quantités d'antigène Rhésus, c'est à dire : hématies O+ normales (10-20 000 sites) et hématies sur-exprimant l'antigène rhésus (>60000 sites).The same sequence coding for an IgG1 specific for the Rhesus D antigen is transfected into CHO and YB2 / 0. The cytotoxic activity of the anticoφs is compared with respect to Rhesus positive red cells expressing on their surface different quantities of Rhesus antigen, ie: normal O + red cells (10-20,000 sites) and red cells over-expressing the antigen rhesus (> 60,000 sites).
Les résultats sont présentés à la Figure 1 :The results are presented in Figure 1:
L'activité ADCC des anticoφs exprimés dans CHO (triangle) ou YB2/0 (carré) sur des hématies normales (N, vide) ou sur-exprimant l'antigène Rhésus (GR6, plein) sont comparées.The ADCC activity of the anticoφs expressed in CHO (triangle) or YB2 / 0 (square) on normal red cells (N, empty) or overexpressing the Rhesus antigen (GR6, full) are compared.
La différence d'activité ADCC entre l'anticoφs exprimé dans CHO et l'anticoφs exprimé dans YB2/0 est moindre sur les hématies sur-exprimant l'antigène Rhésus surtout aux fortes quantités d'anticoφs et augmente au fur et à mesure que le nombre de site antigénique décroît. Ainsi, plus la densité antigénique baisse, plus la différence d'activité ADCC entre l'anticoφs produit dans YB2/0 et l'anticoφs produit dans CHO augmente. Exemple 2 : ADCC induit par des anticorps anti-HLA DR en fonction de la quantité d'effecteursThe difference in ADCC activity between the anticoφs expressed in CHO and the anticoφs expressed in YB2 / 0 is less on red cells over-expressing the Rhesus antigen especially at high amounts of anticoφs and increases as the number of antigenic sites decreases. Thus, the more the antigen density decreases, the more the difference in ADCC activity between the anticoφs produced in YB2 / 0 and the anticoφs produced in CHO increases. Example 2 ADCC Induced by Anti-HLA DR Antibodies as a Function of the Amount of Effectors
La même séquence codant pour une IgGl spécifique de l'antigène HLA-DR est transfectée dans CHO et YB2/0. L'activité cytotoxique des anticoφs est comparée vis à vis de la cellule Raji en présence de différents ratios effecteurs/cibles (voir Figure 2).The same sequence coding for an IgG1 specific for the HLA-DR antigen is transfected into CHO and YB2 / 0. The cytotoxic activity of the anticoφs is compared with respect to the Raji cell in the presence of different effector / target ratios (see Figure 2).
La différence d'activité cytotoxique entre l'anticoφs optimisé exprimé par YB2/0 etThe difference in cytotoxic activity between the optimized anticoφs expressed by YB2 / 0 and
CHO s'accroît au fur et à mesure que le ratio E/T diminue. Ainsi, pour les ratios suivants, 20/1 ; 10/1 ; 5/1 ; et 2/1, le pourcentage relatif de lyse induit par l'anticoφs exprimé dans CHO (100% étant la valeur de l'anticoφs exprimé dans YB2/0 pour chaque ratio) est de 61%, 52%, 48% et 36% respectivement.CHO increases as the E / T ratio decreases. Thus, for the following ratios, 20/1; 10/1; 5/1; and 2/1, the relative percentage of lysis induced by the anticoφs expressed in CHO (100% being the value of the anticoφs expressed in YB2 / 0 for each ratio) is 61%, 52%, 48% and 36% respectively.
L'anticoφs exprimé dans YB2/0 s'avère plus cytotoxique que lorsqu'il est produit parThe anticoφs expressed in YB2 / 0 proves to be more cytotoxic than when it is produced by
CHO dans des conditions de faibles quantités d'effecteurs.CHO under conditions of small quantities of effectors.
Exemple 3 : ADCC induit par des anticorps anti-HLA DR en fonction de la quantité d'antigènes accessiblesExample 3: ADCC Induced by Anti-HLA DR Antibodies as a Function of the Quantity of Antigens Accessible
La même séquence codant pour une IgGl spécifique de l'antigène HLA-DR est transfectée dans CHO et YB2/0. L'activité cytotoxique des anticoφs est comparée vis à vis de la cellule Raji en présence de différents ratios effecteurs/cibles (ratio E/T).The same sequence coding for an IgG1 specific for the HLA-DR antigen is transfected into CHO and YB2 / 0. The cytotoxic activity of the anticoφs is compared with respect to the Raji cell in the presence of different effector / target ratios (E / T ratio).
L'activité cytotoxique des anticoφs est comparée vis à vis de cellules Raji dont les sites antigéniques ont été préalablement bloqués avec des quantités croissantes d'un anticoφs murin inactif (non cytotoxique) anti-HLA-DR, de façon à avoir un nombre décroissant d'antigènes HLA-DR disponibles vis à vis des anticoφs à évaluer (voir Figure 3).The cytotoxic activity of the anticoφs is compared with respect to Raji cells whose antigenic sites have been previously blocked with increasing amounts of an inactive (non-cytotoxic) murine antico DRs anti-HLA-DR, so as to have a decreasing number of 'HLA-DR antigens available with regard to the anticoφs to be evaluated (see Figure 3).
Moins il y a de sites antigéniques disponibles, plus la différence d'activité cytotoxique entre l'anticoφs optimisé produit dans YB2/0 et l'anticoφs produit dans CHO augmente. Cela indique qu'une des applications de Panticoφs optimisé peut concerner des cellules cibles exprimant à leur surface un antigène peu exprimé reconnu par l'anticoφs thérapeutique. Ceci procure un net avantage thérapeutique vis à vis d'un anticoφs exprimé dans une cellule de type CHO.The fewer antigenic sites available, the greater the difference in cytotoxic activity between the optimized anticoφs produced in YB2 / 0 and the anticoφs produced in CHO increases. This indicates that one of the applications of optimized Panticoφs can relate to target cells expressing on their surface a poorly expressed antigen recognized by therapeutic anticoφs. This provides a clear therapeutic advantage over an anticoφs expressed in a CHO type cell.
Exemple 4 : Production d'IL2 par Jurkat CD16 induite par des anticorps anti- HLA DR en fonction de la quantité d'antigènes accessiblesExample 4 Production of IL2 by Jurkat CD16 Induced by Anti-HLA DR Antibodies as a Function of the Quantity of Antigens Accessible
La même séquence codant pour une IgGl spécifique de l'antigène HLA-DR est transfectée dans CHO et YB2/0. L'activation de la cellule effectrice (sécrétion d'IL2 par Jurkat CD 16) induite par les anticoφs est comparée vis à vis de cellules Raji dont les sites antigéniques ont été préalablement bloqués avec des quantités croissantes d'un anticoφs murin anti-HLA-DR, de façon à avoir un nombre décroissant d'antigène HLA-DR disponible vis à vis des anticoφs à évaluer (voir Figure 4).The same sequence coding for an IgG1 specific for the HLA-DR antigen is transfected into CHO and YB2 / 0. The activation of the effector cell (secretion of IL2 by Jurkat CD 16) induced by anticoφs is compared with respect to Raji cells whose antigenic sites have been previously blocked with increasing amounts of a murine anti-HLA anticoφs DR, so as to have a decreasing number of HLA-DR antigen available vis-à-vis the anticoφs to be evaluated (see Figure 4).
Ces résultats montrent également que moins il y a de sites antigéniques disponibles, plus la différence d'activation des cellules effectrices entre l'anticoφs optimisé produit par YB2/0 et l'anticoφs produit dans CHO augmente.These results also show that the fewer antigenic sites available, the greater the difference in activation of effector cells between the optimized anticoφs produced by YB2 / 0 and the anticoφs produced in CHO.
Exemple 5 : ADCC induit par des anticorps anti-CD20 en fonction de la quantité d'antigènes.Example 5: ADCC induced by anti-CD20 antibodies as a function of the quantity of antigens.
Les résultats obtenus avec l'anti-CD20 en ADCC confirment ceux obtenus avec les anti-HLADR, c'est à dire que moins il y a de sites antigéniques disponibles et exprimés à la surface des cellules cibles, plus la différence d'activation des cellules effectrices entre l'anticoφs optimisé produit par YB2/0 et l'anticoφs produit dans CHO augmente. Exemple 6 : Production d'IL2 par Jurkat CD16 induite par des anticorps anti- CD20 en fonction de la quantité d'antigènes accessiblesThe results obtained with the anti-CD20 in ADCC confirm those obtained with the anti-HLADR, that is to say that the fewer antigenic sites available and expressed on the surface of the target cells, the greater the difference in activation of the effector cells between the optimized anticoφs produced by YB2 / 0 and the anticoφs produced in CHO increases. Example 6 Production of IL2 by Jurkat CD16 Induced by Anti-CD20 Antibodies as a Function of the Quantity of Antigens Accessible
La même séquence codant pour une IgGl spécifique de l'antigène CD20 est transfectée dans CHO et YB2/0. L'activation de la cellule effectrice (sécrétion d'IL2 par Jurkat CD 16) induite par les anticoφs est comparée vis à vis de cellules Raji dont les sites antigéniques ont été préalablement bloqués avec des quantités croissantes d'un anticoφs murin inactif anti-CD20, de façon à avoir un nombre décroissant d'antigène CD20 disponibles vis à vis des anticoφs à évaluer (voir Figure 5).The same sequence coding for an IgG1 specific for the CD20 antigen is transfected into CHO and YB2 / 0. The activation of the effector cell (secretion of IL2 by Jurkat CD 16) induced by anticoφs is compared with respect to Raji cells whose antigenic sites have been previously blocked with increasing amounts of an inactive murine anticoφs anti-CD20 , so as to have a decreasing number of CD20 antigens available with respect to the anticoφs to be evaluated (see Figure 5).
Moins il y a de sites antigéniques disponibles, plus la différence d'activation des cellules Jurkat CD 16 induite par l'anticoφs optimisé produit par YB2/0 et l'anticoφs produit dans CHO augmente. Cela indique qu'une cellule exprimant une faible densité antigénique peut néanmoins induire l'activation d'une cellule effectrice via un anticoφs optimisé. Cette capacité est beaucoup plus restreinte voire nulle avec un anticoφs exprimé dans CHO.The fewer antigenic sites available, the greater the difference in activation of Jurkat CD 16 cells induced by the optimized anticoφs produced by YB2 / 0 and the anticoφs produced in CHO. This indicates that a cell expressing a low antigenic density can nevertheless induce the activation of an effector cell via an optimized anticoφs. This capacity is much more limited or even zero with an anticoφs expressed in CHO.
Les applications thérapeutiques de Panticoφs optimisé c'est à dire produit dans YB2/0, peuvent ainsi concerner des cellules cibles exprimant à leur surface un antigène peu exprimé.The therapeutic applications of Panticoφs optimized, that is to say produced in YB2 / 0, can thus relate to target cells expressing on their surface a poorly expressed antigen.
En conclusion, les anticoφs optimisés s'avèrent particulièrement utiles pour des applications thérapeutiques lorsque les cellules cibles expriment à leur surface peu d'antigènes, et ceci quel que soit l'antigène.In conclusion, the optimized anticoφs prove to be particularly useful for therapeutic applications when the target cells express on their surface few antigens, and this whatever the antigen.
Exemple 7 : Corrélation in vitro entre ADCC et libération d'IL-2 par la cellule Jurkat CD16.Example 7: Correlation in vitro between ADCC and release of IL-2 by the Jurkat CD16 cell.
Pour cette étude, 3 anticoφs monoclonaux anti-D ont été comparés. L'anticoφs monoclonal (Mab) DF5-EBV a été produit par des Lymphocytes B humain obtenus chez un donneur immunisé D-négatif et immortalisés par transformation avec l'EBV. Cet anticoφs a été utilisé comme contrôle négatif étant donné que lors d'un essai clinique, il s'est montré incapable d'éliminer les globules rouges Rhésus positif de la circulation.For this study, 3 monoclonal anti-D antibodies were compared. The monoclonal antibody (Mab) DF5-EBV was produced by human B Lymphocytes obtained from an immunized D-negative donor and immortalized by transformation with EBV. This antibody was used as a negative control since in a clinical trial it was unable to remove Rhesus positive red blood cells from the circulation.
L'anticoφs monoclonal (Mab) DF5-YB2/0 a été obtenu en exprimant la séquence primaire de DF5-EBV dans la lignée YB2/0. L'anticoφs monoclonal R297 et d'autres anticoφs recombinants ont également été exprimés dans YB2/0.The monoclonal antico Ms (Mab) DF5-YB2 / 0 was obtained by expressing the primary sequence of DF5-EBV in the line YB2 / 0. The monoclonal antibody R297 and other recombinant antibodies were also expressed in YB2 / 0.
Les anticoφs sont testés in vitro pour leur capacité à induire une lyse des globules rouges traités à la papaïne en utilisant des cellules mononucléées (PBL) comme effecteur.Anticoφs are tested in vitro for their ability to induce lysis of red blood cells treated with papain using mononuclear cells (PBL) as an effector.
Tous les tests ont été effectués en présence d'immunoglobulines humaines (IVIg) de sorte à reconstituer les conditions physiologiques.All the tests were carried out in the presence of human immunoglobulins (IVIg) so as to reconstitute the physiological conditions.
On pense que les IVIg se lient avec une haute affinité au FcgammaRI (CD64). Les deux Mab DF5-YB2/0 et R297 induisent une lyse des globules rouges à un niveau comparable à celui des anticoφs polyclonaux WinRho. En revanche, le Mab DF5-IVIg is thought to bind with high affinity to FcgammaRI (CD64). The two Mab DF5-YB2 / 0 and R297 induce lysis of red blood cells at a level comparable to that of WinRho polyclonal antibodies. In contrast, the Mab DF5-
EBV est complètement inefficace.EBV is completely ineffective.
Dans une deuxième série d'expérience, des cellules NK purifiées et des globules rouges non traités ont été utilisés comme effecteurs et cibles respectivement. Après 5 heures d'incubation, les Mabs antiD-R297 et DF5-YB2/0 se sont montrés capables de provoquer la lyse des globules rouges, alors que DF5-EBV reste inefficace. Dans ces deux expériences, la lyse des globules rouges a été inhibée par le Mab 3G8 dirigé contre le FcgammaRIII (CD 16).In a second series of experiments, purified NK cells and untreated red blood cells were used as effectors and targets respectively. After 5 hours of incubation, the antiD-R297 and DF5-YB2 / 0 Mabs have been shown to be able to cause lysis of red blood cells, while DF5-EBV remains ineffective. In these two experiments, the lysis of red blood cells was inhibited by Mab 3G8 directed against FcgammaRIII (CD 16).
En résumé, ces résultats démontrent que l'ADCC provoquée par le Mab R297 et le Mab DF5-YB2/0 implique le FcgammaRIII exprimé à la surface des cellules NK.In summary, these results demonstrate that the ADCC caused by Mab R297 and Mab DF5-YB2 / 0 involves FcgammaRIII expressed on the surface of NK cells.
Dans le cadre de l'invention, une troisième série d'expériences a été réalisée en utilisant un test in vitro à l'aide des cellules Jurkat CD 16 pour évaluer l'efficacité d'anticoφs anti-D. Les Mab ont été incubés pendant la nuit avec des globules rouges Rhésus positif et des cellules Jurkat CD 16. La libération d'IL-2 dans les surnageants a été évaluée par ELIS A.In the context of the invention, a third series of experiments was carried out using an in vitro test using Jurkat CD 16 cells to evaluate the effectiveness of anti-D antibodies. Mab were incubated overnight with red blood cells Rhesus positive and Jurkat CD 16 cells. The release of IL-2 in the supernatants was evaluated by ELIS A.
Une forte corrélation entre l'ADCC et l'activation des cellules Jurkat (production d'I12) a été observée, ce qui implique que ce test peut être utilisé pour faire la discrimination des Mabs anti-D en fonction de leur réactivité envers FcgammaRIII ( CD16).A strong correlation between ADCC and activation of Jurkat cells (production of I12) has been observed, which implies that this test can be used to discriminate anti-D Mabs according to their reactivity towards FcgammaRIII ( CD16).
Les mêmes échantillons sont évalués en ADCC et dans le test Jurkat IL2. Les résultats sont exprimés en pourcentage par rapport à l'anticoφs de référence "anti-D R297". La courbe de corrélation entre les 2 techniques a un coefficient r2=0.9658 (figure 6).The same samples are evaluated in ADCC and in the Jurkat IL2 test. The results are expressed as a percentage relative to the reference anticoφs "anti-D R297". The correlation curve between the two techniques has a coefficient r2 = 0.9658 (Figure 6).
En conclusion, ces données montrent l'importance des modifications post- traductionnelles de la structure des anticoφs et leur impact sur l'activité ADCC spécifique du FcgammaRIII (CD 16). La libération de cytokines telles que IL-2 par les cellules Jurkat CD 16 reflète cette activité.In conclusion, these data show the importance of post-translational modifications of the structure of the antibodies and their impact on the specific ADCC activity of FcgammaRIII (CD 16). The release of cytokines such as IL-2 from Jurkat CD 16 cells reflects this activity.
Exemple 8 : activation de cellules NK et production d'IL2 et d'IFNγEXAMPLE 8 Activation of NK Cells and Production of IL2 and IFNγ
Modèle de mise au point : lignée cellulaire Jurkat transfectée avec le gène codant pour le récepteur CD16. Applications : renforcement d'une réponse anti-tumorale. L'IL2, produite par les cellules effectrices activées par des immuns complexes antigène- anticoφs, induit une activation des lymphocytes T et des cellules NK pouvant aller jusqu'à une stimulation de la prolifération cellulaire. L'IFNγ stimule l'activité desDevelopment model: Jurkat cell line transfected with the gene coding for the CD16 receptor. Applications: strengthening of an anti-tumor response. IL2, produced by effector cells activated by complex antigen-anticoφs immune cells, induces activation of T lymphocytes and NK cells, which can go as far as stimulating cell proliferation. IFNγ stimulates the activity of
CTLs et peut renforcer l'activité des macrophages.CTLs and can enhance the activity of macrophages.
Exemple 9 : activation de monocytes macrophages et production de TNF et d'IL- lRaExample 9 Activation of Macrophage Monocytes and Production of TNF and IL-1Ra
Applications : renforcement de la phagocytose et induction de propriétés anti- inflammatoires. Le TNF, produit par les cellules effectrices activées par des immuns complexes antigène-anticoφs, stimule la prolifération des macrophages et des lymphocytes infiltrant les tumeurs. LTL-IRa est une cytokine qui entre en compétition avec l'ILl au niveau de son récepteur et exerce ainsi un effet anti-inflammatoire.Applications: strengthening of phagocytosis and induction of anti-inflammatory properties. TNF, produced by effector cells activated by immune systems antigen-anticoφs complexes, stimulates the proliferation of macrophages and lymphocytes infiltrating tumors. LTL-IRa is a cytokine that competes with IL1 at its receptor and thus exerts an anti-inflammatory effect.
Exemple 10 : activation de cellules dendritiques et production d'ILlOEXAMPLE 10 Activation of Dendritic Cells and Production of IL10
Applications : induction d'une tolérance spécifique à certains antigènes. LTL10 est une molécule inhibitrice de l'activation de différentes cellules effectrices et de la production de cytokines. Ainsi l'ILl 0 produite par les cellules effectrices activées par des immuns complexes antigène-anticoφs peut avoir un rôle régulateur de l'activité cytotoxique des anticoφs vis à vis de cellules normales, mais exprimant des antigènes communs avec les cellules cibles visées, et également moduler les effets du TNF alpha.Applications: induction of specific tolerance to certain antigens. LTL10 is a molecule that inhibits the activation of different effector cells and the production of cytokines. Thus the IL10 produced by the effector cells activated by antigen-anticoφs complex immune systems can have a regulatory role in the cytotoxic activity of the anticoφs with respect to normal cells, but expressing antigens common with the target cells targeted, and also modulate the effects of TNF alpha.
Exemple 11 : Induction de la sécrétion de cytokines par différentes cellules effectrices.Example 11: Induction of the secretion of cytokines by different effector cells.
Trois populations cellulaires ont été étudiées : les polynucléaires, les cellules mononuclées et les cellules NK. L'induction de la synthèse de cytokines par des anticoφs est dépendante de la présence de la cible. Il y a peu de différences dans la capacité de l'anticoφs anti-D R297 et de l'anticoφs polyclonal à induire la production de différentes cytokines. Par contre, AD1 est très souvent non inducteur de sécrétion de cytokines.Three cell populations were studied: polymorphonuclear cells, mononuclear cells and NK cells. The induction of cytokine synthesis by antico ants is dependent on the presence of the target. There are few differences in the ability of anti-D R297 and polyclonal antibodies to induce the production of different cytokines. On the other hand, AD1 is very often not inducing secretion of cytokines.
Résultats :Results:
11.1 L'anticoφs monoclonal R297 et Panticoφs polyclonal WinRho induisent une sécrétion importante d'IL8 en présence de cellules mononucléées. Cette sécrétion est dépendante de la concentration d'anticoφs et de la présence de la cible antigénique, c'est à dire des hématies Rh positif. L'anticoφs AD1 est beaucoup moins apte à induire la production d'IL8 (figure 7). En présence de cellules mononucléées et d'hématies Rhésus positif, l'anticoφs monoclonal R297 et l'anticoφs polyclonal anti-D WinRho induisent une sécrétion importante de TNF alpha, moins forte bien que supérieure à celles induites par AD1 d'IL6, d'IFN gamma, d'IPlO, de TNF alpha et TGF Beta. En présence de plus forte concentration d'anticoφs, la sécrétion dIL6, d'IFN gamma, d'IPlO augmente, mais celle de TNF alpha et le TGF Beta décroît (figure 8).11.1 The monoclonal anticoφs R297 and Panticoφs polyclonal WinRho induce an important secretion of IL8 in the presence of mononuclear cells. This secretion is dependent on the concentration of anticoφs and the presence of the antigenic target, that is to say Rh positive red cells. Anticoφs AD1 is much less able to induce the production of IL8 (Figure 7). In the presence of mononuclear cells and Rhesus positive red cells, the monoclonal antibody R297 and the anti-D polyclonal antibody anti-D WinRho induce an important secretion of TNF alpha, less strong but greater than those induced by AD1 of IL6, of IFN gamma, IP10, TNF alpha and TGF Beta. In the presence of a higher concentration of anticoφs, the secretion dIL6, of IFN gamma, of IP10 increases, but that of TNF alpha and the TGF Beta decreases (FIG. 8).
11.2 L'anticoφs monoclonal R297 et l'anticoφs polyclonal anti-D WinRho induisent une sécrétion très faible, mais supérieure à AD1, d'IL2, d'IFN gamma, d'IPlO et de TNF par les polynucléaires. Cette sécrétion est dépendante de la concentration d'anticoφs (figure 9).11.2 The monoclonal anticoφs R297 and the polyclonal anti-D anti-D WinRho induce a very weak secretion, but superior to AD1, of IL2, of IFN gamma, of IPlO and of TNF by the polymorphonuclear cells. This secretion is dependent on the concentration of anticoφs (Figure 9).
11.3 L'anticoφs monoclonal R297 et l'anticoφs polyclonal anti-D WinRho induisent une sécrétion importante d'IFN gamma, d'IPlO et de TNF par les cellules NK. Cette sécrétion est dépendante de la concentration d'anticoφs (figure 10).11.3 The monoclonal antibody R297 and the anti-D polyclonal antibody anti-D WinRho induce significant secretion of IFN gamma, IP10 and TNF by the NK cells. This secretion is dependent on the concentration of anticoφs (Figure 10).
Exemple 11 : Anticorps anti-CD 20 et anti-HLA DR chimériques optimisés produits dans YB2/0Example 11: Optimized chimeric anti-CD 20 and anti-HLA DR antibodies produced in YB2 / 0
IntroductionIntroduction
Nos premiers résultats ont montré que les anticoφs anti-D produits dans YB2/0 ainsi que les anticoφs polyclonaux utilisés en clinique induisaient la production de cytokines, en particulier de TNF alpha et d'interféron gamma (IFN gamma) à partir de cellules NK purifiées ou de cellules mononucléées. Par contre d'autres anticoφs anti- D, produits dans d'autres lignées cellulaires sont négatifs en ADCC et se sont révélés incapables d'induire une sécrétion de cytokines.Our first results showed that the anti-D antibodies produced in YB2 / 0 as well as the polyclonal antico cliniques used in the clinic induced the production of cytokines, in particular TNF alpha and gamma interferon (IFN gamma) from purified NK cells. or mononuclear cells. In contrast, other anti-D antibodies produced in other cell lines are ADCC negative and have been shown to be incapable of inducing cytokine secretion.
Les résultats complémentaires ci dessous montrent que ce mécanisme n'est pas exclusif aux anti-D en présence d'hématies Rhésus positif mais s'applique également aux anticoφs anti-CD20 et anti-HLA DR exprimés dans YB2/0. L'expression dans CHO confère à l'anticorps des propriétés activatrices moins importantes. Ceci est en corrélation avec les résultats obtenus en ADCC.The additional results below show that this mechanism is not exclusive to anti-D in the presence of Rhesus positive red cells but also applies to anti-CD20 and anti-HLA DR anticoφs expressed in YB2 / 0. The expression in CHO gives the antibody less important activating properties. This is correlated with the results obtained in ADCC.
Matériel Anticorps.Antibody material.
Anti-CD20 : l'anticoφs chimérique anti-CD20 transfecté dans YB2/0 est comparé à un anticoφs commercial anti-CD20 produit dans CHO (Rituxan).Anti-CD20: the chimeric anti-CD20 anticoφs transfected in YB2 / 0 is compared to a commercial anti-CD20 antico (s produced in CHO (Rituxan).
Anti-HLA DR : la même séquence codant pour l'anticoφs chimérique anti-HLA DR est transfectée dans CHO (Bl 1) ou YB2/0 (4B7). Cellules cibles : cellules Raji exprimant à leur surface l'antigène CD20 et HLA-DRAnti-HLA DR: the same sequence encoding the chimeric anti-HLA DR antibody is transfected into CHO (Bl 1) or YB2 / 0 (4B7). Target cells: Raji cells expressing on their surface the CD20 antigen and HLA-DR
Cellule effectrices : cellules NK humaines purifiées par sélection négative à partir de poche de sang humain.Effector cells: human NK cells purified by negative selection from a human blood bag.
Méthode Différentes concentrations d'anticoφs anti-CD20 ou anti-HLA DR sont incubées avec les cellules Raji et les cellules NK. Après 16 heures d'incubation, les cellules sont centrifugées. Les surnageants sont dosés en TNF alpha et en IFN gamma.Method Different concentrations of anti-CD20 or anti-HLA DR antibodies are incubated with Raji cells and NK cells. After 16 hours of incubation, the cells are centrifuged. The supernatants are assayed in TNF alpha and in IFN gamma.
Résultats :Results:
1) TNF alpha : Les résultats sont exprimés en pg/ml de TNF alpha dosé dans les surnageants. En abscisse figurent les différentes concentrations d'anticoφs ajoutées dans le mélange réactionnel (figure 11).1) TNF alpha: The results are expressed in pg / ml of TNF alpha assayed in the supernatants. On the abscissa are the different concentrations of anticoφs added to the reaction mixture (Figure 11).
Les anticoφs anti-CD 20 et anti-HLA DR chimériques produits dans YB2/0 induisent des taux plus importants de TNF en présence de leur cible (Raji) par rapport aux mêmes anticoφs produits dans CHO. La quantité de TNF alpha est bien dose dépendante de la concentration d'anticoφs ajouté. A lOng/ml d'anticoφs on induit 5 fois plus de TNF alpha avec les anticoφs produits dans YB2/0 par rapport aux anticoφs produits dans CHO. 2) IFN gamma: Les résultats sont exprimés en pg/ml d'IFN gamma dosé dans les surnageants. En abscisse figurent les différentes concentrations d'anticoφs ajoutées dans le mélange réactionnel (figure 12).The chimeric anti-CD 20 and anti-HLA DR anticoφs produced in YB2 / 0 induce higher levels of TNF in the presence of their target (Raji) compared to the same anticoφs produced in CHO. The amount of TNF alpha is dose dependent on the concentration of added anticoφs. At 10 ng / ml of anticoφs, 5 times more TNF alpha is induced with the anticoφs produced in YB2 / 0 compared to the anticoφs produced in CHO. 2) IFN gamma: The results are expressed in pg / ml of IFN gamma assayed in the supernatants. On the abscissa are the different concentrations of anticoφs added to the reaction mixture (Figure 12).
Les anticoφs anti-CD 20 et anti-HLA DR chimériques produits dans YB2/0 induisent des taux plus importants d' IFN gamma en présence de leur cible (Raji) par rapport aux mêmes anticoφs produits dans CHO. La quantité d'IFN gamma est bien dose dépendante de la concentration d'anticoφs ajouté. A toutes les concentrations utilisées (10 à 200ng/ml) l'anticoφs anti-HLA DR produit dans CHO n'induit pas de sécrétion d'IFN gamma, alors que 40ng/ml de l'anticoφs produit dans YB2/0 induit environ lOOOpg/ml d'IFN gamma.The chimeric anti-CD 20 and anti-HLA DR antibodies produced in YB2 / 0 induce higher levels of gamma IFN in the presence of their target (Raji) compared to the same antibodies produced in CHO. The amount of IFN gamma is dose dependent on the concentration of added anticoφs. At all the concentrations used (10 to 200 ng / ml) the anti-HLA DR anticoφs produced in CHO does not induce IFN gamma secretion, while 40 ng / ml of the anticoφs produced in YB2 / 0 induces approximately lOOOpg / ml of IFN gamma.
Pour l'anticoφs anti-CD20, il faut pour induire 300pg/ml d'IFN gamma moins de lOng/ml de l'anticoφs produit dans YB2/0 et 200ng/ml de l'anticoφs produit dans CHO (figure 12). For the anti-CD20 anticoφs, it is necessary to induce 300pg / ml of gamma IFN less than 10 ng / ml of the anticoφs produced in YB2 / 0 and 200 ng / ml of the anticoφs produced in CHO (FIG. 12).

Claims

REVENDICATIONS
1. Utilisation d'un anticoφs monoclonal chimérique, humanisé ou humain optimisé caractérisé en ce que : a) il est produit dans une lignée, cellulaire sélectionnée pour ses propriétés de glycosylation du fragment Fc d'un anticoφs, ou b) la structure glycannique du Fcgamma a été modifiée ex vivo, et/ou c) sa séquence primaire a été modifiée de façon à augmenter sa réactivité vis à vis des récepteurs Fc ; ledit anticoφs présentant i) un taux d'ADCC dépendant du FcγRIII (CD 16) supérieur à1. Use of an optimized chimeric, humanized or human monoclonal anticoφs characterized in that: a) it is produced in a cell line selected for its glycosylation properties of the Fc fragment of an anticoφs, or b) the glycan structure of the Fcgamma has been modified ex vivo, and / or c) its primary sequence has been modified so as to increase its reactivity towards Fc receptors; said anticoφs having i) a rate of ADCC dependent on FcγRIII (CD 16) greater than
50 %, de préférence supérieur à 100 % pour un ratio E/T (cellules effectrices/cellules cibles) inférieur à 5/1, de préférence inférieur à 2/1, comparé au même anticoφs produit dans une lignée CHO ; et ii) un taux de production d'au moins une cytokine par une cellule effectrice de type Jurkat CD 16 ou issue du système immunitaire exprimant le récepteur CD 16 supérieur à 50 %, 100 % ou de préférence supérieur à 200 % comparé au même anticoφs produit dans une lignée CHO; pour la préparation d'un médicament destiné au traitement de pathologies pour lesquelles le nombre de sites antigéniques, la densité antigénique sont faibles ou les antigènes sont peu accessibles aux anticoφs, ou encore pour lesquelles le nombre de cellules effectrices activées ou recrutées est limité.50%, preferably greater than 100% for an E / T ratio (effector cells / target cells) less than 5/1, preferably less than 2/1, compared to the same anticoφs produced in a CHO line; and ii) a production rate of at least one cytokine by an effector cell of the Jurkat CD 16 type or from the immune system expressing the CD 16 receptor greater than 50%, 100% or preferably greater than 200% compared to the same anticoφs produced in a CHO line; for the preparation of a medicament intended for the treatment of pathologies for which the number of antigenic sites, the antigenic density are low or the antigens are hardly accessible to the anticoφs, or for which the number of effector cells activated or recruited is limited.
2. Utilisation selon la revendication 1 caractérisée en ce que le nombre de sites antigéniques est inférieur à 250 000, de préférence inférieur à 100 000 ou 50 000 par cellule cible.2. Use according to claim 1 characterized in that the number of antigenic sites is less than 250,000, preferably less than 100,000 or 50,000 per target cell.
3. Utilisation selon l'une des revendications 1 et 2, caractérisée en ce que lesdites cytokines libérées par les anticoφs optimisés sont choisies parmi des interleukines, des interférons et des facteurs de nécrose tissulaire (TNF). 3. Use according to one of claims 1 and 2, characterized in that said cytokines released by the optimized anticoφs are chosen from interleukins, interferons and tissue necrosis factors (TNF).
4. Utilisation selon l'une des revendications 1 et 2, caractérisée en ce que l'anticoφs optimisé induit la sécrétion d'au moins une cytokine choisie parmi IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,... TNFa, TGFβ, IP10 et IFNγ par les cellules effectrices du système immunitaire en particulier celles exprimant le récepteur CD 16.4. Use according to one of claims 1 and 2, characterized in that the optimized anticoφs induces the secretion of at least one cytokine chosen from IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, ... TNFa, TGFβ, IP10 and IFNγ by the effector cells of the immune system, in particular those expressing the CD 16 receptor.
5. Utilisation selon l'une des revendications 1 et 2, caractérisée en ce que l'anticoφs induit la sécrétion d'IL-2 par la cellule Jurkat CD 16 ou d' IFNγ et d'IL2 par les cellules effectrices du système immunitaire exprimant le récepteur CD 16 pour un faible nombre de sites antigéniques présents à la surface des cellules cibles ou pour un faible nombre d'antigènes accessibles aux anticoφs ou pour un faible nombre de cellules effectrices.5. Use according to one of claims 1 and 2, characterized in that the anticoφs induces the secretion of IL-2 by the Jurkat CD 16 cell or of IFNγ and IL2 by the effector cells of the immune system expressing the CD 16 receptor for a small number of antigenic sites present on the surface of the target cells or for a low number of antigens accessible to the antibodies or for a low number of effector cells.
6. Utilisation selon l'une des revendications 1 à 5, caractérisée en ce que la cellule effectrice est une cellule leucocytaire, en particulier de la famille des NK (natural killer) ou une cellule du groupe monocytes-macrophages.6. Use according to one of claims 1 to 5, characterized in that the effector cell is a leukocyte cell, in particular of the family of NK (natural killer) or a cell of the monocyte-macrophage group.
7. Utilisation selon l'une des revendications 1 à 5, caractérisée en ce que la cellule effectrice est une cellule Jurkat transfectée avec un vecteur d'expression codant pour le récepteur CD 16.7. Use according to one of claims 1 to 5, characterized in that the effector cell is a Jurkat cell transfected with an expression vector coding for the CD 16 receptor.
8. Utilisation selon l'une des revendications 1 à 5, caractérisée en ce que l'anticoφs optimisé est préparé après avoir été purifié et/ou modifié ex vivo par modification de la structure glycannique du f agment Fc.8. Use according to one of claims 1 to 5, characterized in that the optimized anticoφs is prepared after having been purified and / or modified ex vivo by modification of the glycan structure of the agent Fc.
9. Utilisation selon l'une des revendications 1 à 5, caractérisée en ce que Panticoφs optimisé est produit par des cellules de lignées de myélomes de rat, en particulier YB2/0 et ses dérivés. 9. Use according to one of claims 1 to 5, characterized in that Panticoφs optimized is produced by cells of rat myeloma lines, in particular YB2 / 0 and its derivatives.
10. Utilisation selon l'une des revendications 1 à 10, caractérisée en ce que l'anticoφs optimisé présente une structure glycannique générale de type biantenné, avec des chaînes courtes, une faible sialylation, des mannoses et GlcNAc du point d'attache terminaux non intercalaires, et une faible fucosylation.10. Use according to one of claims 1 to 10, characterized in that the optimized anticoφs has a general glycan structure of biantenné type, with short chains, low sialylation, mannoses and GlcNAc of the non-terminal attachment point inserts, and low fucosylation.
11. Utilisation selon la revendication 10, caractérisée en ce que Panticoφs optimisé présente un taux de GlcNac intermédiaire non nul.11. Use according to claim 10, characterized in that Panticoφs optimized has a non-zero intermediate GlcNac level.
12. Utilisation d'un anticoφs selon l'une des revendications 1 à 11 pour la préparation d'un médicament destiné au traitement d'une pathologie choisie parmi la maladie hémolytique du nouveau-né, le Syndrome de Sézary, les leucémies myéloides chroniques, les cancers dont les cibles antigéniques sont faiblement exprimées, notamment le cancer du sein, les pathologies liées à l'environnement visant notamment les personnes exposées aux biphényles polychlorinés, les maladies infectieuses, notamment la tuberculose, le syndrome de la fatigue chronique (CFS), les infections parasitaires comme par exemple les schistosomules. 12. Use of an anticoφs according to one of claims 1 to 11 for the preparation of a medicament intended for the treatment of a pathology chosen from hemolytic disease of the newborn, Sézary Syndrome, chronic myeloid leukemias, cancers whose antigenic targets are weakly expressed, in particular breast cancer, pathologies linked to the environment targeting in particular people exposed to polychlorinated biphenyls, infectious diseases, in particular tuberculosis, chronic fatigue syndrome (CFS), parasitic infections such as schistosomules.
EP03775438A 2002-09-13 2003-09-15 Treatment of pathologies which escape the immune response, using optimised antibodies Ceased EP1545614A2 (en)

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FR0211415 2002-09-13
FR0211416A FR2844521B1 (en) 2002-09-13 2002-09-13 MEASUREMENT OF THE PRODUCTION OF CYTOKINES AS A MARKER FOR ACTIVATION OF EFFECTOR CELLS
FR0211415A FR2844520B1 (en) 2002-09-13 2002-09-13 USE OF ANTIBODY INDUCING THE SECRETION OF CYTOKINES IN THERAPY
FR0211416 2002-09-13
FR0307066A FR2844455B1 (en) 2002-09-13 2003-06-12 TREATMENT OF PATHOLOGIES EXCLUDING IMMUNE RESPONSE BY OPTIMIZED ANTIBODIES
FR0307066 2003-06-12
PCT/FR2003/002714 WO2004028564A2 (en) 2002-09-13 2003-09-15 Treatment of pathologies which escape the immune response, using optimised antibodies

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IL230101A (en) 2016-05-31
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CA2498383C (en) 2015-04-28
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JP6238743B2 (en) 2017-11-29
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AU2003283469C1 (en) 2010-05-20
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US20050271652A1 (en) 2005-12-08

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