EP1969368A2 - Potentiation of apoptosis by monoclonal antibodies - Google Patents
Potentiation of apoptosis by monoclonal antibodiesInfo
- Publication number
- EP1969368A2 EP1969368A2 EP06841970A EP06841970A EP1969368A2 EP 1969368 A2 EP1969368 A2 EP 1969368A2 EP 06841970 A EP06841970 A EP 06841970A EP 06841970 A EP06841970 A EP 06841970A EP 1969368 A2 EP1969368 A2 EP 1969368A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- apoptosis
- antibody
- antibodies
- cell
- 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
Links
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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
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- 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
- A61P31/18—Antivirals for RNA viruses for HIV
-
- 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
-
- 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
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- 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
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- 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
- C07K2317/71—Decreased effector function due to an Fc-modification
Definitions
- the present invention relates to the use of an antibody composition having a fucose content of less than 65% to induce apoptosis in vitro.
- antibodies are also tools of choice in diagnosis and therapy, where they are an alternative to conventional treatments.
- NK cell deficits also occur in patients with lupus erythematosus (Green 2005), patients with congestive cardiac myopathy (Anderson 1982), patients with histiocytosis with hemophagocytosis (perforin deficiency) (Clementi R, 2005) and HIV-infected patients (perforin and granzyme deficiency)
- the Applicant has therefore sought to provide tools whose activity is independent of the cytotoxic functions of immune effector cells, and thus may be administered to patients with a decrease in the activity of immune effector cells.
- the Applicant has shown, in WO 01/77181, the importance of selecting cell lines for producing antibodies having high ADCC activity via the FcgammaRIII receptor (CD16). It was then shown that modification of the Fc region glycosylation of antibodies produced in rat myeloma lines such as YB2 / 0 led to improved ADCC activity.
- the glycan structures of such antibody compositions impart low fucosylation to the antibody composition.
- the invention thus relates to the use of an antibody composition, the fucose content of which is less than 65%, for inducing, in vitro, ex vivo or in vivo, apoptosis.
- a first subject of the invention relates in particular to a method for inducing, in vitro, ex vivo or in vivo, the apoptosis of a target cell by an antibody composition, comprising contacting the composition of antibodies and the target cell, in the presence of cells expressing CD16 on their surface, the antibodies being directed against the target cell and the antibody composition having a fucose content of less than 65%.
- the antibodies consist of heavy chains and light chains, linked together by disulfide bridges.
- Each chain is constituted, in the N-terminal position, of a variable region (or domain) specific for the antigen against which the antibody is directed, and in the C-terminal position, of a constant region, consisting of a single CL domain for light chains and several domains (CHi, CH 2 and CH 3 ) for heavy chains.
- the combination of the variable domains and the CH1 and CL domains of the heavy and light chains forms the Fab portions of the antibody, which are connected to the Fc region by a very flexible hinge region, allowing each Fab to bind to the target antigen.
- the Fc region which mediates the effector properties of the antibody, remains accessible to effector molecules such as Fc ⁇ R receptors (FcgammaR).
- FcgammaR Fc ⁇ R receptors
- the Fc region consisting of 2 CH 2 globular domains and CH 3 , is glycosylated at the CH 2 domain with the presence, on each of the 2 chains, of a biantennal N-glycan linked to asparagine 297 (Asn 297).
- Such an N-glycan is in the following general form (presented form "GO"), to which other sugars can be added:
- fucose is understood to mean the fucose borne by these N-oligosaccharides.
- the fucose molecule when present, is linked to the N-acetylglucosamine (GIcNAc) of the N-oligosaccharide, this GIcNAc itself being bound to the Asn 297.
- GIcNAc N-acetylglucosamine
- each of the N-glycans carried by either of the 2 heavy chains of each antibody may carry a fucose molecule or not carry one.
- each antibody may comprise 0, 1 or 2 fucose molecules, respectively, according to that none of its N-glycans carry fucose, that only one of its N-glycans carries a fucose molecule, or that its 2 N glycans each carry a molecule of fucose.
- “antibody composition whose fucose content is less than 65%” is understood to mean an antibody composition of which, among the totality of the glycan structures carried by each glycosylation site (Asn 297), antibodies of the composition less than 65% contain a molecule of fucose.
- such glycan structures are more particularly selected from the following forms:
- the fucose content is less than 65%.
- ADCC Antibody-Dependent Cell-mediated Cytotoxicity
- the antibody compositions according to the invention have the particularity and advantage of triggering a strong apoptosis reaction.
- Apoptosis plays an essential role in triggering cell death. Many factors intervene to induce apoptosis, but all lead to a common pathway through mitochondria, Bcl-2 protein, and caspases.
- the main mechanisms initiating the process of programmed cell death are stress (for example by hypo-oxygenation), treatment with cytotoxic substances or corticosteroids, deprivation of growth factors, damage to DNA, and transmission of a death signal (Fas receptor of cytotoxic lymphocytes and natural killers, TNF- ⁇ necrosis factor). Following the cell death signal, the caspases are activated, causing the activation of proteins associated with apoptosis.
- Phosphatidylserines are then translocated from the inner side of the cell membrane to the outer surface. They can be visualized "in vitro" by the attachment of annexin V to the. surface of the cell. Condensation of the cytoplasm, nucleus and chromatin, fragmentation of the DNA, budding of the plasma membrane and loss of membrane asymmetry are then visible. At this stage, the cells fix "in vitro" propidium iodide
- the potentiation of the apoptosis induced by the antibody compositions according to the invention is not due to the cytotoxic functions of the effector immune cell, but is induced by a strong aggregation of the Fc region of the antibodies with the CD16 receptor expressed at the same time. the surface of the effector immune cells.
- the antibody composition according to the invention comprises a content greater than 60%, preferably greater than 80%, of forms G0 + G1 + G0F + G1F, it being understood that the content of G0F + G1F forms is lower than at 50%, preferably less than 30%.
- the "in vitro" demonstration of the induction of apoptosis by the antibody compositions according to the invention was carried out with Jurkat cells CD16 transfected with no cytolytic activity, as opposed to NK cells. This is a model in which cell death can only be induced by apoptosis.
- the method of the invention intended to induce in vitro the apoptosis of a target cell, is carried out by means of cells expressing CD16 on their surface, but which are not effector cells. These cells therefore have no or more cytotoxic activity against the target cells.
- the potentiation of apoptosis by the compositions according to the invention is established with all effector cells expressing CD16, and in particular peripheral blood cells expressing CD16, such as monocytes-macrophages, neutrophils, NK cells and certain subpopulations of T cells.
- the compositions according to the invention can be used in the presence of a quantitative or qualitative deficit (absence of ADCC) in a population of effector immune cells (cytotoxic cells). Indeed, in this case, this potentiation is possible thanks to the other cells expressing CD16 on their surface (CD16 + cells), in particular cells expressing CD16 on their surface, but which have no or more cytotoxic activity.
- the antibody composition of the invention has a fucose content of less than 55%, or even less than 40%, 30% or 20%.
- the fucose content of the antibody composition is between 20% and 45%, or between 25% and
- the antibody composition according to the invention also has a fucose / galactose ratio ratio of less than 0.6.
- this ratio is less than 0.5; less than 0.4; less than 0.2 or even more advantageously less than 0.1.
- the term "fucose ratio / galactose content” means the ratio between the fucose content of the antibody composition and the galactose content of the antibody composition, these two sugars being susceptible to to be carried by each glycan structure bound to each glycosylation site. (Asn 297) antibodies of the composition.
- the antibody composition used according to the invention is produced by the YB2 / 0 cell line, or any cell derived from YB2 / 0 and conferring the same characteristics in terms of post-translational modifications of the proteins, in particular on the plan of glycosylation.
- This line was chosen because of its ability to produce antibody compositions some of which, after selection, exhibit low fucosylation, for which the Applicant has shown in the present invention that it is useful in the production of antibody compositions capable of strongly inducing apoptosis.
- Another object of the invention is the use of the YB2 / 0 cell line to produce antibodies with a high apoptosis capacity.
- Another subject of the invention is a method for selecting antibody compositions with a high apoptotic capacity, comprising the following steps:
- a negative control can be obtained for example in the presence of cells not expressing CD16.
- the measurement of the induction of apoptosis can be carried out in different ways, by using specific conventional techniques for measuring apoptosis, for example the measurement of annexin V (early stage). apoptosis measuring phosphatityserines exposed to the cell surface) associated with propidium iodide (advanced stage of apoptosis measuring the degradation of DNA) or other markers such as YO Pro-1 (intercalating agent of DNA measuring the fragmentation of the latter).
- the term "high apoptotic capacity" means an ability to induce apoptosis of at least 20% or even 50% or more, in the presence of the cell expressing the CD16 compared to the negative control.
- the cells expressing CD16 on their surface may be CD16 transfected cells, in particular Jurkat cells, peripheral blood cells expressing CD16, for example monocytes-macrophages, neutrophils, NK cells. and certain sub-populations of T cells, this list not being limiting.
- cells expressing CD16 on their surface are cells that have no or more cytotoxic activity.
- the method of the invention further comprises a step of determining the fucose content of the selected antibody compositions. This determination can take place before the step of contacting the antibody to be evaluated with the antigen, or after the selection of the antibody compositions showing an induction of apoptosis greater than that of the negative control.
- the determination of the fucose content of the antibody composition is carried out before the step of contacting the antibody to be evaluated with the antigen, it is advantageous to select the antibody compositions having a fucose content. less than 65%.
- the antibodies selected are anti-idiotypic anti-factor VIII inhibitor antibodies, antibodies directed against autoantibodies to target memory B cells,
- any antibody can be selected recognizing an antigen preferentially expressed on the surface of tumor cells, including hematopoietic proliferations.
- Another subject of the invention relates to the use of monoclonal antibody compositions whose fucose content is less than 65%, preferably less than 30%, particularly advantageously between 20% and 45%, or between 25% and 40%, or of antibody compositions selected by the selection method described above, for the preparation of a medicament for treatment, without the cytotoxic functions of the immune effector cells being involved, of a quantitative or qualitative deficiency (absence of ADCC) in NK cells, chosen in particular from CLB-B (chronic B cell lymphocytic leukemia), lupus erythematosus, congestive cardiac myopathies, histiocytosis with haemophagocytosis, and HIV and AIDS. hemophilia A or B.
- Said antibody compositions exhibit an interaction for CD16 at least 2-fold higher, or even advantageously 10-fold greater than the therapeutic antibody Rituxan.
- the interaction between the Fc region of the antibody and the CD16 receptor can be measured by direct or indirect binding by competition with a monoclonal anti-CDl ⁇ antibody type 3G8 directed against the binding site of the Fc part of the antibodies, on CD16 positive cells.
- the antibody composition used according to the invention comprises a content greater than 60%, preferably greater than 80%, of forms G0 + G1 + G0F + G1F, it being understood that the content of G0F + G1F forms is less than 50%, preferably less than 30%.
- the antibody composition used according to the invention also has a fucose / galactose ratio ratio of less than 0.6.
- this ratio is less than 0.5; less than 0.4; less than 0.2 or even more advantageously less than 0.1.
- this use is intended for the preparation of a medicament for the treatment of pathologies in which one or more population (s) of effector cells is decreased or even zero, said cells thus inducing low cytotoxic activity by ADCC.
- Another subject of the invention relates to the use of anti-CD20 monoclonal antibodies having a strong interaction for CD1 ⁇ , selected according to the process of the invention, or answering the fucosylation characteristics mentioned above, for the manufacture of a drug for treatment, without the cytotoxic functions of effector immune cells, patients with B-CLL and in which a decrease in the lytic functions of NK cells may be observed.
- the antibody of the invention may be used to treat any hematopoietic proliferation.
- Another subject of the invention is the use of said monoclonal antibody compositions for the manufacture of a medicament for treatment, without involving the cytotoxic functions of the effector immune cells, of patients who have an NK cell deficiency, and who could not be treated with antibodies whose cytotoxicity is expressed only by NK .
- patients with lupus erythematosus Green 2005
- patients with congestive cardiac myopathy Anderson 1982
- patients with histiocytosis with hemophagocytosis perforin deficiency
- NK cell-related cytotoxicity deficiency has been described in patients with acute myeloid leukemia (Nasrallah 1983) and hairy cell leukemia (Trentin 1990). More generally, smoking populations (Takeuchi 2001) show decreased NK activity compared to non-smokers.
- the antibodies according to the invention may be particularly advantageous in the treatment of patients with hemophilia A or B, possibly to target memory B lymphocytes.
- Figure 3 Diagram of potentiation of apoptosis via CDl ⁇ .
- Figure 4 A: Potentiation of apoptosis of Daudi cells by an anti-human IgG antibody.
- B potentiation of apoptosis of B cells by an anti-human IgG antibody.
- Figure 5 A: Potentiation of apoptosis of Daudi cells by Jurkat CDl ⁇ .
- B potentiation of B cell apoptosis by Jurkat CDl ⁇ .
- Antibody An anti-HLA-DR antibody was used for the development of a study model. It is a recombinant antibody in which the variable domains are of murine origin whereas the constant part is of human origin. The same sequence was expressed in the CHO line and in the YB2 / 0 line (these antibodies expressed in YB2 / 0 are called "EMABling® Antibodies").
- Jurkat clone E6.1 cells come from I 1 ECACC (European Collection of Ceil Culture). These same cells were transfected with a vector encoding expression for the gamma chain (Y) as well as a vector encoding Fc ⁇ RIIIa (L48F158 haplotypes). These cells transfected with a DNA molecule so as to express CD16 can not induce cytotoxicity via an ADCC mechanism.
- Daudi cells come from the ATCC (American Type Culture Collection).
- NK (Natural Killer) cells are purified from peripheral blood samples by negative selection on Myltenyi magnetic beads.
- Daudi cells (2.5 ⁇ 10 5 cells) are incubated with anti-HLA-DR (1 ⁇ g / ml) in 24-well plates (P24) for 24 hours at 37 ° C.
- Daudi cells (2.5 x 10 5 cells) are incubated with the anti-HLA-DR antibodies (1 ⁇ g / ml) in the presence of a human anti-IgG (20 ⁇ g / ml), in plates of 24 well (P24) for 24 hours at 37 ° C.
- Daudi cells (2.5 x 10 5 cells) are incubated with anti-HLA-DR (1 ⁇ g / ml) in the presence of Jurkat cells (2.5 x 10 5 cells) in 24-well plates (P24) for 24 hours at 37 ° C.
- Daudi cells (2.5 x 10 5 cells) are incubated with anti-HLA-DR (1 ⁇ g / ml) in the presence of Jurkat cells transfected with the
- CD16 (2.5 ⁇ 10 5 ), in 24-well plates (P24) for 24 h at 37 ° C. (FIG. 3).
- Daudi cells (2.5 x 10 5 cells) are incubated with anti-HLA-DR (1 ⁇ g / ml) in the presence of NK cells (2.5 x 10 5 cells) and an EGTA (4 mM) / MgCl 2 (2 mM) mixture, in 24-well plates (P24) for 24 hours at 37 ° C.
- the cells are then collected, washed twice and then incubated with annexin V-FITC and propidium iodide (PI) according to the supplier's instructions (BD Biosciences).
- the cells are analyzed by flow cytometry (Beckman C ⁇ ulter EPICS XL cytometer) using the software Expo 32 from Beckman Coulter.
- the annexin V binding corresponds to an early signal of apoptosis whereas the fixation of the PI being later (DNA fragmentation), the percentage of so-called apoptotic cells is arbitrarily defined as the sum of the Annexin V positive cells and the doubled cells. positive Annexine V / PI.
- the apoptosis of Daudi cells observed in the absence of antibody (natural death) is of the order of 10%. This basic value is deduced from the results obtained during the different experiments and is thus arbitrarily at 0%.
- Figure 4 B showing the percentage of apoptotic cells, shows that apoptosis of B cells from peripheral blood, is obtained in the presence of an anti-human IgG antibody. Under these conditions, this study shows that there are no significant differences between the potentiation of the apoptosis induced by the antibody expressed in CHO (DR CHO) and the anti-HLA-DR EMABling® antibody (DR YB2 / 0).
- Jurkat (untransfected) induce respectively 3% and 2% apoptosis on Daudi (condition 3).
- condition 4 which can interact via CD16 with the Fc portion of the anti-HLA-DR antibodies attached to the surface of the Daudi cell, the apoptosis is potentiated.
- the final percentage of apoptosis is respectively
- the potentiation of apoptosis in The presence of the EM ⁇ Bling® antibody is approximately 100% greater than that induced with the antibody produced in CHO.
- the results are expressed as the percentage of apoptotic cells observed, the EMABling® antibody arbitrarily representing 100% for each condition studied. Under these conditions the statistical study shows that the difference in potentiation of apoptosis is very significant between the two antibodies (p ⁇ 0.0001) and this in favor of the EMABling® antibody.
- apoptosis of B cells derived from peripheral blood is studied after substituting the anti-human IgG antibody (crosslinking agent or cross-linker) by a Jurkat cell transfected with CD16.
- Untransfected wild Jurkat cells (WT) are used as a negative control.
- the results expressed as a percentage of apoptotic cells indicate that apoptosis induced by anti-HLA-DR EMABling® (DR YB2 / 0) is strongly potentiated compared to that induced by the antibody produced in CHO (DR CHO). This property is probably due to the better binding of the anti-HLA-DR EMABling® (DR-YB2 / 0) on the CD16 compared to that of the anti-HLA-DR CHO (DR CHO).
- CD16 results not only in an increase in ADCC and secretion of cytokines (see EP document
- Nasrallah AG, Miale TD Decreased natural killer cell activity in children with acute leukemia.Natural killer cell activity in children with acute lymphocytic and acute myelogenous leukemia. Cancer Res. 1983 Nov; 43 (11): 5580-5.
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Abstract
The present invention relates to the use of a composition of anti-MLA-DR antibodies expressed in cell line YB210, the fucose content of which is less than 65%, for the in vitro induction of apoptosis.
Description
Potentialisation de l' apoptose par des anticorps monoclonauxPotentiation of apoptosis by monoclonal antibodies
La présente invention se rapporte à l'utilisation d'une composition d'anticorps dont la teneur en fucose est inférieure à 65%, pour induire in vitro 1' apoptose.The present invention relates to the use of an antibody composition having a fucose content of less than 65% to induce apoptosis in vitro.
De plus en plus utilisés en recherche, les anticorps constituent également des outils de choix en diagnostic et en thérapeutique, où ils sont une alternative aux traitements conventionnels .Increasingly used in research, antibodies are also tools of choice in diagnosis and therapy, where they are an alternative to conventional treatments.
De nombreux anticorps à usage thérapeutique, d'origine plasmatique ou monoclonale, sont actuellement sur le marché, ou en phase de développement clinique. Leurs propriétés sont exploitées pour obtenir des outils thérapeutiques capables de se lier de manière spécifique à leur cible, et de recruter de manière efficace les cellules immunitaires effectrices, provoquant ainsi la destruction de la cellule cible grâce aux fonctions cytotoxiques de ces cellules.Many antibodies for therapeutic use, of plasma or monoclonal origin, are currently on the market, or in the clinical development phase. Their properties are exploited to obtain therapeutic tools capable of specifically binding to their target, and to efficiently recruit the effector immune cells, thus causing the destruction of the target cell through the cytotoxic functions of these cells.
Toutefois, dans certaines pathologies, comme la LLCHowever, in some pathologies, like the LLC
(leucémie lymphoïde chronique) , on observe chez les patients un déficit de l'activité des cellules immunitaires effectrices en particulier les cellules NK, qui peut être à l'origine d'une incidence accrue des pathologies secondaires chez ces patients (Ziegler et al., 1981). D'autre part, lorsque ces patients sont traités dans le cadre de leur LLC par l'anticorps monoclonal anti-CD20 Rituxan, on observe une faible efficacité de ce traitement qui pourrait s'expliquer en partie par la faible activité ADCC de leurs cellules NK (Farag 2003).(Chronic lymphocytic leukemia), there is a deficit in the activity of effector immune cells, particularly NK cells, which may be at the origin of an increased incidence of secondary pathologies in these patients (Ziegler et al. , nineteen eighty one). On the other hand, when these patients are treated as part of their CLL with Rituxan anti-CD20 monoclonal antibody, there is a low efficacy of this treatment which could be explained in part by the low ADCC activity of their NK cells. (Farag 2003).
On rencontre également des déficits en cellules NK chez les patients atteint de lupus érythémateux
(Green 2005) , les patients avec myopathie cardiaques congestives (Anderson 1982), les patients souffrants d'histiocytoses avec hémophagocytose (déficit en perforine) (Clementi R, 2005) ainsi que les patients infectés par le VIH (déficit en perforine et granzyme)NK cell deficits also occur in patients with lupus erythematosus (Green 2005), patients with congestive cardiac myopathy (Anderson 1982), patients with histiocytosis with hemophagocytosis (perforin deficiency) (Clementi R, 2005) and HIV-infected patients (perforin and granzyme deficiency)
(Portales, 2003) . On peut donc craindre, par analogie à ce qui est décrit pour les patients atteints de LLC, que le traitement de ces malades avec des anticorps monoclonaux conventionnels ne soit peu ou pas efficace.(Portales, 2003). It is therefore to be feared, by analogy with what is described for patients with CLL, that the treatment of these patients with conventional monoclonal antibodies is not very or not effective.
Le Demandeur a donc cherché à fournir des outils dont l'activité est indépendante des fonctions cytotoxiques des cellules immunitaires effectrices, et ainsi susceptibles d'être administrés aux patients présentant une diminution de l'activité des cellules immunitaires effectrices .The Applicant has therefore sought to provide tools whose activity is independent of the cytotoxic functions of immune effector cells, and thus may be administered to patients with a decrease in the activity of immune effector cells.
Le Demandeur avait montré, dans le document WO 01/77181, l'importance de sélectionner des lignées cellulaires permettant de produire des anticorps présentant une forte activité ADCC via le récepteur FcgammaRIII (CDl6) . Il avait alors été montré que la modification de la glycosylation de la région Fc des anticorps produits dans des lignées de myélomes de rat telle que YB2/0 conduisait à améliorer l'activité ADCC. Les structures glycanniques de telles compositions d'anticorps confèrent à la composition d'anticorps une faible fucosylation.The Applicant has shown, in WO 01/77181, the importance of selecting cell lines for producing antibodies having high ADCC activity via the FcgammaRIII receptor (CD16). It was then shown that modification of the Fc region glycosylation of antibodies produced in rat myeloma lines such as YB2 / 0 led to improved ADCC activity. The glycan structures of such antibody compositions impart low fucosylation to the antibody composition.
Or, le Demandeur a découvert de manière surprenante que de telles compositions d'anticorps, outre leur forte activité cytotoxique, induisent, en présence de cellules exprimant le CDl6 à leur surface, une apoptose importante alors que le même anticorps produit dans CHO potentialise beaucoup moins l' apoptose.
DescriptionHowever, the Applicant has surprisingly found that such antibody compositions, in addition to their high cytotoxic activity, induce, in the presence of cells expressing CD16 on their surface, significant apoptosis whereas the same antibody produced in CHO potentiates much less. apoptosis. Description
L'invention se rapporte ainsi à l'utilisation d'une composition d'anticorps, dont la teneur en fucose est inférieure à 65%, pour induire, in vitro, ex vivo ou in vivo, l'apoptose.The invention thus relates to the use of an antibody composition, the fucose content of which is less than 65%, for inducing, in vitro, ex vivo or in vivo, apoptosis.
Un premier objet de l'invention se rapporte en particulier à une méthode pour induire, in vitro, ex vivo ou in vivo, l'apoptose d'une cellule cible par une composition d'anticorps, comprenant la mise en contact de la composition d'anticorps et de la cellule cible, en présence de cellules exprimant le CDl6 à leur surface, les anticorps étant dirigés contre la cellule cible et la composition d'anticorps possédant une teneur en fucose inférieure à 65%.A first subject of the invention relates in particular to a method for inducing, in vitro, ex vivo or in vivo, the apoptosis of a target cell by an antibody composition, comprising contacting the composition of antibodies and the target cell, in the presence of cells expressing CD16 on their surface, the antibodies being directed against the target cell and the antibody composition having a fucose content of less than 65%.
Les anticorps sont constitués de chaînes lourdes et de chaînes légères, liées entre elles par des ponts disulfures. Chaque chaîne est constituée, en position N-terminale, d'une région (ou domaine) variable spécifique de l'antigène contre lequel l'anticorps est dirigé, et en position C-terminale, d'une région constante, constituée d'un seul domaine CL pour les chaînes légères et de plusieurs domaines (CHi, CH2 et CH3) pour les chaînes lourdes. L'association des domaines variables et des domaines CHl et CL des chaînes lourdes et légères forme les parties Fab de l'anticorps, qui sont connectées à la région Fc par une région charnière très flexible, permettant à chaque Fab de se fixer à l'antigène cible. La région Fc, médiatrice des propriétés effectrices de l'anticorps, reste accessible aux molécules effectrices telles que les récepteurs FcγR (FcgammaR) . La région Fc, constituée de 2 domaines globulaires CH2
et CH3, est glycosylée au niveau au domaine CH2 avec la présence, sur chacune des 2 chaînes, d'un N-glycanne biantenné, lié à l'asparagine 297 (Asn 297) . Un tel N-glycanne se présente sous la forme générale suivante (forme présentée « GO ») , à laquelle d'autres sucres peuvent s ' aj outer :The antibodies consist of heavy chains and light chains, linked together by disulfide bridges. Each chain is constituted, in the N-terminal position, of a variable region (or domain) specific for the antigen against which the antibody is directed, and in the C-terminal position, of a constant region, consisting of a single CL domain for light chains and several domains (CHi, CH 2 and CH 3 ) for heavy chains. The combination of the variable domains and the CH1 and CL domains of the heavy and light chains forms the Fab portions of the antibody, which are connected to the Fc region by a very flexible hinge region, allowing each Fab to bind to the target antigen. The Fc region, which mediates the effector properties of the antibody, remains accessible to effector molecules such as FcγR receptors (FcgammaR). The Fc region, consisting of 2 CH 2 globular domains and CH 3 , is glycosylated at the CH 2 domain with the presence, on each of the 2 chains, of a biantennal N-glycan linked to asparagine 297 (Asn 297). Such an N-glycan is in the following general form (presented form "GO"), to which other sugars can be added:
liaison à 1ΑSN297 B GIcNAc © Mannose binding to 1ΑSN297 B GIcNAc © Mannose
Ainsi, dans la composition d'anticorps selon l'invention, on entend par « fucose » le fucose porté par ces N-oligosaccharides . La molécule de fucose, lorsqu'elle est présente, est liée à la N- acétylglucosamine (GIcNAc) du N-oligosaccharide, cette GIcNAc étant elle-même liée à l 'Asn 297.Thus, in the antibody composition according to the invention, "fucose" is understood to mean the fucose borne by these N-oligosaccharides. The fucose molecule, when present, is linked to the N-acetylglucosamine (GIcNAc) of the N-oligosaccharide, this GIcNAc itself being bound to the Asn 297.
Chacun des N-glycannes porté par l'une ou l'autre des 2 chaînes lourdes de chaque anticorps, peut porter une molécule de fucose ou ne pas en porter. Ainsi, chaque anticorps peut comporter 0, 1 ou 2 molécules de fucose, selon respectivement qu'aucun de ses N- glycannes ne porte de fucose, qu'un seul de ses N- glycannes porte une molécule de fucose, ou que ses 2 N-glycannes portent chacun une molécule de fucose. Ainsi, on entend par « composition d'anticorps dont la teneur en fucose est inférieure à 65% » une composition d'anticorps, dont, parmi la totalité des structures glycanniques portées par chaque site de glycosylation (Asn 297) des anticorps de la composition, moins de 65% comportent une molécule de fucose.
De manière avantageuse, de telles structures glycanniques sont plus particulièrement sélectionnées parmi les formes suivantes :Each of the N-glycans carried by either of the 2 heavy chains of each antibody may carry a fucose molecule or not carry one. Thus, each antibody may comprise 0, 1 or 2 fucose molecules, respectively, according to that none of its N-glycans carry fucose, that only one of its N-glycans carries a fucose molecule, or that its 2 N glycans each carry a molecule of fucose. Thus, "antibody composition whose fucose content is less than 65%" is understood to mean an antibody composition of which, among the totality of the glycan structures carried by each glycosylation site (Asn 297), antibodies of the composition less than 65% contain a molecule of fucose. Advantageously, such glycan structures are more particularly selected from the following forms:
Ainsi, de manière avantageuse", parmi les structures glycanniques de forme GO, GOF, Gl et GIF, portées par chaque site de glycosylation (Asn 297) des anticorps de la composition selon l'invention, la teneur en fucose est inférieure à 65%.Thus, advantageously, among the glycan structures of GO, GOF, Gl and GIF form, carried by each glycosylation site (Asn 297), antibodies of the composition according to the invention, the fucose content is less than 65%. .
Une telle composition d'anticorps, ainsi que ses caractéristiques avantageuses quant à l'induction de l'ADCC (Antibody-Dependent Cell-mediated Cytotoxicity) , sont décrites dans le document WO 01/77181.Such an antibody composition, as well as its advantageous characteristics as to the induction of ADCC (Antibody-Dependent Cell-mediated Cytotoxicity), are described in WO 01/77181.
Les compositions d'anticorps selon l'invention ont pour particularité et avantage de déclencher une forte réaction d'apoptose. Or, l'apoptose a un rôle essentiel dans le déclenchement de la mort cellulaire. De nombreux facteurs interviennent pour induire l'apoptose, mais tous aboutissent à une voie commune passant par la mitochondrie, la protéine Bcl-2 et les caspases . Les principaux mécanismes mettant en route le processus de mort cellulaire programmée sont le stress (par exemple par hypo-oxygénâtion) , le traitement par des substances cytotoxiques ou des corticoïdes, la
privation de facteurs de croissance, l'atteinte de l'ADN, et la transmission d'un signal de mort (récepteur Fas des lymphocytes cytotoxiques et des natural killer, du facteur de nécrose TNF-α) . A la suite du signal de mort cellulaire, les caspases sont activées, provoquant l'activation des protéines associées à l'apoptose. Les phosphatidylsérines sont ensuite transloquées de la face interne de la membrane cellulaire à la face externe. Elles peuvent être visualisées « in vitro » par la fixation de l'annexine V à la. surface de la cellule. La condensation du cytoplasme, du noyau et de la chromatine, une fragmentation de l'ADN, un bourgeonnement de la membrane plasmique ainsi qu'une perte de l'asymétrie membranaire sont alors visibles. A ce stade, les cellules fixent « in vitro » l'iodure de propidiumThe antibody compositions according to the invention have the particularity and advantage of triggering a strong apoptosis reaction. Apoptosis plays an essential role in triggering cell death. Many factors intervene to induce apoptosis, but all lead to a common pathway through mitochondria, Bcl-2 protein, and caspases. The main mechanisms initiating the process of programmed cell death are stress (for example by hypo-oxygenation), treatment with cytotoxic substances or corticosteroids, deprivation of growth factors, damage to DNA, and transmission of a death signal (Fas receptor of cytotoxic lymphocytes and natural killers, TNF-α necrosis factor). Following the cell death signal, the caspases are activated, causing the activation of proteins associated with apoptosis. Phosphatidylserines are then translocated from the inner side of the cell membrane to the outer surface. They can be visualized "in vitro" by the attachment of annexin V to the. surface of the cell. Condensation of the cytoplasm, nucleus and chromatin, fragmentation of the DNA, budding of the plasma membrane and loss of membrane asymmetry are then visible. At this stage, the cells fix "in vitro" propidium iodide
(agent intercalant de l'ADN) . Il y a ensuite formation de corps apoptotiques qui sont digérés par les macrophages environnants . La potentialisation de l'apoptose induite par les compositions d'anticorps selon l'invention n'est pas due aux fonctions cytotoxiques de la cellule immunitaire effectrice, mais est induite par une forte agrégation de la région Fc des anticorps avec le récepteur CD16 exprimé à la surface des cellules immunitaires effectrices .(intercalating agent of DNA). There is then formation of apoptotic bodies that are digested by the surrounding macrophages. The potentiation of the apoptosis induced by the antibody compositions according to the invention is not due to the cytotoxic functions of the effector immune cell, but is induced by a strong aggregation of the Fc region of the antibodies with the CD16 receptor expressed at the same time. the surface of the effector immune cells.
De manière avantageuse, la composition d'anticorps selon l ' invention comprend une teneur supérieure à 60%, de préférence supérieure à 80%, de formes G0+G1+G0F+G1F, étant entendu que la teneur en formes G0F+G1F est inférieure à 50%, de préférence inférieure à 30%.Advantageously, the antibody composition according to the invention comprises a content greater than 60%, preferably greater than 80%, of forms G0 + G1 + G0F + G1F, it being understood that the content of G0F + G1F forms is lower than at 50%, preferably less than 30%.
La démonstration « in vitro » de l'induction de l'apoptose par les compositions d'anticorps selon l'invention a été effectuée avec des cellules Jurkat
transfectées CDl6 dépourvues d'activité cytolytique, à l'opposé des cellules NK. Ceci constitue un modèle dans lequel la mort cellulaire ne peut être induite que par apoptose. Ainsi, la méthode de l'invention, destinée à induire in vitro l' apoptose d'une cellule cible, est réalisée au moyen de cellules exprimant le CD16 à leur surface, mais qui ne sont pas des cellules effectrices . Ces cellules n'ont donc pas ou plus d'activité cytotoxique à l ' encontre des cellules cibles. A titre d'exemple, on peut citer la cellule Jurkat CDl6.The "in vitro" demonstration of the induction of apoptosis by the antibody compositions according to the invention was carried out with Jurkat cells CD16 transfected with no cytolytic activity, as opposed to NK cells. This is a model in which cell death can only be induced by apoptosis. Thus, the method of the invention, intended to induce in vitro the apoptosis of a target cell, is carried out by means of cells expressing CD16 on their surface, but which are not effector cells. These cells therefore have no or more cytotoxic activity against the target cells. By way of example, mention may be made of the Jurkat CD16 cell.
« In vivo », la potentialisation de l' apoptose par les compositions selon l'invention s'établit avec toutes cellules effectrices exprimant le CDl6, et en particulier les cellules du sang périphériques exprimant le CDl6, comme les monocytes-macrophages, les neutrophiles, les cellules NK et certaines sous- populations de cellules T. Ainsi, les compositions selon l'invention peuvent être utilisées en présence d'un déficit quantitatif ou qualitatif (absence d'ADCC) en une population de cellules immunitaires effectrices (cellules cytotoxiques ) . En effet, dans ce cas, cette potentialisation est possible grâce aux autres cellules exprimant le CDl6 à leur surface (cellules CD16+) , en particulier des cellules exprimant le CDl6 à leur surface, mais qui n'ont pas ou plus d'activité cytotoxique."In vivo", the potentiation of apoptosis by the compositions according to the invention is established with all effector cells expressing CD16, and in particular peripheral blood cells expressing CD16, such as monocytes-macrophages, neutrophils, NK cells and certain subpopulations of T cells. Thus, the compositions according to the invention can be used in the presence of a quantitative or qualitative deficit (absence of ADCC) in a population of effector immune cells (cytotoxic cells). Indeed, in this case, this potentiation is possible thanks to the other cells expressing CD16 on their surface (CD16 + cells), in particular cells expressing CD16 on their surface, but which have no or more cytotoxic activity.
De tels déficits se rencontrent chez les patients ayant une LLC-B (Leucémie Lymphoïde Chronique à cellules B) , chez lesquels on observe une baisse d'activité des cellules NK (Foa 1986, Ziegler 1981) ou plus spécifiquement un défaut de molécules cytolytiques (Katrinakis 1996) , ainsi que les patients atteint de lupus erythromateux chez lesquels il a été décrit un déficit en cellules NK (Green 2005) . Les compositions d'anticorps pourront être
utilisées selon l'invention dans de telles pathologies .Such deficits are found in patients with B-CLL (Chronic Lymphocytic Leukemia B-cell), in whom there is a decrease in NK cell activity (Foa 1986, Ziegler 1981) or more specifically a defect of cytolytic molecules ( Katrinakis 1996), as well as patients with erythematous lupus who have been diagnosed with NK cell deficiency (Green 2005). Antibody compositions may be used according to the invention in such pathologies.
Avantageusement, la composition d'anticorps de l'invention possède une teneur en fucose inférieure à 55%, ou encore inférieure à 40%, à 30% ou à 20%.Advantageously, the antibody composition of the invention has a fucose content of less than 55%, or even less than 40%, 30% or 20%.
De manière particulièrement avantageuse, la teneur en fucose de la composition d'anticorps est comprise entre 20% et 45%, ou entre 25% etParticularly advantageously, the fucose content of the antibody composition is between 20% and 45%, or between 25% and
De manière particulièrement avantageuse, la composition d'anticorps selon l'invention présente en outre un ratio taux de fucose/taux de galactose inférieur à 0,6. De manière avantageuse, ce ratio est inférieur à 0,5 ; inférieur à 0,4 ; inférieur à 0,2 ou de manière encore plus avantageuse inférieur à 0,1. Aux fins de la présente invention, on entend par « ratio taux de fucose/taux de galactose » le ratio entre la teneur en fucose de la composition d'anticorps et la teneur en galactose de la composition d'anticorps, ces 2 sucres étant susceptibles d'être portés par chaque structure glycannique liée à chaque site de glycosylation. (Asn 297) des anticorps de la composition.In a particularly advantageous manner, the antibody composition according to the invention also has a fucose / galactose ratio ratio of less than 0.6. Advantageously, this ratio is less than 0.5; less than 0.4; less than 0.2 or even more advantageously less than 0.1. For the purpose of the present invention, the term "fucose ratio / galactose content" means the ratio between the fucose content of the antibody composition and the galactose content of the antibody composition, these two sugars being susceptible to to be carried by each glycan structure bound to each glycosylation site. (Asn 297) antibodies of the composition.
Avantageusement, la composition d'anticorps utilisée selon l'invention est produite par la lignée cellulaire YB2/0, ou toute cellule dérivée d'YB2/0 et conférant les mêmes caractéristiques sur le plan des modifications post-traductionnelles des protéines, notamment sur le plan de la glycosylation. Cette lignée a été choisie en raison de sa capacité à produire des compositions d'anticorps dont certaines, après sélection, présentent une faible fucosylation, pour laquelle le Demandeur a montré dans la présente invention qu'elle est utile dans la production de
compositions d'anticorps capables .d'induire fortement l'apoptose.Advantageously, the antibody composition used according to the invention is produced by the YB2 / 0 cell line, or any cell derived from YB2 / 0 and conferring the same characteristics in terms of post-translational modifications of the proteins, in particular on the plan of glycosylation. This line was chosen because of its ability to produce antibody compositions some of which, after selection, exhibit low fucosylation, for which the Applicant has shown in the present invention that it is useful in the production of antibody compositions capable of strongly inducing apoptosis.
Ainsi, un autre objet de l'invention est l'utilisation de la lignée cellulaire YB2/0 pour produire des anticorps à forte capacité d'apoptose.Thus, another object of the invention is the use of the YB2 / 0 cell line to produce antibodies with a high apoptosis capacity.
Un autre objet de l'invention est un procédé de sélection de compositions d'anticorps à forte capacité apoptotigue, comprenant les étapes suivantes :Another subject of the invention is a method for selecting antibody compositions with a high apoptotic capacity, comprising the following steps:
1) mise en contact de l'anticorps à évaluer avec l'antigène dudit anticorps (ou d'une cellules exprimant cet antigène) en présence de cellules exprimant le récepteur CD16 à leur surface. 2) mesure de l'induction de l'apoptose1) contacting the antibody to be evaluated with the antigen of said antibody (or a cell expressing this antigen) in the presence of cells expressing the CD16 receptor on their surface. 2) measurement of the induction of apoptosis
3) sélection des compositions d'anticorps montrant une induction de l'apoptose supérieure d'au moins 20%, voire 50% ou plus, en présence de la cellule exprimant le CDl6 par rapport au contrôle négatif.3) selection of antibody compositions showing greater apoptosis induction of at least 20%, or even 50% or more, in the presence of the CD16-expressing cell relative to the negative control.
Lors de la mise en œuvre de l'étape 1) de ce procédé, un contrôle négatif peut être par exemple obtenu en présence de cellules n'exprimant pas le CDl6. Lors de l'étape 2, la mesure de l'induction de l'apoptose peut s'effectuer de différentes manières, en utilisant des techniques classiques spécifiques pour mesurer l'apoptose comme par exemple la mesure de l'annexine V (stade précoce d'apoptose mesurant les phosphatitylsérines exposées à la surface cellulaire) associée au iodure de propidium (stade avancé d'apoptose mesurant la dégradation de l'ADN) ou d'autres marqueurs comme le YO Pro-1 (agent intercalant de l'ADN mesurant la fragmentation de ce dernier) . Aux fins de l'invention, on entend par « forte capacité apoptotique » une capacité d'induction de l'apoptose supérieure d'au moins 20%, voire 50% ou
plus, en présence de la cellule exprimant le CD16 par rapport au contrôle négatif.During the implementation of step 1) of this method, a negative control can be obtained for example in the presence of cells not expressing CD16. In step 2, the measurement of the induction of apoptosis can be carried out in different ways, by using specific conventional techniques for measuring apoptosis, for example the measurement of annexin V (early stage). apoptosis measuring phosphatityserines exposed to the cell surface) associated with propidium iodide (advanced stage of apoptosis measuring the degradation of DNA) or other markers such as YO Pro-1 (intercalating agent of DNA measuring the fragmentation of the latter). For the purposes of the invention, the term "high apoptotic capacity" means an ability to induce apoptosis of at least 20% or even 50% or more, in the presence of the cell expressing the CD16 compared to the negative control.
Dans la mise en œuvre de ce procédé, les cellules exprimant le CD16 à leur surface peuvent être des cellules transfectées CDl6, notamment des cellules Jurkat, des cellules du sang périphériques exprimant CDl6, par exemple les monocytes-macrophages, les neutrophiles, les cellules NK et certaines sous- populations de cellules T, cette liste n'étant pas limitative. De préférence, les cellules exprimant le CDl6 à leur surface sont des cellules qui n'ont pas, ou plus, d'activité cytotoxique.In the implementation of this method, the cells expressing CD16 on their surface may be CD16 transfected cells, in particular Jurkat cells, peripheral blood cells expressing CD16, for example monocytes-macrophages, neutrophils, NK cells. and certain sub-populations of T cells, this list not being limiting. Preferably, cells expressing CD16 on their surface are cells that have no or more cytotoxic activity.
De manière avantageuse, le procédé de l'invention comporte en outre une étape de détermination de la teneur en fucose des compositions d'anticorps sélectionnées. Cette détermination peut avoir lieu avant l'étape de mise en contact de l'anticorps à évaluer avec l'antigène, ou bien après la sélection des compositions d'anticorps montrant une induction d'apoptose supérieure à celle du contrôle négatif.Advantageously, the method of the invention further comprises a step of determining the fucose content of the selected antibody compositions. This determination can take place before the step of contacting the antibody to be evaluated with the antigen, or after the selection of the antibody compositions showing an induction of apoptosis greater than that of the negative control.
Si la détermination de la teneur en fucose de la composition d'anticorps est réalisée avant l'étape de mise en contact de l'anticorps à évaluer avec l'antigène, il est avantageux de sélectionner les compositions d'anticorps possédant une teneur en fucose inférieure à 65%. Préférentiellement, on sélectionne les compositions d'anticorps dont la teneur en fucose est inférieure à 30%, ou bien comprise entre 20% et 45%, ou entre 25% et 40%. De manière avantageuse, les anticorps sélectionnés sont des anticorps anti-idiotypiques anti-inhibiteurs du facteur VIII, des anticorps dirigés contre des autoanticorps pour cibler les lymphocytes B mémoires,If the determination of the fucose content of the antibody composition is carried out before the step of contacting the antibody to be evaluated with the antigen, it is advantageous to select the antibody compositions having a fucose content. less than 65%. Preferably, the antibody compositions whose fucose content is less than 30%, or between 20% and 45%, or between 25% and 40%, are selected. Advantageously, the antibodies selected are anti-idiotypic anti-factor VIII inhibitor antibodies, antibodies directed against autoantibodies to target memory B cells,
' ainsi que des anticorps dirigés contre des protéines virales et/ou bactériennes exprimées à la surface de cellules infectées, dans le but d'induire leur élimination. On peut aussi sélectionner tout anticorps
reconnaissant un antigène exprimé préférentiellement à la surface de cellules tumorales, ceci incluant les proliférations hématopoïétiques .as well as antibodies directed against viral and / or bacterial proteins expressed on the surface of infected cells, with the aim of inducing their elimination. Any antibody can be selected recognizing an antigen preferentially expressed on the surface of tumor cells, including hematopoietic proliferations.
Un autre objet de l'invention se rapporte à l'utilisation de compositions d'anticorps monoclonaux dont la teneur en fucose est inférieure à 65%, de préférence inférieure à 30%, de manière particulièrement avantageuse comprise entre 20% et 45%, ou entre 25% et 40%, ou de compositions d'anticorps sélectionnées par le procédé de sélection décrit ci-dessus, pour la préparation d'un médicament destiné au traitement, sans mise en jeu des fonctions cytotoxiques des cellulaires immunitaires effectrices, d'un déficit quantitatif ou qualitatif (absence d'ADCC) en cellules NK, notamment choisi parmi la LLC- B (leucémie lymphoïde chronique à cellules B) , le lupus érythémateux, les myopathies cardiaques congestives, des histiocytoses avec hémophagocytose ainsi que le VIH et l'hémophilie A ou B.Another subject of the invention relates to the use of monoclonal antibody compositions whose fucose content is less than 65%, preferably less than 30%, particularly advantageously between 20% and 45%, or between 25% and 40%, or of antibody compositions selected by the selection method described above, for the preparation of a medicament for treatment, without the cytotoxic functions of the immune effector cells being involved, of a quantitative or qualitative deficiency (absence of ADCC) in NK cells, chosen in particular from CLB-B (chronic B cell lymphocytic leukemia), lupus erythematosus, congestive cardiac myopathies, histiocytosis with haemophagocytosis, and HIV and AIDS. hemophilia A or B.
Lesdites compositions d'anticorps présentent une interaction pour le CDl6 au moins 2 fois supérieure, voire avantageusement 10 fois supérieure à l'anticorps thérapeutique Rituxan. L'interaction entre la région Fc de l'anticorps et le récepteur CDl6 peut être mesurée par la fixation directe ou indirecte par compétition avec un anticorps monoclonal anti-CDlβ de type 3G8 dirigé contre le site de fixation de la partie Fc des anticorps, sur des cellules CD16 positives.Said antibody compositions exhibit an interaction for CD16 at least 2-fold higher, or even advantageously 10-fold greater than the therapeutic antibody Rituxan. The interaction between the Fc region of the antibody and the CD16 receptor can be measured by direct or indirect binding by competition with a monoclonal anti-CDlβ antibody type 3G8 directed against the binding site of the Fc part of the antibodies, on CD16 positive cells.
De manière avantageuse, la composition d'anticorps utilisée selon l'invention comprend une teneur supérieure à 60%, de préférence supérieure à 80%, de formes G0+G1+G0F+G1F, étant entendu que la teneur en formes G0F+G1F est inférieure à 50%, de préférence inférieure à 30%.
De manière particulièrement avantageuse, la composition d'anticorps utilisée selon l'invention présente en outre un ratio taux de fucose/taux de galactose inférieur à 0,6. De manière avantageuse, ce ratio est inférieur à 0,5 ; inférieur à 0,4 ; inférieur à 0,2 ou de manière encore plus avantageuse inférieur à 0,1.Advantageously, the antibody composition used according to the invention comprises a content greater than 60%, preferably greater than 80%, of forms G0 + G1 + G0F + G1F, it being understood that the content of G0F + G1F forms is less than 50%, preferably less than 30%. In a particularly advantageous manner, the antibody composition used according to the invention also has a fucose / galactose ratio ratio of less than 0.6. Advantageously, this ratio is less than 0.5; less than 0.4; less than 0.2 or even more advantageously less than 0.1.
De manière avantageuse, cette utilisation est destinée à la préparation d'un médicament destiné au traitement de pathologies dans lesquelles une ou plusieurs population (s) de cellules effectrices est diminuée voire nulle, lesdites cellules induisant ainsi une faible activité cytotoxique par ADCC.Advantageously, this use is intended for the preparation of a medicament for the treatment of pathologies in which one or more population (s) of effector cells is decreased or even zero, said cells thus inducing low cytotoxic activity by ADCC.
Un autre objet de l'invention se rapporte à l'utilisation d'anticorps monoclonaux anti-CD20 ayant une forte interaction pour le CDlβ, sélectionnés selon le procédé de l'invention, ou répondant aux caractéristiques de fucosylation précitées, pour la fabrication d'un médicament destiné au traitement, sans mise en jeu des fonctions cytotoxiques des cellules immunitaires effectrices, des patients atteints de LLC-B et chez lesquels on peut éventuellement observer une baisse des fonctions lytiques des cellules NK.Another subject of the invention relates to the use of anti-CD20 monoclonal antibodies having a strong interaction for CD1β, selected according to the process of the invention, or answering the fucosylation characteristics mentioned above, for the manufacture of a drug for treatment, without the cytotoxic functions of effector immune cells, patients with B-CLL and in which a decrease in the lytic functions of NK cells may be observed.
D'une manière générale, l'anticorps de l'invention peut être utilisé pour traiter toute prolifération hématopoïétique .In general, the antibody of the invention may be used to treat any hematopoietic proliferation.
Un autre objet de l'invention est l'utilisation desdites compositions d'anticorps monoclonaux pour la fabrication d'un médicament destiné au traitement, sans mise en jeu des fonctions cytotoxiques des cellules immunitaires effectrices, de patients qui ont un déficit en cellules NK, et qui ne pourraient
pas être traités par des anticorps dont la cytotoxicité s'exprime uniquement grâce aux NK.. On peut citer à titre d'exemple, les patients atteint de lupus érythémateux (Green 2005), les patients avec myopathie cardiaques congestives (Anderson 1982), les patients souffrants d'histiocytoses avec hémophagocytose (déficit en perforine) (Clementi R,Another subject of the invention is the use of said monoclonal antibody compositions for the manufacture of a medicament for treatment, without involving the cytotoxic functions of the effector immune cells, of patients who have an NK cell deficiency, and who could not be treated with antibodies whose cytotoxicity is expressed only by NK . . For example, patients with lupus erythematosus (Green 2005), patients with congestive cardiac myopathy (Anderson 1982), patients with histiocytosis with hemophagocytosis (perforin deficiency) (Clementi R,
2005) ainsi que les patients infectés par le VIH2005) as well as patients infected with HIV
(déficit en perforine et granzyme) (Portales, 2003). On peut également inclure les personnes atteintes de trisomie 21 (Nurmi 1982) et les patients receveurs d'un greffon chez lesquels les traitements associés induisent un déficit des cellules NK et une augmentation du risque de développer des tumeurs (Alamartine 1997) . Dans les pathologies hématopoïétiques , en plus des patients atteints de LLC-B, un déficit de cytotoxicité lié aux cellules NK a été décrit chez les patients atteints de leucémie myéloïde aiguë (Nasrallah 1983) et de leucémie à tricholeucocytes (Trentin 1990) . D'une façon plus générale, les populations de fumeurs (Takeuchi 2001) montrent une activité NK diminuée par rapport aux individus non fumeurs. De plus, les anticorps selon l'invention peuvent être particulièrement avantageux dans le traitement des patients atteints d'hémophilie A ou B, éventuellement pour cibler les lymphocytes B mémoires .(perforin and granzyme deficiency) (Portales, 2003). Persons with trisomy 21 (Nurmi 1982) and graft recipients may also be included in whom the associated treatments induce NK cell deficiency and increased risk of developing tumors (Alamartine 1997). In hematopoietic pathologies, in addition to patients with B-CLL, NK cell-related cytotoxicity deficiency has been described in patients with acute myeloid leukemia (Nasrallah 1983) and hairy cell leukemia (Trentin 1990). More generally, smoking populations (Takeuchi 2001) show decreased NK activity compared to non-smokers. In addition, the antibodies according to the invention may be particularly advantageous in the treatment of patients with hemophilia A or B, possibly to target memory B lymphocytes.
D'autres aspects et avantages de l'invention seront décrits dans les exemples qui suivent, qui doivent être considérés comme illustratifs et ne limitent pas l'étendue de l'invention.Other aspects and advantages of the invention will be described in the examples which follow, which should be considered as illustrative and do not limit the scope of the invention.
Description des FiguresDescription of the Figures
Figure 1 : Tableau 1
Figure 2 : Tableau 2Figure 1: Table 1 Figure 2: Table 2
Figure 3 : Schéma de potentialisation de l'apoptose via le CDlβ.Figure 3: Diagram of potentiation of apoptosis via CDlβ.
Figure 4 : A : potentialisation de l'apoptose des cellules Daudi par un anticorps anti-IgG humaine.Figure 4: A: Potentiation of apoptosis of Daudi cells by an anti-human IgG antibody.
B : potentialisation de l'apoptose des cellules B par un anticorps anti-IgG humaine. Apoptose des cellules B purifiées à partir de sang périphérique, induite par les anticorps anti-HLA-DR EMABling (DR YB2/0) et anti-HLA DR CHO (DR CHO) à 24h en présence ou non d'un agent réticulant (cross- linker) (F(ab')2 anti-fragment Fc d'Ig humaine) .B: potentiation of apoptosis of B cells by an anti-human IgG antibody. Apoptosis of purified B cells from peripheral blood, induced by anti-HLA-DR EMABling antibodies (DR YB2 / 0) and anti-HLA DR CHO (DR CHO) at 24 h with or without a crosslinking agent (cross - linker) (F (ab ') 2 anti-Fc fragment of human Ig).
Figure 5 : A : potentialisation de l'apoptose des cellules Daudi par Jurkat CDlβ.Figure 5: A: Potentiation of apoptosis of Daudi cells by Jurkat CDlβ.
B : potentialisation de l'apoptose des cellules B par Jurkat CDlβ. Apoptose des cellules B purifiées à partir de sang périphérique, induite par les anticorps anti-HLA-DR EMABling (DR YB2/0) et anti- HLA DR CHO (DR CHO) à 24h en présence de cellules Jurkat transfectées (Jurkat CDlβ) ou non (Jurkat WT) par la molécule CDlβ.B: potentiation of B cell apoptosis by Jurkat CDlβ. Apoptosis of purified B cells from peripheral blood, induced by anti-HLA-DR EMABling antibodies (DR YB2 / 0) and anti-HLA DR CHO (DR CHO) at 24 h in the presence of transfected Jurkat cells (Jurkat CDlβ) or no (Jurkat WT) by the molecule CDlβ.
ExemplesExamples
Anticorps : Un anticorps anti-HLA-DR a été utilisé pour la mise au point d'un modèle d'étude. C'est un anticorps recombinant dans lequel les domaines variables sont d'origine murine alors que la partie constante est d' origine humaine . La même séquence a été exprimée dans la lignée CHO et dans la lignée YB2/0 (ces anticorps exprimés dans YB2/0 sont appelés « Anticorps EMABling® ») .Antibody: An anti-HLA-DR antibody was used for the development of a study model. It is a recombinant antibody in which the variable domains are of murine origin whereas the constant part is of human origin. The same sequence was expressed in the CHO line and in the YB2 / 0 line (these antibodies expressed in YB2 / 0 are called "EMABling® Antibodies").
Cellules : Les cellules Jurkat clone E6.1 proviennent de I1ECACC (European Collection of CeIl Culture). Ces mêmes cellules ont été transfectées par un vecteur
d'expression codant pour la chaîne gamma (Y) ainsi que par un vecteur codant le FcγRIIIa (haplotypes L48F158) . Ces cellules transfectées avec une molécule d'ADN de manière à exprimer le CDl6 ne peuvent pas induire de cytotoxicité via un mécanisme d'ADCC.Cells: Jurkat clone E6.1 cells come from I 1 ECACC (European Collection of Ceil Culture). These same cells were transfected with a vector encoding expression for the gamma chain (Y) as well as a vector encoding FcγRIIIa (L48F158 haplotypes). These cells transfected with a DNA molecule so as to express CD16 can not induce cytotoxicity via an ADCC mechanism.
Les cellules Daudi proviennent de l'ATCC (American Type Culture Collection) .Daudi cells come from the ATCC (American Type Culture Collection).
Les cellules NK (Natural Killer) sont purifiées à partir d'échantillons de sang périphériques par sélection négative sur billes magnétiques Myltenyi .NK (Natural Killer) cells are purified from peripheral blood samples by negative selection on Myltenyi magnetic beads.
Induction de l' apoptose :Induction of apoptosis:
Condition 1 : les cellules Daudi (2,5 x 105 cellules) sont incubées avec les anti-HLA-DR (lμg/ml) , dans des plaques de 24 puits (P24) pendant 24h à 370C.Condition 1: Daudi cells (2.5 × 10 5 cells) are incubated with anti-HLA-DR (1 μg / ml) in 24-well plates (P24) for 24 hours at 37 ° C.
Condition 2 : les cellules Daudi (2,5 x 105 cellules) sont incubées avec les anticorps anti-HLA-DR (lμg/ml) en présence d'une anti-IgG humaine (20 μg/ml) , dans des plaques de 24 puits (P24) pendant 24h à 370C.Condition 2: Daudi cells (2.5 x 10 5 cells) are incubated with the anti-HLA-DR antibodies (1 μg / ml) in the presence of a human anti-IgG (20 μg / ml), in plates of 24 well (P24) for 24 hours at 37 ° C.
Condition 3 : les cellules Daudi (2,5 x 105 cellules) sont incubées avec les anti-HLA-DR (lμg/ml) en présence de cellules Jurkat (2,5 x 105 cellules), dans des plaques de 24 puits (P24) pendant 24h à 370C.Condition 3: Daudi cells (2.5 x 10 5 cells) are incubated with anti-HLA-DR (1 μg / ml) in the presence of Jurkat cells (2.5 x 10 5 cells) in 24-well plates (P24) for 24 hours at 37 ° C.
Condition 4 : les cellules Daudi (2,5 x 105 cellules) sont incubées avec les anti-HLA-DR (lμg/ml) en présence de cellules Jurkat transfectées avec leCondition 4: Daudi cells (2.5 x 10 5 cells) are incubated with anti-HLA-DR (1 μg / ml) in the presence of Jurkat cells transfected with the
CD16 (2,5 x 105) , dans des plaques de 24 puits (P24) pendant 24h à 370C (figure 3).CD16 (2.5 × 10 5 ), in 24-well plates (P24) for 24 h at 37 ° C. (FIG. 3).
Condition 5 : les cellules Daudi (2,5 x 105 cellules) sont incubées avec les anti-HLA-DR (lμg/ml) en présence de cellules NK (2,5 x 105 cellules) et
d'un mélange EGTA (4 mM) /MgCl2 (2 mM) , dans des plaques de 24 puits (P24) pendant 24h à 370C.Condition 5: Daudi cells (2.5 x 10 5 cells) are incubated with anti-HLA-DR (1 μg / ml) in the presence of NK cells (2.5 x 10 5 cells) and an EGTA (4 mM) / MgCl 2 (2 mM) mixture, in 24-well plates (P24) for 24 hours at 37 ° C.
Mesure de l'apoptose par la -technique Annexine V/PI :Measurement of apoptosis by the Annexin V / PI technique:
Les cellules sont ensuite collectées, lavées 2 fois puis incubées avec l' annexine V-FITC et l'iodure de propidium (PI) en accord avec les instructions du fournisseur (BD Biosciences). Les cellules sont analysées par cytométrie en flux (cytomètre EPICS XL Beckman Cόulter) en utilisant le logiciel Expo 32 de chez Beckman Coulter. La fixation de l' annexine V correspondant à un signal précoce d'apoptose alors que la fixation du PI étant plus tardive (fragmentation ADN) , le pourcentage de cellules dites apoptotiques est défini arbitrairement comme la somme des cellules Annexine V positives et des cellules doublement positives Annexine V/PI.The cells are then collected, washed twice and then incubated with annexin V-FITC and propidium iodide (PI) according to the supplier's instructions (BD Biosciences). The cells are analyzed by flow cytometry (Beckman Cόulter EPICS XL cytometer) using the software Expo 32 from Beckman Coulter. The annexin V binding corresponds to an early signal of apoptosis whereas the fixation of the PI being later (DNA fragmentation), the percentage of so-called apoptotic cells is arbitrarily defined as the sum of the Annexin V positive cells and the doubled cells. positive Annexine V / PI.
Exemple 1: Potentialisation de l'apoptose par un anticorps anti-IgG humaine (cross linker)Example 1 Potentiation of apoptosis by an anti-human IgG antibody (cross linker)
L'apoptose des cellules Daudi observée en absence d'anticorps (mort naturelle) est de l'ordre de 10%. Cette valeur de base est déduite des résultats obtenus lors des différentes expériences et se situe donc arbitrairement à 0%.The apoptosis of Daudi cells observed in the absence of antibody (natural death) is of the order of 10%. This basic value is deduced from the results obtained during the different experiments and is thus arbitrarily at 0%.
Dans le tableau 1 (cf figure 1) , les résultats sont exprimés en pourcentage de cellules apoptotiques (AnnexineV et PI positive) . Les anticorps anti-HLA-DR EMABling® et CHO utilisés seuls (condition 1) à la concentration de lμg/ml induisent peu d'apoptose sur Daudi (<3%) . Par contre en présence d'un anticorps dirigé contre la région Fc d'une immunoglobuline humaine (condition 2) qui agrège les anticorps anti- HLA-DR à la surface de la cellule Daudi, l'apoptose
est potentialisée d'une façon identique pour les deux anticorps, passant de moins de 3% à 19%. Sur la figure 4 A, les résultats sont exprimés en pourcentage de cellules apoptotiques observées, l'anticorps EMABling® représentant arbitrairement 100% pour chaque condition étudiée. Dans ces conditions l'étude statistique montre qu'il n'y a pas de différences significatives entre la potentialisation de l'apoptose induite par l'anticorps exprimé dans CHO et l'anticorps anti-HLA-DR EMABling® (p=0.85).In Table 1 (see FIG. 1), the results are expressed as a percentage of apoptotic cells (AnnexinV and PI positive). The anti-HLA-DR EMABling® and CHO antibodies used alone (condition 1) at a concentration of 1 μg / ml induce little apoptosis on Daudi (<3%). On the other hand, in the presence of an antibody directed against the Fc region of a human immunoglobulin (condition 2) which aggregates anti-HLA-DR antibodies on the surface of the Daudi cell, apoptosis is potentiated in an identical manner for both antibodies, from less than 3% to 19%. In FIG. 4A, the results are expressed as the percentage of apoptotic cells observed, the EMABling® antibody arbitrarily representing 100% for each condition studied. Under these conditions, the statistical study shows that there are no significant differences between the potentiation of the apoptosis induced by the antibody expressed in CHO and the anti-HLA-DR EMABling® antibody (p = 0.85). .
La figure 4 B indiquant le pourcentage de cellules apoptotiques , montre que l'apoptose des cellules B issues de sang périphérique, n'est obtenue qu'en présence d'un anticorps anti-IgG humaine. Dans ces conditions, cette étude montre qu'il n'y a pas de différences significatives entre la potentialisation de l'apoptose induite par l'anticorps exprimé dans CHO (DR CHO) et l'anticorps anti-HLA-DR EMABling® (DR YB2/0) .Figure 4 B showing the percentage of apoptotic cells, shows that apoptosis of B cells from peripheral blood, is obtained in the presence of an anti-human IgG antibody. Under these conditions, this study shows that there are no significant differences between the potentiation of the apoptosis induced by the antibody expressed in CHO (DR CHO) and the anti-HLA-DR EMABling® antibody (DR YB2 / 0).
Exemple 2 : Potentialisation de l'apoptose par Jurkat CDl6Example 2 Potentiation of Apoptosis by Jurkat CD16
Les résultats sur cellules Daudi (tableau 2, cf figure 2) sont exprimés en pourcentage de cellules apoptotiques (AnnexineV et PI positives) . Les anticorps anti-HLA-DR ΞMABling® et CHO utilisés à la concentration de Iμg/ml en présence de cellules témoinThe results on Daudi cells (Table 2, cf. FIG. 2) are expressed as a percentage of apoptotic cells (AnnexineV and PI positive). The anti-HLA-DR antibodies ΞMABling® and CHO used at the concentration of Iμg / ml in the presence of control cells
Jurkat (non transfectées) induisent respectivement 3% et 2% d'apoptose sur Daudi (condition 3) . En présence de la cellule Jurkat CD16 (condition 4) , qui peut interagir via le CD16 avec la partie Fc des anticorps anti-HLA-DR fixés à la surface de la cellule Daudi, l'apoptose est potentialisée. En présence des anticorps produits par les cellules CHO et EMABling® le pourcentage final d'apoptose est respectivement deJurkat (untransfected) induce respectively 3% and 2% apoptosis on Daudi (condition 3). In the presence of the Jurkat CD16 cell (condition 4), which can interact via CD16 with the Fc portion of the anti-HLA-DR antibodies attached to the surface of the Daudi cell, the apoptosis is potentiated. In the presence of the antibodies produced by the CHO and EMABling® cells, the final percentage of apoptosis is respectively
8% et 19%. Ainsi la potentialisation de l'apoptose en
présence de l'anticorps EMΑBling® est supérieure d'environ 100% par rapport à celle induite obtenue avec l'anticorps produit dans CHO. Sur la figure 5 A les résultats sont exprimés en pourcentage de cellules apoptotiques observées, l'anticorps EMABling® représentant arbitrairement 100% pour chaque condition étudiée. Dans ces conditions l'étude statistique montre que la différence de potentialisation de l'apoptose est très significative entre les deux anticorps (p<0.0001) et ceci en faveur de l'anticorps EMABling®.8% and 19%. Thus the potentiation of apoptosis in The presence of the EMΑBling® antibody is approximately 100% greater than that induced with the antibody produced in CHO. In FIG. 5A, the results are expressed as the percentage of apoptotic cells observed, the EMABling® antibody arbitrarily representing 100% for each condition studied. Under these conditions the statistical study shows that the difference in potentiation of apoptosis is very significant between the two antibodies (p <0.0001) and this in favor of the EMABling® antibody.
Par ailleurs, l'apoptose de cellules B issues de sang périphérique est étudiée après avoir substitué l'anticorps anti-IgG humaine (agent réticulant, ou cross-linker) par une cellule Jurkat transfectée par le CDl6. Les cellules Jurkat sauvages non transfectées (WT) sont utilisées en tant que témoin négatif. Les résultats exprimés en pourcentage de cellules apoptotiques (figure 5 B), indiquent que l'apoptose induite par l' anti-HLA-DR EMABling® (DR YB2/0) est fortement potentialisée par rapport à celle induite par l'anticorps produit dans CHO (DR CHO) . Cette propriété est probablement due à la meilleure fixation de l' anti-HLA-DR EMABling® (DR-YB2/0) sur le CDl6 par rapport à celle de l'anti HLA-DR CHO (DR CHO).Furthermore, apoptosis of B cells derived from peripheral blood is studied after substituting the anti-human IgG antibody (crosslinking agent or cross-linker) by a Jurkat cell transfected with CD16. Untransfected wild Jurkat cells (WT) are used as a negative control. The results expressed as a percentage of apoptotic cells (FIG. 5B) indicate that apoptosis induced by anti-HLA-DR EMABling® (DR YB2 / 0) is strongly potentiated compared to that induced by the antibody produced in CHO (DR CHO). This property is probably due to the better binding of the anti-HLA-DR EMABling® (DR-YB2 / 0) on the CD16 compared to that of the anti-HLA-DR CHO (DR CHO).
La forte interaction des anticorps EMABling® avec leThe strong interaction of EMABling® antibodies with
CD16 se traduit non seulement par une augmentation de l'ADCC et de la sécrétion de cytokines (cf document EPCD16 results not only in an increase in ADCC and secretion of cytokines (see EP document
1 537 419) mais également par une potentialisation de l'apoptose. Ceci confère aux anticorps EMABling® un avantage fonctionnel supplémentaire important par rapport au même anticorps produit dans CHO.
Références1,537,419) but also by potentiation of apoptosis. This confers on EMABling® antibodies an important additional functional advantage over the same antibody produced in CHO. References
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Natural killer cell activity in families of patients with systemic lupus erythematosus : démonstration of a killing defect in patients. Clin Exp Immunol. 2005 JuI; 141(1) :165-73.Natural killer cell activity in families of patients with systemic lupus erythematosus: demonstration of a killing defect in patients. Clin Exp Immunol. 2005 JuI; 141 (1): 165-73.
Katrinakis G, Kyriakou D, Papadaki H, Kalokyri I, Markidou F, Eliopoulos GD. Defective natural killer cell activity in B-cell chronic lymphocytic leukaemia is associated with impaired release of natural killer cytotoxic factor(s) but not of tumour necrosis factor-alpha. Acta Haematol. 1996 ; 96 (1) : 16-23.Katrinakis G, Kyriakou D, Papadaki H, Kalokyri I, Markidou F, Eliopoulos GD. Defective natural killer cell activity in B-cell chronic lymphocytic leukemia is associated with impaired release of natural killer cytotoxic factor (s) but not of tumor necrosis factor-alpha. Acta Haematol. 1996; 96 (1): 16-23.
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Claims
1. Méthode pour induire in vitro l'apoptose d'une cellule cible par une composition d'anticorps monoclonaux, comprenant la mise en contact de ladite composition d'anticorps et de ladite cellule cible, en présence de cellules exprimant le CD16 à leur surface, lesdits anticorps étant dirigés contre ladite cellule cible et ladite composition d'anticorps possédant une teneur en fucose inférieure à 65%.A method for inducing apoptosis of a target cell by a monoclonal antibody composition in vitro comprising contacting said antibody composition with said target cell in the presence of cells expressing CD16 on their surface said antibodies being directed against said target cell and said antibody composition having a fucose content of less than 65%.
2. Méthode selon la revendication 1, caractérisée en ce que ladite teneur en fucose est inférieure à 30%.2. Method according to claim 1, characterized in that said fucose content is less than 30%.
3. Méthode selon l'une quelconque des revendications 1 ou 2, caractérisée en ce que ladite teneur en fucose est comprise entre 20% et 45% , ou entre 25% et 40%.3. Method according to any one of claims 1 or 2, characterized in that said fucose content is between 20% and 45%, or between 25% and 40%.
4. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que ladite composition d'anticorps est produite par la lignée cellulaire YB2/0.4. Method according to any one of the preceding claims, characterized in that said antibody composition is produced by the YB2 / 0 cell line.
5. Utilisation de la lignée cellulaire YB2/0 pour produire des anticorps à forte capacité d'apoptose.5. Use of the YB2 / 0 cell line to produce antibodies with high apoptosis capacity.
6. Procédé de sélection de compositions d'anticorps à forte capacité apoptotique, comprenant les étapes suivantes : i. mise en contact de l'anticorps à évaluer avec l'antigène dudit anticorps (ou d'une cellules exprimant cet antigène) en présence de cellules exprimant le CDl6 à leur surface. ii . mesure de l'induction de l'apoptose iii . sélection des compositions d'anticorps montrant une induction d'apoptose supérieure d'au moins 20%, voire 50% ou plus, en présence de la cellule exprimant le CD16 par rapport au contrôle négatif (absence de COl6) .A method of selecting high apoptotic capacity antibody compositions, comprising the steps of: i. contacting the antibody to be evaluated with the antigen of said antibody (or a cell expressing that antigen) in the presence of cells expressing CD16 on their surface. ii. measurement of induction of apoptosis iii. selecting antibody compositions showing greater apoptosis induction of at least 20%, or even 50% or more, in the presence of the cell expressing CD16 compared to the negative control (absence of COl6).
7. Procédé selon la revendication 6, caractérisé en ce que les anticorps sélectionnés sont des anticorps anti-idiotypiques anti-inhibiteurs du facteur VIII, des anticorps dirigés contre des autoanticorps, ainsi que des anticorps dirigés contre des protéines virales et/ou bactériennes exprimées à la surface de cellules infectées .7. Method according to claim 6, characterized in that the antibodies selected are anti-idiotypic anti-factor VIII inhibitory antibodies, antibodies directed against autoantibodies, as well as antibodies directed against viral and / or bacterial proteins expressed in vitro. the surface of infected cells.
8. Utilisation d'une composition d'anticorps monoclonaux, laquelle composition possède une teneur en fucose inférieure à 65%, ou est obtenue par le procédé selon l'une des revendications 6 ou 7, pour la préparation d'un médicament destiné au traitement, sans mise en jeu des fonctions cytotoxiques des cellules immunitaires effectrices, d'un déficit quantitatif ou qualitatif (absence d'ADCC) en cellules NK.8. Use of a composition of monoclonal antibodies, which composition has a fucose content of less than 65%, or is obtained by the method according to one of claims 6 or 7, for the preparation of a medicament for the treatment without involvement of the cytotoxic functions of the effector immune cells, a quantitative or qualitative deficit (absence of ADCC) in NK cells.
9. Utilisation- selon la revendication 8, dans laquelle la teneur en fucose de ladite composition d'anticorps monoclonaux est inférieure à 30%.Use according to claim 8, wherein the fucose content of said monoclonal antibody composition is less than 30%.
10. Utilisation selon la revendication 8, dans laquelle la teneur en fucose de ladite composition d'anticorps monoclonaux est comprise entre 20% et 45%, ou entre 25% et 40%. Use according to claim 8, wherein the fucose content of said monoclonal antibody composition is between 20% and 45%, or between 25% and 40%.
11. Utilisation selon la revendication 8, dans laquelle ladite composition d'anticorps monoclonaux est produite par la lignée cellulaire YB2/0.The use of claim 8, wherein said monoclonal antibody composition is produced by the YB2 / 0 cell line.
12. Utilisation selon l'une quelconque des revendications 8 à 11, pour la préparation d'un médicament destiné au traitement de la LLC-B.12. Use according to any one of claims 8 to 11 for the preparation of a medicament for the treatment of LLC-B.
13. Utilisation selon l'une quelconque des revendications 8 à 11, pour la préparation d'un médicament destiné au traitement du lupus érythémateux, des myopathies cardiaques congéstives, des histiocytoses avec hémophagocytose ainsi que du VIH et de l'hémophilie A ou B. 13. Use according to any one of claims 8 to 11, for the preparation of a medicament for the treatment of lupus erythematosus, cardiac myopathies, histiocytosis haemophagocytosis as well as HIV and haemophilia A or B.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0512815A FR2895086B1 (en) | 2005-12-16 | 2005-12-16 | POTENTIATION OF APOPTOSIS BY MONOCLONAL ANTIBODIES |
| PCT/FR2006/002767 WO2007071839A2 (en) | 2005-12-16 | 2006-12-15 | Potentiation of apoptosis by monoclonal antibodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1969368A2 true EP1969368A2 (en) | 2008-09-17 |
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|---|---|---|---|
| EP06841970A Ceased EP1969368A2 (en) | 2005-12-16 | 2006-12-15 | Potentiation of apoptosis by monoclonal antibodies |
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| US (1) | US20090305228A1 (en) |
| EP (1) | EP1969368A2 (en) |
| JP (1) | JP2009519302A (en) |
| KR (1) | KR20080106164A (en) |
| CN (1) | CN101375160A (en) |
| AU (1) | AU2006328798B2 (en) |
| BR (1) | BRPI0619994A2 (en) |
| CA (1) | CA2633331A1 (en) |
| FR (1) | FR2895086B1 (en) |
| IL (1) | IL192195A0 (en) |
| WO (1) | WO2007071839A2 (en) |
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| US5783186A (en) * | 1995-12-05 | 1998-07-21 | Amgen Inc. | Antibody-induced apoptosis |
| AU3827099A (en) * | 1998-05-08 | 1999-11-29 | Universite Catholique De Louvain | Apoptosis of naive human nk cells by crosslinking of their fcgammariiia molecules wiht a rat igg2b (lo-cd2a/bti-322) or its igg1 humanized monoclonal antibody |
| FR2807767B1 (en) * | 2000-04-12 | 2005-01-14 | Lab Francais Du Fractionnement | MONOCLONAL ANTIBODIES ANTI-D |
| US7595165B2 (en) * | 2002-09-13 | 2009-09-29 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Method of measuring activation of effector cells |
| FR2844455B1 (en) * | 2002-09-13 | 2007-12-14 | Lab Francais Du Fractionnement | TREATMENT OF PATHOLOGIES EXCLUDING IMMUNE RESPONSE BY OPTIMIZED ANTIBODIES |
| FR2844513B1 (en) * | 2002-09-13 | 2007-08-03 | Lab Francais Du Fractionnement | ANTIBODIES FOR ADCC AND INDUCING PRODUCTION OF CYTOKINS. |
| FR2858235B1 (en) * | 2003-07-31 | 2006-02-17 | Lab Francais Du Fractionnement | USE OF ANTIBODIES OPTIMIZED IN ADCC TO TREAT LOW PATIENT PATIENTS |
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2005
- 2005-12-16 FR FR0512815A patent/FR2895086B1/en not_active Expired - Fee Related
-
2006
- 2006-12-15 US US12/097,686 patent/US20090305228A1/en not_active Abandoned
- 2006-12-15 KR KR1020087017266A patent/KR20080106164A/en not_active Application Discontinuation
- 2006-12-15 WO PCT/FR2006/002767 patent/WO2007071839A2/en active Application Filing
- 2006-12-15 CA CA002633331A patent/CA2633331A1/en not_active Abandoned
- 2006-12-15 BR BRPI0619994-1A patent/BRPI0619994A2/en not_active IP Right Cessation
- 2006-12-15 EP EP06841970A patent/EP1969368A2/en not_active Ceased
- 2006-12-15 AU AU2006328798A patent/AU2006328798B2/en not_active Ceased
- 2006-12-15 JP JP2008545047A patent/JP2009519302A/en active Pending
- 2006-12-15 CN CNA2006800529235A patent/CN101375160A/en active Pending
-
2008
- 2008-06-15 IL IL192195A patent/IL192195A0/en unknown
Non-Patent Citations (2)
| Title |
|---|
| DA SILVA ANTONIO J. ET AL: "Alefacept, an Immunomodulatory recombinant LFA-3/IgG1 fusion protein, induces CD16 signaling and CD2/CD16-dependent apoptosis of CD2+ cells", THE JOURNAL OF IMMUNOLOGY, vol. 168, 27 February 2002 (2002-02-27), pages 4462 - 4471 * |
| DE ROMEUF CHRISTOPHE ET AL: "Chronic lymphocytic leukaemia cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved engagement of FcgammaRIIIA/CD16", BRITISH JOURNAL OF HAEMATOLOGY, vol. 140, 2008, pages 635 - 643 * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2006328798A1 (en) | 2007-06-28 |
| CA2633331A1 (en) | 2007-06-28 |
| BRPI0619994A2 (en) | 2011-10-25 |
| CN101375160A (en) | 2009-02-25 |
| FR2895086A1 (en) | 2007-06-22 |
| FR2895086B1 (en) | 2012-10-05 |
| JP2009519302A (en) | 2009-05-14 |
| IL192195A0 (en) | 2008-12-29 |
| WO2007071839A3 (en) | 2007-08-23 |
| KR20080106164A (en) | 2008-12-04 |
| AU2006328798B2 (en) | 2012-02-09 |
| US20090305228A1 (en) | 2009-12-10 |
| WO2007071839A2 (en) | 2007-06-28 |
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