NON-RADIOACTIVE METHOD FOR DETERMINING THE CYTOLYTIC ACTION OF AN AGENT FOR TARGET CELLS, USE THEREO

专利摘要:
Non-radioactive method for direct in vitro determination (control and quantification) of the cytolytic action of an agent active against target cells and / or a target cell environment, comprising the steps of genetic transformation of target cells for expressing an enzyme exogenous to said target cells, exposing said target cells genetically transformed to the active agent and / or said environment to be tested, which can lead to the lysis of at least a portion of the target cells by releasing said exogenous enzyme in the extracellular medium, and measuring the activity of the exogenous enzyme released during the lysis of said target cells, characterized in that said exogenous enzyme is an enzyme of molar mass less than or equal to 45 kDa , and whose activity is detectable by luminescence or fluorescence. Application to measurement of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or measurement of apoptosis.
公开号:FR3033335A1
申请号:FR1557175
申请日:2015-07-28
公开日:2016-09-09
发明作者:Veronique Bonnaudet；Laurent Bretaudeau；Alexis Rossignol
申请人:Clean Cells；
IPC主号:

专利说明:

[0001] The present invention relates to the field of measurement of cell death and more particularly to a method for determining the cytolytic action of active substances with respect to target cells and / or a medium surrounding said target cells. In the industrial field, the regulation imposes to characterize as completely as possible any phenomenon or product that may have consequences for the health of human or animal populations. In the broad context of the implementation of tests measuring cell death, called cytotoxicity tests, these phenomena or products may include environmental phenomena, such as exposure to an irradiation or pollution source, chemicals or non-health biological or therapeutic products. It is in the field of biologically active substances, and more specifically in the context of substances for therapeutic purposes, that the characterization requirements are the most advanced. One of the main criteria to characterize is that of biological activity, also called "potency". Indeed, according to the regulatory definition of the European Medical Agency (CHMP / BWP / 157653/2007), the potency is the quantitative measurement of a biological activity based on an attribute of the pharmaceutical product related to a relevant biological property of the latter, this measure having to reflect its biological activity in the clinical situation for which the product is designed. According to the regulations, a potency test must therefore be based on the direct measurement of the expected biological function of the test product, consistent with its known or assumed mode of action. In other words, and in the case of a product whose purpose is to cause the death of a given cell population (the death of tumor cells in the case of anti-cancer treatment, for example), a cytotoxicity test must be based on the direct measurement of the death of the target cell population. Similarly, the establishment of a test seeking the absence (or existence) of cytotoxic effects on a product not intended for therapeutic use must also be based on a method specifically detecting the death of the target cells. . In the case of biological drugs, in particular that of antibodies (or immunoglobulins, labeled Ig) for therapeutic use, cytotoxicity tests are among the long list of quality control tests required by the regulation before placing them on the market. molecules.
[0002] According to the regulatory texts, these tests should reflect as much as possible the biological activity of the drug in its clinical use, based on its known or suspected mechanisms of action. However, the modes of action of the antibodies are varied: Mechanisms dependent on the Fab region of the antibody: direct action of the antibodies by their antigen binding activity. These mechanisms are specific to each antibody and may correspond to i) the neutralization of an antigen, ii) the neutralization of a membrane antigen by antagonism or iii) the agonist action on a membrane antigen. In the latter two cases, the agonist or antagonist action on a membrane antigen may lead to inhibition of growth of the target cell population and / or induction of death by apoptosis and / or necrosis. - Effector mechanisms mediated by the Fc region: mechanisms common to all antibodies, their intensity depends on the class and the subclass of the antibodies as well as their physicochemical structure (amino acid sequence, structure and composition of the glycosylated chains, etc. .). These effector functions mediated by the Fc region will depend on the interaction of said Fc region with two major types of immune system specific receptors: i) the complement system via the interaction between the Fc region and the first C1 component of the proteolytic cascade of complement; ii) the Fc region receptors of antibodies for Ig (FcR). In many cases, particularly when the antibody is directed against a membrane molecule, the mechanism sought to observe the therapeutic effect is of the lytic type (for example, in the case of onco-hematology use). Lysis can be obtained, for example, by induction of apoptosis / necrosis or inhibition of cell proliferation via agonism / antagonism phenomena, activation of the complement system or recruitment of cytotoxic cells expressing on the surface. one of the FcRs 25 (such as natural killer cells (NK), macrophages, cytotoxic lymphocytes, polynuclear cells, etc.). In such cases, regulatory texts therefore require the establishment of a potency test measuring cell lysis in a biologically relevant manner and consistent with the mode of action envisaged in vivo. A first object of the invention is therefore to propose a method for directly measuring cell death (cytotoxicity) by measuring the amount released in the medium of an enzyme artificially introduced into said cells.
[0003] BACKGROUND OF THE INVENTION Cytotoxicity assays, or cytolysis, are widely used in biological, clinical and pharmaceutical research to measure cell death induced by any biologically active substance, by any biologically relevant mechanism, in order to screen a bank. candidate molecules, identify a mechanism of action or characterize the biological activity of a pharmaceutical product for example (potency test). A cytotoxicity test performed as part of a potency measurement consists of measuring the death of cells of interest (generally called target cells) under experimental conditions consistent with the biological mechanism under consideration. In the context of the therapeutic antibodies, three main mechanisms of action can be explored by this test: 1) an induction of apoptosis by agonism or antagonism of a membrane molecule by the antibody; 2) Activation of the complement system (CDC for "ComplementDependent Cytotoxicity") by the antibody adsorbed to the target cell; 3) a lysis mechanism, by ADCC (for "Antibody-Dependent Cell-mediated Cytotoxicity") or by phagocytosis, mediated by cellular effectors recruited via the interaction between the FcR they express on their surface and the Fc region antibodies adsorbed to the target cells. To carry out each of these tests, target cells are incubated with the antibody in question in the presence, respectively, of 1) the culture medium alone, 2) the culture medium containing a source of complement or 3) the culture medium. containing appropriate effector cells.
[0004] To date, there are several methods of direct measurement of cell death, that is to say whose measured data are directly and effectively derived from the death of the target cells: a). method of releasing a radioactive isotope (51 Cr, 1111n, 3H). H). This method involves incubating, prior to the cytolysis experiment itself, the target cells in a sodium chromate solution, which enters the cells to bind to the intracellular proteins. When the cells die, their intracellular content is released into the supernatant. A measurement of the radioactivity of the supernatant thus allows a direct measurement of the quantity of dead target cells, even if other types of cells (for example effector cytotoxic cells) were mixed with the target cells during the test because only the target cells were radiolabelled. The background of the method (i.e., the signal intensity in the absence of cell death) is minimal due to a very low 51 Cr spontaneous release on the mean time a cytotoxicity test lasts. (3 to 4 hours minimum). In addition, by its radioactive nature, the signal generated is very intense, which leads to a high signal-to-noise ratio (of the order of 5 to 12). The method is very sensitive. Developed at the end of the 1960s, this method is still, to this day, the reference method, because of its specificity with regard to the death of the target cells and its high performances, particularly in terms of sensitivity. . It also has 5 good high-throughput analysis capabilities because of signal stability over long periods of time (several hours to several days). On the other hand, it has the disadvantage of requiring an incompressible time of marking with 51Cr (incubation and washes) of approximately one to two hours according to the protocols, significantly lengthening the duration of the experiment. In addition, the labeling adds variability to the assay, related to the amount and quality of the reagents added, to the different incubation times and experimental steps. Finally, regulatory constraints related to the use of radioactivity are increasingly burdensome and costly, in terms of administrative authorization, source management, waste disposal, labor law, medical follow-up or exposure of manipulators. Several variants of this method have also been described, using other radioisotopes, for example based on the use of tritium (3H) or indium (1111n) but involving the same radiation protection constraints and therefore subject to the same limitations. b). calcein-acetoxymethyl (calcein-AM) method. This compound is permeable to lipid bilayers (thus to the membranes of eukaryotic cells) thanks to its acetoxymethyl radical and will therefore penetrate the cells. Once inside, the acetoxymethyl radical is cleaved by intracellular enzymes (esterases), thus restoring the fluorescence properties of calcein. The calcein released into the supernatant is used to measure the amount of lysed cells. This method has interesting high-throughput analysis capabilities avoiding radioactivity issues. In contrast, the spontaneous release of calcein is very high (about 40% of the maximum release) reflecting a high permeability of the plasma membrane to calcein, which leads to a high background of the method. Since the fluorescent signal emitted by calcein is weak, the signal-to-noise ratio is unfavorable, around 2.5. This method therefore has low sensitivity and performance and has a target cell labeling step with calcein-AM which increases the overall test duration from one to two hours and further enhances its variability. vs). method based on the use of lanthanides, such as europium (Eu3 +) or terbium (Tb3 +), complexed with a chelate or fluorescence enhancer such as diethylenetriaminopentaacetate (DTPA) or 2,2 'acid: 6 ', 2 "-Terpyridine-6,6" -dicarboxylic acid (TDA), similar in principle to the 51 Cr and calcein-AM methods. For example, in DELFIA technology (commercially available from Perkin Elmer, Boston, MA), target cells are loaded with a fluorescent enhancer ligand, BATDA (bis [acetoxymethyl] 2,2 ': 6', 2 "-terpyridine- 6.6 "-dicarboxylate) which penetrates through the plasma membranes. In the cell, ester bonds are hydrolyzed by esterases to form a hydrophilic ligand, TDA, in theory poorly permeable to the membrane (after addition of probenecid, an inhibitor of MDR, "Mufti Drug Resistance" transporter) and released into the extracellular medium during cytolysis. After removal of the supernatant, a solution of europium (Eu) is added so that it complexes with the free TDA to form a fluorescent chelate (EuTDA). The measurement of this signal, of the TRF ("Time-Resolved Fluorescence") type, is indicative of the quantity of lysed cells. In another case, the target cells are labeled with europium and its release into the supernatant is measured by the addition of the DTPA chelate. These methods are non-radioactive and have high throughput analysis capability. On the other hand, the spontaneous release of TDA or free europium is quite high and variable according to cell type: the maximum release for Eu3 + is twice that with 51Cr. In some cases (Figure 1), the intensity of the spontaneous release is almost equal to that of the specific signal and has a variability in the signal intensity related to the cellular type considered or the physiological state of the cell (which conditions for example, the nature and activity of its esterases). This method also requires an experimental time for labeling the target cells with europium or BATDA, as well as an incubation time of the supernatants with the second component (TDPA or Eu3 +, respectively) increasing the overall experimental duration of the test. and its variability by adding additional steps. All of these limitations result in a rather high variability of the Eu3 + assay, as well as an inability to employ it with a number of cell types, which makes its use and validation difficult in an industrial context. . d). methods using the flow cytometry technique (and its derivative of flow cytometry imaging) to measure the frequency of living and / or dead cells during the test. These methods have in common the use of a combination of one or more fluorescent markers to distinguish target and effector cell populations. Alternatively, some authors have used definitive labeling of target cells by genetically transforming them to stably express a fluorescent protein. This labeling of target populations is coupled with detection of cell viability by the use of appropriate markers (usually interleaving DNA agents impermeable to living cell membranes but entering apoptotic or dead cells). The combination of these markers makes it possible to determine a frequency and / or a number of dead or living cells within a given population (usually the target cells). These methods, although specific to the mechanism of cell death, do not allow a high-throughput analysis because of the technical constraints related to flow cytometry. For example, certain probes may induce cross-labeling during the reaction (by exchange between the different cell types of probes linked by non-covalent bonds). Depending on the methods and equipment, the sample acquisition time in flow cytometry can be from several seconds to tens of seconds. The resulting non-simultaneous acquisition of the samples has the consequence that it can take several tens of minutes between the first and the last sample of a series, during which time the physiological conditions of the cells can evolve. These different elements have important consequences on the variability of the tests and, to achieve with these methods performances compatible with the standards of the pharmaceutical industry, the analysis can not exceed a few samples (in practice not more than three) tested simultaneously. , which corresponds to a low flow. e). method based on a microscope count by an operator or an automated system. The distinction between dead and living cells and their enumeration is achieved through the use of a vital dye, for example trypan blue or eosin. Nevertheless, these microscopic methods do not make it possible to discriminate between several distinct cell types which would have been mixed for the purposes of the test (in the case of a measurement of ADCC for example), except to use fluorescence microscopy methods. . We then return to methods similar to those described for flow cytometry but which implement laborious counting methods and whose disadvantages already mentioned are further amplified. These methods, slow, not very reproducible and not at all adapted to a high-throughput analysis, are in practice not used in the context of potency tests. f). impedance variation: a number of electronic methods exist for measuring the detachment of cells adhering to a support (this phenomenon may be from 30 to the death of the cells, but not exhaustively). These methods are generally based on the measurement of an impedance variation induced by the quantity and / or the physiological state of the cells present on a suitable support. Nevertheless, such methods are not suitable when several different cell types are mixed and the death of a single type has to be measured (for example ADCC tests). In addition, these methods require the use of adherent target cells, expensive equipment, often associated with a complex experimental device. And above all, the measure of cellular detachment does not correspond to cytolysis mechanisms; it can, at best, be correlated with it. Thus, their implementation in an industrial setting is therefore difficult. boy Wut). methods based on the detection of components naturally expressed in the cytoplasm of eukaryotic cells. Such molecules must be free in the cytoplasm (i.e. not included in vesicles or bound to organelles) in order to be released into the supernatant if the cell is lysed, but they must not be secreted In the extracellular medium when the cells are in their physiological state. Their presence in the supernatant must be easily measurable by current methods and they must be strongly expressed (i.e. a large number of molecules per cell) and this by the most possible different cell types (ubiquitous molecules). There are a very large number of described solutions or commercially available kits claiming a measure of cytotoxicity by such methods. The revelation technologies are generally based on the addition to the supernatant of one or more buffers, substrates, enzymes and / or reagents for carrying out the assay by generating a final molecule measurable by bioluminescence, chemiluminescence, colorimetry or fluorescence. It should be noted that in most cases these tests can also be used "backwards" to determine the overall viability of the cell population. These methods have the advantage of not requiring specific labeling of the target cells (reducing the risk of variability and the handling time) and allow, by their nature, high throughput analysis. Nevertheless, the major and unacceptable disadvantage of all these tests based on the release of a ubiquitous molecule for measuring cell lysis is that they are no longer relevant when several cell types are mixed for carrying out the test. . Indeed, it is impossible to discriminate which of the cell types participated, and in what proportion, the release of the molecule. This is for example the case of ADCC assays in which target cells and effector cells must be mixed to observe the cytotoxic effect. The effector cells that died during the test, in connection with their usual mortality rate or by the mechanisms related to the test itself (such as exhaustion or redirected lysis, for example), will also participate in the signal. In this sense, it is not therefore specific methods for lysis of the target cells. In addition, some molecules to be measured are not sufficiently stable during the duration of the test (for example ATP has a very short half-life in the extracellular medium), others have a high background and / or a weak signal intensity leading to a lack of sensitivity of the method. In view of these elements, another object of the invention is therefore to provide a non-radioactive cytotoxicity method, in order to overcome the many regulatory and health constraints. Another object of the invention is to propose a method of cytotoxicity that does not require extemporaneous labeling of the target cell (in order not to imply a loss of experimental time and an increase in the overall variability of the test by adding several steps. variables), while maintaining a direct and specific measurement of the lysis of said cell. To overcome the aforementioned drawbacks and circumvent the need for extemporaneous labeling of the target cell, while maintaining a direct and specific measurement of the lysis of said cell, Schafer et al. described in 1997 (Journal of Immunological Methods, 204 pp. 89-98, 1997) a method of genetic transformation to obtain target cell lines stably expressing an exogenous intracytoplasmic enzyme. In this method, the death of said cells theoretically should allow the release of this enzyme in the extracellular medium, the amount of enzyme being then measured by an appropriate method. These authors first of all carried out the transformation of a target line by the F-Luc gene (Firely Luciferase, firefly luciferase) under the control of the beta-actin promoter.
[0005] However, the lifetime of F-Luc in the extracellular medium is very short (half-life of 30 minutes) and incompatible with a test of ADCC or CDC whose mechanism of action requires 2 to 4 hours to reach the maximum of lysed cells. Then they evaluated the transformation of the target cells by beta-galactosidase and compared these performances to that of the traditional radioactive chromium labeling method. Although beta- galactosidase is more stable than F-Luc, and the signal-to-noise ratio is better with beta-galactosidase than with the radioactive chromium method, the betagalactosidase method underestimates about 30 to 40% the amount of dead cells, probably due to an incomplete release of this enzyme during cell death. It is therefore not applicable to effectively measure the death of target cells in a non-radioactive therapeutic antibody characterization test aimed at meeting regulatory requirements, for example in the characterization of ADCC and CDC activities.
[0006] More recently, other authors have described the use of target cells constitutively expressing F-Luc in an ADCC test, based on a different principle (Alpert et al., J. Virol 86: 12039, 2012). Fu et al., PLoS ONE 5: e11867, 2010): quantification of the luminescent signal used to evaluate the amount of living cells. It is therefore not a direct measurement method of target cell death, which is also subject to certain limitations. Thus, the half-life of the F-Luc being short, and the test being relatively long (8 hours), the F-Luc activity is sensitive to variations such as a possible disturbance of the physiological state of the cells during the test (decreased synthesis, increased catabolism), without this variation being directly related to a change in cell mortality and thus generating a first level of variability of the method. Similarly, since the measurement is homogeneous over the lysed cell suspension after the test, it is highly likely that the F-Luc produced during the test (or at least in the last few hours, given its short half-life) and released by dead cells into the supernatant will generate a second level of variability by adding its signal to that of purely intracellular F-Luc. The presence of residual F-Luc activity in the supernatant was also noted by the authors. Finally, this method requires very long experimental times (at least 8 hours) for the loss of signal to be sufficiently significant. This method is therefore not applicable to the functional characterization of therapeutic antibodies in the context of "potency" tests that comply with regulatory requirements.
[0007] Another object of the invention is therefore to overcome the aforementioned drawbacks and to propose a method including a genetic transformation step to obtain target cell lines expressing, stably throughout the duration of the test, an exogenous enzyme. intra-cytoplasmic, the death of said cells allowing the release (almost total) of this enzyme in the extracellular medium.
[0008] It is another object of the invention to provide a direct, sensitive and specific measurement method for the death of target cells (and not a measure of the disappearance of living cells), which is particularly applicable to the functional characterization of antibodies. therapeutics in the context of potency trials. These and other objects are achieved by the non-radioactive method of the present invention for direct in vitro determination (control and quantification) of the cytolytic action of an agent active against target cells and or a medium surrounding the target cells, comprising the following successive steps: i) genetic transformation of target cells to express an exogenous enzyme to said target cells; ii) exposure of said target cells genetically transformed to the target cells; active agent and / or or said environment to be tested, which can lead to the lysis of at least a part of the target cells by releasing said exogenous enzyme in the extracellular medium, iii) Measurement of the activity of the exogenous enzyme released upon lysis of said target cells characterized in that said exogenous enzyme is an enzyme of molar mass less than or equal to 45 kDa, and whose activity is detectable by luminescen this or fluorescence. Thus, the release of this exogenous enzyme, particularly because of its reduced size, is representative of the lysis of the target cells and gives results, in terms of background, maximum lysis, specific lysis and overall performance, similar or higher than the 51Cr reference method, as will be demonstrated later in the examples.
[0009] Advantageously, the molar mass of the exogenous enzyme is less than or equal to 30 kDa, preferably less than or equal to 25 kDa, more preferably less than or equal to 20 kDa. Advantageously, said exogenous enzyme has stable enzyme activity in the extracellular medium of steps ii) and iii) of said method for at least 3 hours, preferably for at least 24 hours, and more preferably for at least 48 hours, 20 to 20 hours. temperatures between 34 ° C and 40 ° C. According to a preferred embodiment, said exogenous enzyme is a luciferase. More particularly, said exogenous enzyme has a peptide sequence which can have at least 60% homology, preferably at least 80% homology with the wild-type 19 kDa subunit coding sequence of luciferase produced by shrimp. Oplophorus gracilirostris. Preferably, said exogenous enzyme is in a form with optimized activity and stability, marketed by Promega under the name NanoLuc®, referred to throughout the text as nanoluciferase.
[0010] Such an enzyme is in particular stable in the extracellular medium for periods of time compatible with the test types envisaged, that is to say at least 24 hours. The amount of exogenous enzyme released is measured by cleaving a substrate specific for said enzyme, the substrate preferably being from the coelenterazine family, more particularly from the furimazine family and its derivatives. The medium to be tested surrounding the target cells may comprise a biological agent and / or a chemical agent and / or a physical agent potentially active with respect to said target cells. The method therefore comprises, as a first step i), the genetic transformation of a relevant line of target cells to make it stably express said exogenous enzyme, segregated in the cytoplasm under physiological conditions. According to a first embodiment of the invention, the biological agent is an antibody, preferably chosen from monoclonal antibodies, especially for therapeutic purposes. In the case of antibodies or related molecules, the choice of the target target cell is made according to the target of the antibody to be tested. With each new experimental model (for a given antigenic target), a new target cell is developed, unless the same target naturally or artificially expresses several relevant antigens. Several options are possible for the choice of these target cells. The first option is to select, among the cell lines available from the 20 different banks of biological material deposits, a relevant line for the model to be developed. Some of these lines will be given by way of example in the following paragraph, limited to a few antigenic targets for which there are currently marketed antibodies. It is understood that other lines are possible, for the antigenic molecules mentioned but also for other antigenic targets not mentioned but for which antibodies are to be developed or under development. In the case of antibodies directed against molecules expressed primarily by normal or pathological B cells, such as CD19, CD20 or IL-6R (CD126), all lymphocyte or B lymphoma lines are possible candidates, for example, lines WIL2, WIL2-S, Daudi, Raji, Ramos, JY, MC116, GA-10, DOHH, ARH-77, SU-DHL, Z138 or their derivatives. In the case of antibodies directed against molecules expressed primarily by normal or pathological T lymphocytes, such as CD3, CD25 or LPAM ("Peyer Lymphocyte Patch Adhesion Molecule" or integrin alpha [4] beta [7]), all T lymphocyte or lymphoma type lines are potential candidates, for example the Jurkat, DERL, HD-MAR-2, HH, SR-786, SUP-T1, Loucy, CCRF-CEM, HUT-78 or their 5 lines. derivatives. The antibodies may also be antibodies directed against molecules expressed primarily by cells of normal or pathological lymphoid origin, such as human CTLA-4 (CD152), PD-1 (CD279) or CD30; in this case, all the B and T lymphocyte lines would be potential candidates, therefore for example all the B and T lines already mentioned above. The antibodies may also be antibodies directed against molecules expressed primarily by normal or pathological lymphoid and myeloid cells, such as human CD52, VLA4 (CD49d) or LFA-1 (CD11a); in this case, any lymphoid or myeloid lineage cell line would be suitable, or, in addition to the B and T cell lines already mentioned above, myeloid lines such as THP-1, HL-60 or U-937 or their derivatives, for example . The antibodies can also be antibodies directed against molecules expressed mainly by carcinoid cells, such as anti-EGFR (for "Epidermal Growth Factor Receptor", also noted HER1 or ErbB1), EGFR2 (or HER2, or ErbB2), EGFR -3 (or HER3, or ErbB3) or EpCAM (CD326): any line resulting from carcinoid tumors is then a potential candidate, for example, HCC1954, SKBR3, SKOV3, Caco-2, HeLa, MCF-7, PC-3 or their derivatives. The antibody can also be an antibody directed against the TNF-alpha molecule: the T cell or myeloid cell lines as described above are then potential candidates. The antibody may also be an antibody directed against the VEGF ("Vascular Endothelial Growth Factor") molecule or its VEGFR receptor: lines described to express these molecules would then be appropriate, for example the A375, M21, Hoc-7, Panc lines. -3, D283Med, DAOY, D341Med or their derivatives. The other possible option for the construction of relevant target cell lines is to have a single cell line expressing the selected exogenous enzyme, in which one or more antigenic targets of interest would be integrated, in a second step, by conventional techniques of genetic transformation. In this case, any cell line may be suitable provided it has a non-zero potential for genetic transformation. Preferably, the chosen line will not express human target antigenic potentials that could come to disturb the expression of the molecules introduced by genetic engineering, and thus increase the expression variability of the molecules, and therefore increase the general variability of the 3033335 13 'trial. For example, these cells may be of non-human origin, such as CHO, Sp2 / 0, NSO, NIH3T3 cells or their derivatives, or of embryonic human origin such as HEK293, IM R-90, NTera2 cells or their cells. derivatives. According to a second embodiment of the invention, the chemical and / or physical agent is chosen from chemotherapeutic agents or anti-cancer molecules, preferably cytotoxic molecules or molecules of the family of protein kinase inhibitors. . Advantageously, the method has consisted in expressing the nanoluciferase to the target cells. This nanoluciferase is a protein derived from the 19kDa subunit of a luciferase extracted from the deep-sea shrimp Oplophorus gracilirostris, the second subunit being a 35 kDa protein (proteins described in the article by S. Inouye et al., Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase, in FEBS Letters, 481 (2000) 19-25). This nanoluciferase was optimized and marketed by Promega under the name NanoLuc® (and described in M. Hall et al., Engineered luciferase reporter from a deep sea shrimp using a novel imidazopyrazinone substrate in ACS Chemical Biology 2012, 7, 1848-1857), for the purpose of improving reporter gene systems for systems of i) studying protein-protein or protein-ligand interactions; ii) Protein stability study; iii) energy-donor biosensors to a third acceptor molecule (BRET); iv) in vivo imaging; v) studies of viral load and replication; and vi) reporter assays for cell signaling (gene expression studies or intracellular protein metabolism monitoring). In particular, have been described to date: intracellular use as a reporter gene (signaling pathways, binding to a receptor, for example), imaging on live cells / animals (labeling cells to follow their biological path) or labeling bacteria or viruses for infection tracking; the coupling of nanoluciferase to a protein of interest to measure its internalization or secretion (Norisada et al., Biochem Biophys Res 449: 483, 2014). These systems have in common to couple the coding sequence of the nanoluciferase to that of another protein of interest and / or to the sequence of a genetic promoter activated specifically by the signaling pathway studied and / or to be restricted to intracellular detection of the molecule. In contrast, the inventors' approach has been to provide a method in which the target cells are genetically transformed to transiently or constitutively express said exogenous enzyme in a cytoplasmic and non-secreted form, more particularly to express the nanoluciferase alone. and in free form in the cytoplasm of the target cells and to measure only and specifically the amount of molecules released into the extracellular medium following lysis of cells by mechanisms such as ADCC, CDC, apoptosis or lysis by a detergent. For this purpose, the step of genetic transformation of the target cells advantageously comprises the introduction into said cells of an expression vector carrying the coding sequence of the exogenous enzyme and a constitutive type promoter allowing its transcription in a cell. such as a eukaryotic cell.
[0011] Preferably, the vector may be a viral vector, or a plasmid vector, preferably a plasmid vector. Advantageously, the expression vector of the exogenous enzyme comprises an antibiotic resistance gene. This antibiotic resistance gene allows a selection of eukaryotic target cells that have integrated the vector (so-called "transformed" cells).
[0012] The selective antibiotic is especially capable of eliminating eukaryotic cells not carrying the resistance gene. This antibiotic resistance gene may be, in particular, a geneticin resistance gene (G418), puromycin, blasticidine, hygromycin B, mycophenolic acid or zeocin, preferably a resistance gene to the puromycin.
[0013] Preferably, the introduction of said expression vector into said cells is carried out by infection with viral particles bearing the gene of the exogenous enzyme, when the expression vector is a viral vector or by chemical or physical methods. when the expression vector is a plasmid vector. When the vector is a plasmid vector, introduction into the target cells is advantageously performed by electroporation. These different constitutive elements are assembled according to conventional methods of cloning by molecular biology: use of restriction enzymes coupled or not to polymerase chain reactions (PCR) in order to isolate the DNA sequences of interest and ligation of the various constituents by DNA repair or synthesis enzymes (for example, polymerized ligases or DNAs). A system is then provided for genetic transformation of a target cell to express the exogenous enzyme (e.g., nanoluciferase) in its cytoplasmic compartment. An example of a cell transformation system based on the piggyBac transposase is given in FIG. 5. Although this is not essential for the implementation of the present invention, it is recommended to then proceed to a step of cloning these cellular mixtures. . This provides a target cell line expressing nanoluciferase and derived from a single cell (or clone), which will promote homogeneity and reproducibility of cytotoxicity test results. The method according to the invention finds an interesting use for measuring antibody-dependent cellular cytotoxicity (ADCC), measuring complement-dependent cytotoxicity (CDC) and / or measuring apoptosis. The present invention also relates to a kit for carrying out the method described above, or to its use, comprising: a target cell line expressing the exogenous enzyme; cytotoxic effector cells for carrying out an ADCC test and / or a complement source for carrying out a CDC assay, - a substrate activating said exogenous enzyme to produce light emission and any associated buffer solutions, - an explanatory note. The present invention will now be described in more detail with the aid of the following nonlimiting examples, given by way of illustration, with reference to the figures in which: FIG. 1 shows results obtained with the method of FIG. prior art calcein-AM (DELFIA) (Comparative Example A); Figure 2 shows the comparison between the 51 Cr method and a TR-FRET method using two antibodies, donor and acceptor (comparative example B); Figure 3 shows the preliminary results of a TR-FRET method using a donor antibody and GFP (Comparative Example C); Figure 4 shows the linearity of the luminescent signal generated by the nanoluciferase as well as the linearity of the relationship between the number of cells in the wells after triton X-100 lysis and the signal intensity; Figure 5 shows the map of the plasmids used to transform the target cells; Figure 6 illustrates the intensity of the luminescent signal obtained as a function of the dilution factor applied to the nanoluciferase substrate; Figure 7 shows the correlation between the percentage of mortality measured by flow cytometry and the percentage of specific lysis obtained by the nanoluciferase method; Figure 8 depicts the influence of reaction time on the measurement of specific lysis of target cells in an ADCC assay implementing the method; Figure 9 shows the direct comparison of nanoluciferase ("lumi.") And chromium-51 ("51Cr") methods on the Raji model in an ADCC test (N = 3 experiments, performed by 2 different operators: 2 experiments by Operator # 1 and Experiment # 1 by Operator # 2) Figure 10 shows the dispersion of the Emax and Emin data from the ADCC experiments described in Figure 9; Figure 11 shows the dispersion of the EC50 data of the three ADCC experiments described in Figure 9; Figure 12 shows the results of the potency calculation for the 3 ADCC experiments described in Figure 9; Figure 13 shows the direct comparison of the nanoluciferase ("Iumi") and chromium-51 ("51 Cr") methods on the Raji model in a CDC assay (N = 3 experiments, performed by 2 different operators: 2 experiments per Operator # 1 and Experiment # 1 by Operator # 2) Figure 14 shows the dispersion of the Emax and Emin data from the CDC experiments described in Figure 13; Figure 15 shows the dispersion of the EC50 data of the three CDC experiments described in Figure 13; Figure 16 shows the results of the potency calculation for the 3 CDC experiments described in Figure 13; Figure 17 shows the direct comparison of the nanoluciferase ("lumi.") And chromium-51 ("51 Cr") methods on the SKOV3 model in an ADCC test (N = 3 experiments, performed by 2 different operators: 2 Experiments by Operator # 1 and Experiment # 1 by Operator # 2); Figure 18 shows the dispersion of the Emax and Emin data from the ADCC experiments described in Figure 17; Figure 19 shows the dispersion of EC50 data from the three ADCC experiments described in Figure 17; Figure 20 shows the results of the potency calculation for the three ADCC experiments described in Figure 17; Figure 21 shows the direct comparison of nanoluciferase and chromium-51 methods on the CHO-TNF-alpha model in a CDC assay; Figure 22 shows the dispersion of the Emax and Emin data from the CDC experiments described in Figure 21; Figure 23 shows the dispersion of the EC50 data of the three CDC experiments described in Figure 21; Figure 24 shows the results of the potency calculation for the 3 CDC experiments described in Figure 21; Figure 25 shows the results of the potency calculation for 3 ADCC experiments performed in the CHO-TNF-alpha model; Figure 26 summarizes the experiments evaluating the influence of cellular interactions on the measurement of target cell specific lysis in an ADCC or CDC assay using the nanoluciferase method; Figure 27 illustrates the ability of the nanoluciferase release method to measure target cell death induced by pro-apoptotic treatment of physical type; Figure 28 illustrates the ability of the nanoluciferase release method to measure target cell death induced by chemical-based pro-apoptotic treatment. EXAMPLES In the method according to the present invention described hereinafter, the target cells were permeabilized by a series of electric shocks (electroporation) in the presence of the two plasmids of interest shown in FIG. 5, one carrying the transgene. nanoluciferase and the other that of the enzyme allowing its integration into the genome, transposase PiggyBac.
[0014] Legend to Figure 5: AmpR: ampicillin resistance gene; - pUC (ori): origin of bacterial replication; Lac (F): gene coding for the lactose-specific enzyme S. aureus III; - 3'PB TR: homologous recombination element 3 'of the PiggyBac system; 10 - Core insulator: "chicken hypersensitivity site 4 core insulator"; SV40-polyA: "Simian virus 40 Poly-Adenosine"; - WPRE: "Woodchuck Hepatitis Posttranscriptional Regulatory Element Virus"; PuroR: puromycin resistance gene; - EF1A: promoter of the EF1A; Nanoluciferase: nanoluciferase gene; CMV: CMV promoter - 5'PB TR: 5 'homologous recombination element of the PiggyBac system; - PiggyBac: PiggyBac gene.
[0015] Several target cell lines have undergone this genetic transformation: the Raji line, the SKOV3 line and a CHO line expressing human TNF-alpha. After genetic transformation of the chosen target cells, they are cultured under appropriate conditions (generally, for mammalian cells, the temperature is 37 ± 2 ° C in a humidified atmosphere containing between 5 and 10% of CO2) in a culture medium suitable for the selected cell type containing the selection antibiotic whose resistance gene was introduced into the expression vector (puromycin in Example 3033335 shown in Figure 5). This antibiotic makes it possible to select the cells effectively transformed by not allowing the growth of the cells for which the genetic transformation has not worked. A long culture (a few weeks) provides a mixture of cells that have all stably integrated the transgene into their DNA.
[0016] Although not essential to the practice of the present invention, it is recommended that a cloning step of these cell mixtures be further followed. This makes it possible to obtain a target cell line expressing nanoluciferase and derived from a single cell (or clone), which will promote the homogeneity and reproducibility of the results of the cytotoxicity test.
[0017] In order to guarantee the stability and durability of the supply of transformed target cells, it is preferable to produce a bank of these cells under conditions that allow them to grow statically or with agitation in suitable containers. For example, to produce the cell libraries that confirmed the validity of the method of the present invention, the cells were statically cultured in cell culture-treated flasks at 37 ± 2 ° C in a humid atmosphere at 5 ± 1%. CO2 and in a suitable medium: RPMI-1640 containing 10% fetal calf serum (FCS) and 0.25 g / mL puromycin for the Raji line; in McCoy 5A medium, 10% FCS and 5 μg / mL puromycin for SKOV3 cells; in F12-Ham medium, 10% FCS, 1 mg / mL G418 and 20 μg / mL puromycin for CHO cells. After growth of a sufficient quantity of cells, they are distributed in suitable containers and frozen according to the usual techniques for eukaryotic cells for their cryopreservation. Figure 4 illustrates the type of signal that can be obtained with such target cells (CHO cells expressing TNF-alpha in this example) expressing nanoluciferase after genetic transformation. These results show, on the one hand, that the signal generated by the free nanoluciferase in solution (after production by the cells) is perfectly linear between 101 and 10 6 relative units of luminescence (RLU). They show, on the other hand, that the ratio between maximum signal (after lysis of the cells by a detergent) and spontaneous release is always at least equal to 2 under the conditions tested, and stabilizes around 6 when the number of cells per well is between 250 and 5000 cells. The amount of target cells used in the tests presented below is therefore within this range. The conditions of use and the sensitivity of this system therefore allow great flexibility in the implementation of the test. The very small number of cells required to obtain a satisfactory signal is particularly remarkable when compared to the quantity of cells required by the techniques of the prior art (see Table 1 comparing the performances of the different methods). Specific measures of the direct lysis [rior art and various examples of the invention described hereinafter with the nanoluciferase]). Method Ratio Amount Radio-Sensitivity Reproducti Capacity Signal / Noise Required Assistive analysis of high-throughput cells Chromium 51Cr 5-12 2500 to 10000 Calcein-AM 2.5 2500 to -1+ -1+ 10000 Eu3 + / Tb3 + 1-3 5000 to -1 10000 Flow cytometry NA 1000 to -1000 5000 Beta-galactosidase: 2x105 -1+ No data available Nanoluciferase 4-12 250-5000 (present invention) Legend. "NA": not applicable; "-F": yes; "-" : no ; "- / +": weak. Table 1 Among the methods of the prior art: the current method of reference is the 51Cr chromium method, the calcein-AM and Eu3 + / Tb3 + methods are insensitive and variable depending on the cell type, and the method at the beta-galactosidase underestimates by 30-40% the proportion of dead cells. Execution of the Cytotoxicity Test or Test According to the Invention The target cells prepared as previously described are formed into a homogeneous suspension and counted by any suitable method. The amount of cells needed to carry out the test is deposited in a centrifuge tube and the culture medium is removed by centrifugation between 1 and 30 minutes at an acceleration of 100 g to 1500 g. Preferably, this centrifugation is conventionally carried out between 120 g and 600 g for 5 to 10 minutes, the supernatant culture medium is removed and the cells are taken up in new culture medium (preferably not containing the antibiotic (s)). selection) at the desired concentration for the completion of the final test. The cytotoxicity assay can be performed in any type of container (tubes or plates) to maintain living eukaryotic cells for a few hours, including 96 or 384 well flat-bottom plates, conical or round. Each test well contains a mixture including the amount of target cells chosen, the product whose therapeutic / cytolytic action is to be tested and, if appropriate, a complementary effector system such as cytotoxic cells (in the case an ADCC-type assay or mixed lymphocyte reaction, for example) or a source of complement (in the case of a CDC assay or the like). On the basis of the data presented in Figure 4, the amount of target cells per well will be between 10 and 10,000, preferably between 200 and 3000. The active substance is tested using different concentrations thereof, a single concentration. per well. When an effector system (of the effector cell or complement source type) is required, the test substance can be added to the target cells either simultaneously or before the addition of the effector system, while respecting a variable pre-incubation time. preferably between 5 and 60 minutes. Several tubes or wells may be used under strictly similar conditions in order to achieve multiple "replicates". In the case of an ADCC or mixed lymphocyte reaction assay, the ratio between the amounts of effector cells and target cells is preferably between 0.1 and 200. The effector cells may be any cells capable of exerting a cytotoxic activity towards the chosen target line, such as primary or immortalized, unmodified or genetically transformed NK cells, T lymphocytes, monocytes, macrophages or polynuclear cells. This cytotoxic activity may be natural (that is to say, trigger directly when target cells and effector cells come into contact) or induced by an activating substance, which may or may not be the test substance (monoclonal antibody solutions). , polyclonal or related molecules, pro-cytotoxic molecules, cytokines, hormones, neurotransmitters, etc.). The effector cells, in culture or from an ampoule from a cell bank thawed extemporaneously, are therefore counted by any suitable method. The amount of cells required for carrying out the assay is then deposited in a centrifuge tube and the culture medium is removed by centrifugation between 1 and 30 minutes at an acceleration of 100 g to 1500 g, preferably between 120 g. and 600 g for 5 to 10 minutes. The supernatant culture medium is then removed and the cells are taken up in new culture medium (preferably the same as for the target cells) at the desired concentration for carrying out the final test. This concentration depends on the number of target cells used by the condition and the effector / target ratio chosen. In the case where the activation of the complement is sought, a source of complement is added in the tubes or wells. This source of complement may be an animal serum (for example derived from cattle, sheep, goats, rodents, rabbits, monkeys or humans) or consist of a mixture of the various molecules constituting the system. Purified and / or recombinant complement. The complement system being very well preserved during Evolution, the species from which the source of complement comes from is of little importance. This experimental system is also suitable for testing the cytolytic effect of the serum itself (without the addition of exogenous antibody), for example in the screening processes of autoimmune or vaccine sera. Overall, the proportion of serum or complement source may be from 0.1% to 100% of the total reaction volume. Alternatively, the test can be performed using a single concentration of the cytotoxic substance to be tested, by varying the amount of effector system in the wells (effector / target ratio or complement amount for example), or a mixture of the two approaches. In any case, the principle of the test remains the same.
[0018] After mixing in the tubes or wells the desired amounts of target cells, test substance (s) and / or effector system (s), the culture tubes or plates are incubated between 34 ° C and 40 ° C, preferably at 37 ° C ± 1 ° C, in an oven or incubator.
[0019] The incubation time may be variable depending on the process studied. In the case of phenomena such as ADCC or CDC, the incubation time will generally be between 1 hour and 8 hours, and preferably between 2 hours and 5 hours. In the case of apoptosis phenomena or long-term cytotoxic effects, the duration of the test may be from 8 hours to 72 hours, preferably from 24h to 48 hours. At the end of the test, the supernatant of each well is recovered. Preferably, and to ensure that cells will not be removed, the plates or tubes containing the cells are centrifuged for 10 s to 30 min at an acceleration of 100 g to 1500 g, preferably 30 to 120 s to a speed of between 200 g and 1000 g. Alternatively, an intermediate step of taking the supernatant directly into the wells or reaction tubes, followed by transfer of this supernatant to a new plate or tubes and centrifugation under the above conditions can be accomplished. The volume of supernatant required for reading is then transferred to a plate suitable for reading in a luminometer, i.e. opaque and of white or black color, and mixed with a substrate of the nanoluciferase. These substrates are from the family of coelenterazine and its analogues, and preferably of the furimazine type commercially available from Promega under the product reference "Nano-Glo® Luciferase Assay Substrate". Depending on the type of plate used, the supernatant is mixed with the substrate for a final reaction volume of 25 μl at 340 μl in standard 96-well plates, 15 μl at 175 μl for 96-well plates at 20 "half-well format. from 15 μl to 110 μl for standard 384-well plates, from 4 μl to 25 μl for 384-well "small volume" plates, from 3 μl to 10 μl for 1536-well plates. Based on the recommendations of the supplier and the results presented in Figure 6, the substrate "Nano-Glo® Luciferase Assay Substrate" is diluted by mixing with the supernatant (itself pure or diluted according to the expected signal intensity and depending on the cellular model) in a ratio of between 1/50 (1 volume of substrate per 50 volumes of supernatant) and 1/1000, and preferably between 1/100 and 1/200. After a minimum incubation time of 3 minutes, the plate is read in a luminometer (for example Mithras model LB 940). The acquisition time of the signals is of the order of 0.05 seconds. As demonstrated in the examples presented hereinafter, the intensity of the light signal is proportional to the lysis of the target cells. The generated signal is strong, resulting in a sensitive method, even with small amounts of target cells (from 250 cells per well). The following examples show that the method according to the invention based on the measurement of the release of nanoluciferase has characteristics and performances quite comparable to those of the current reference test based on the release of 51 Cr for detection. and measurement of cell death (see Table 2 in Example 10). On the other hand, it has the very great advantage of avoiding the use of radioactive elements, and therefore all the constraints and all the risks associated with this use, while allowing a faster experience run because it does not require labeling time of the target cells. The principle of reading by luminometry is also a simple and economical method, widely used in laboratories and does not require the purchase of expensive equipment. Finally, its mechanism of operation is quite similar to that of the chromium test and the measurement is specific for the death of the target cells, even when they are mixed with other types of cells whose death must not to be measured. All of these data demonstrate that the cytotoxicity assay based on the release of nanoluciferase has all the characteristics required to become a reference test, in particular at the industrial level, for any test requiring the specific measurement of the effective death of a nanoluciferase. cell population. In an unrestricted manner, these tests could evaluate the cytotoxic effect of pollutants or contaminants, environmental factors and cytotoxic drugs or drugs produced by a chemical synthesis or a biological system and any other toxic element for a given cell population.
[0020] Comparative Example A: Three different cell lines were labeled according to the DELFIA BATDA kit method (Perkin-Elmer), strictly according to the manufacturer's recommendations. 10,000 cells per well (3 wells per condition) are then incubated for 2 hours at 37 ° C in culture medium alone (RPMI [Roswel Park Memorial Institute] containing 10% fetal calf serum [FCS]) to determine release. spontaneously or in culture medium containing 1% Triton X-100 detergent to determine the maximum release (final well volume = 200 μl). The background is determined on culture medium alone, without cell or triton X-100. At the end of the incubation, 20 μl of supernatant is transferred to specific plates (provided in the kit) and 200 μl of europium solution is added. After 15 minutes of incubation, the 30 signals are read by TRF method on a suitable reader (Mithras LB 940, Berthold Technologies, Thoiry, France). In FIG. 1A are presented the absolute values measured for the various parameters in counts per second for the different lines. FIG. 3033335 shows the ratios between the maximum signal and the background noise (white bars) on the one hand or the spontaneous signal (black bars) on the other hand. This figure shows the results of a representative experiment of about ten of experiments in which different signal strength optimization approaches were attempted unsuccessfully, with signal / noise ratios still below 3. Example Comparative B: A first artificial reporter molecule was tested by the inventors, combining two distinct specific antigenic motifs (called "flags"), each recognized by an antibody carrying, for one, a lanthanide donor energy and, for another, an energy acceptor.
[0021] The assembly represents a protein weighing 47 kDa. The presence of the released molecule is detected by adding the two antibodies simultaneously in the supernatant. If the free molecule is present, the two antibodies attach to it, thus finding themselves in close proximity to one another, which allows the transfer of energy and the emission of a TR-type signal. FREIGHT. In the example, SKBR3 target cells expressing this reporter molecule are incubated for 4 hours at 37 ° C. in a 96-well plate in the presence of increasing concentrations (indicated on abscissas) of trastuzumab and of cytotoxic cells (T lymphocytes expressing the CD16 receptor). ). At the end of the 4 hours of incubation, the revealing antibodies are added to the medium and the TR-FRET signal is read in fluorimetry on a Mithras reader LB 940 (Berthold) (reading offset of 300 μs, 665/620 nm ratio). ).
[0022] In parallel, a fraction of the same SKBR3 cells expressing the reporter molecule is labeled with 51 Cr (100 μCi / 106 cells). The rest of the experimental conditions are strictly identical: number of target cells (3000) and effector cells (30000) per well, culture medium (RPMI and 10% fetal calf serum), two wells in duplicate for each concentration tested, 4 hours of incubation at 37 ° C. At the end of the incubation, the supernatant is recovered, transferred to scintillation plates (Lumaplate, Perkin-Elmer) and read in a scintillation counter (Microbeta Jet, Perkin-Elmer). The results, presented in FIG. 2, are expressed as percentage specific lysis, calculated according to the formula: ## EQU1 ## in which "Signal (X)" is the signal obtained for a given well X, "Maximum signal" is the signal obtained when the cells are lysed with 0.75% triton X-100 and "spontaneous signal" is the signal obtained when the cells are simply incubated in the presence of culture medium alone (without antibodies or effector cells). The graph shows on the ordinate the average specific lysis ± standard deviation for each concentration tested according to the two methods, 51Cr (black circles and solid line) or TR-FRET (black triangles and dashed lines). The results presented in Figure 2 relate to a representative experience of two. These results show the inefficacy of the reporter molecule to be representative of the lysis of the target cells as observed by the 51 Cr method. Positive controls (not shown) 10 being otherwise satisfactory, the detection method itself is not to blame. The reporter molecule is therefore probably retained in the cytoplasm of the target cells and is not released during lysis. The large size of the molecule (47kDa) is the preferred hypothesis to explain this cytoplasmic retention. Comparative Example C: Another reporter protein, a recombinant Green Fluorescent Protein (GFP) smaller than that shown in Comparative Example B above (approximately 27 kDa), and whose presence in the supernatant can measured by the use of an antibody coupled to a lanthanide (which effects a TR-FRET energy transfer to the GFP when attached thereto), was tested in this comparative example.
[0023] A target line of SKOV3 cells genetically transformed to express recombinant GFP was used for these assays, the results of which are shown in Figure 3. The left (A) portion of Figure 3 shows the expression of GFP measured by flow cytometry on wild-type (wt) unprocessed SKOV3 cells, top, or transformed (GFP-positive) SKOV3 cells, at the bottom. The expression of GFP by the transformed cells is approximately 1.5-log, which is a satisfactory level. The right part of FIG. 3 (B) represents the assay in which 30,000 or 200,000 cells were triplicated in wells of a 96-well plate and incubated in the presence of culture medium alone (RPMI containing 10% FCS) (spontaneous release) or culture medium plus 0.75% triton X-100 (maximum release). At the end of incubation, the supernatant is removed and an anti-GFP antibody coupled to a lanthanide (Tb) is added. The measured signal (Mithras reader LB 940, Berthold Technologies) is a TR-FRET signal from lanthanide to GFP (reading shift = 300 μs, ratio 520/620 nm). Figure 3B shows the ratio between the maximum signal and the spontaneous signal for each of the two cell quantities, and is representative of one of two experiments. The results show that even with a large amount of target cells per well (3x104), the maximum spontaneous ratio is only 2, and that a considerable amount of cells (2x105) is required to begin to observe a interesting ratio (of the order of 4). This method is therefore far from the performance of 51Cr in terms of sensitivity (as mentioned above, it is between 8 and 12 with 3000 cells per well). Here again, the size of GFP and / or the mechanisms of its release during cell lysis are not compatible with an effective measure of cell death. Example 1: shows the linearity of the luminescent signal and the influence of the quantity of cells on this signal. The luminescence of a supernatant containing nanoluciferase was measured, after lysis with triton X-100, diluted in series between 1/1 and 1/10000. 25 μl of each dilution are mixed with 25 μl of Nano-Glo® substrate (Promega) in a 96-well "half-well" plate before reading with the luminometer (Mithras LB 940, Berthold Technologies, acquisition time 0). , 05 s). A linear regression of the values obtained is carried out (see FIG. 4A: intensity of the luminescent signal obtained, expressed in RLU (Relative Luminescence Unit) as a function of dilution). On the one hand, it can be seen that this signal is linear (regression coefficient R2 = 0.9997) over the entire measurement range and ranges from about 10 to 106 arbitrary luminescence units on the reader used. On the other hand, the linearity of spontaneous and maximal release signals from CHO-K1 cells expressing nanoluciferase was evaluated by incubating 4 h of varying amounts of target cells (10000, 5000, 2500, 1000, 500, 250, 100, 50 and 25 cells per well) in the presence of medium alone (spontaneous) or 0.25% triton X-100 (maximum). 25 μl of supernatant are then removed and mixed with 25 μl of Nano-Glo® substrate before reading with the luminometer (acquisition time 0.05 s). The results are shown in Figure 4B. The x-axis shows the number of cells per well and the y-axis on the left represents the luminescent signal obtained in the spontaneous (black circles) and maximum (black squares) conditions. A linear regression is performed for each of these two series of points and represented on the graph (respectively, broad dotted line and solid line), as well as the associated correlation factor (R2), which shows a very good linear relationship between these two series of points. settings.
[0024] For each quantity of cells, the ratio between maximum signal and spontaneous signal is calculated and represented by a white triangle on the right ordinate axis, each point being connected by a fine dashed line to facilitate reading. all of these data demonstrate the feasibility of this method, which proves to be sensitive (25 cells per well are sufficient to have a satisfactory signal), linear over a wide range of cell quantities and luminescence (all the regression factors analyzed are greater than 0.94) and flexible (the ratio [maximum release] / [spontaneous release] remains constant between 250 and 2500 cells per well). Example 2: Shows the linear relationship between the intensity of the luminescent signal and the number of cells actually killed during a cytotoxicity assay. A complement lysis assay was performed on Raji cells (expressing nanoluciferase) in the presence of a commercially available anti-CD20 therapeutic antibody, MabThera® (INN: rituximab). These Raji cells expressing nanoluciferase are incubated for 4 h at 37 ° C. in flat-bottomed 96-well plates (3000 cells per well) in RPMI-1640 medium, with variable amounts of anti-CD20 antibody (MabThera®, Roche, 9 concentrations of 1093.5 to 0.167 ng / mL by 1/3 dilution step) and 1 CH50 ("complement haemolytic 50", a common measure of complement activity) of guinea pig complement (Sigma-Aldrich ). Control conditions are also carried out making it possible to measure the spontaneous release (3000 RPMI-1640 target cells, 1 guinea-pig complement CH50 and 1093.5 ng / mL of an antibody that does not bind to Raji cells, Herceptin ®) and the maximum release of nanoluciferase in the presence of 0.01% triton X-100. Each operating condition is performed in triplicate. At the end of the incubation, 25 μl of supernatant is recovered in each well, mixed with 25 μl of Nano-Glo® substrate diluted 1/50 in PBS, incubated for 3 min and read with the luminometer.
[0025] The percentage of specific lysis obtained for each concentration X is calculated from the luminescence measurements (in RLU) according to the formula: (RLU (X) -RLU (spontaneous)) / (RLU (max) - RLU (spontaneous)) x100. After removal of the supernatant, the rest of the cells are resuspended, mixed extemporaneously with 0.25 μM final of the TO-PRO-3 vital marker (Life Technologies) and analyzed by flow cytometry to determine the percent mortality (c). that is, the percentage of TO-PRO-3 labeled cells). Figure 7 shows the results obtained 3033335 for each of the concentrations of MabThera®. Each point represents the average of the triplicates, the vertical and horizontal bars representing the standard deviation. The theoretical line y = x is represented by a dotted line. The results show that the regression line has a slope of 1.08 ± 0.10, an intercept of 3.01 ± 4.21 and a linear regression coefficient of 0.8242. These results show a satisfactory proportional and linear relationship between the two methods of measurement of cytolysis, which confirms that the lysis phenomenon measured by the nanoluciferase method is translated functionally by an effective death of the cells at the biological level. Example 3: Figure 8, in which the specific lysis of adherent (SKOV3) or non-adherent (Raji) cells expressing nanoluciferase was measured in an ADCC assay according to the present invention, shows the dynamic character of said method. Non-adherent (Raji) or adherent (SKOV3) target cells transformed to stably express nanoluciferase were incubated (at 37 ° C in a humidified 5% CO2 atmosphere), in 96-well flat-bottom plates treated for cell culture. (3000 target cells per well), in the presence of increasing concentrations (the logarithms of which are indicated on the abscissa) of the corresponding antibody (rituximab [MabThera®] or trastuzumab [Herceptin®], respectively) and cytotoxic effector cells (T cells expressing CD16, 30000 effector cells per well) in RPMI-1640 supplemented with 5% FCS. Each range is deposited 4 times (for the 4 incubation times tested) with 3 wells per condition (triplicate). After 1 (black circles), 2 (black squares), 3 (white diamonds) or 4 (black triangles) hours of incubation, 25u.L of supernatant are recovered in the corresponding wells, transferred to a "half-well" plate. white, mixed with 25 μl of NanoGlo® substrate diluted 1/50 to D-PBS, incubated for 3 minutes and read on a Mithras LB940 luminometer. The percentage of specific lysis obtained for each concentration X is calculated from the luminescence measurements (in RLU) according to the formula (RLU (X) -RLU (spontaneous)) / (RLU (max) - RLU (spontaneous)) x100 , the maximum and spontaneous conditions being obtained by incubation of the same cells in a medium containing an irrelevant antibody (3000 ng / ml of trastuzumab or 5000 ng / ml of rituximab, respectively, to constitute the measure of spontaneous release) and 0.01 % Triton X-100 (maximum release). The specific lysis percentages are plotted on the ordinate, with each point representing the average of the triplicate and the vertical bars 3033335 representing its standard deviation. The data was then modeled using a 4-parameter sigmoidal regression using the GraphPad Prism software. A gradual increase in the amount of lysed cells is observed, concomitantly with the increase in the cytolytic agent concentration (the therapeutic antibody) and on the other hand with the incubation time. In both models, 1 and 2 hours are times too short to observe the maximum lytic effect while 3 and 4 hours show a saturation of the cytotoxicity phenomenon. In the latter case, an incubation time greater than 3 hours does not make it possible to obtain a higher lysis of the target cells. The present method is therefore further capable of evaluating the kinetic aspects of cytolysis.
[0026] EXAMPLE 4 In order to evaluate their performance under strictly comparable conditions, the 51 Cr and nanoluciferase methods were compared in parallel in the same CD20-specific ADCC assay, repeated 3 times independently (one tests being performed by a second operator). The same Raji cells expressing the nanoluciferase were labeled or not labeled with 51 Cr and used in parallel in the assay. Raji target cells transformed to stably express the nanoluciferase were incubated or not for 1 hour in a saline solution of Na 2 CrO 4 equivalent to 100 μCi of 51 Cr per million cells, then washed 4 times with RPMI-1640, 10% FCS and counted. The cells (3000 per well), labeled or not labeled with 51 Cr, were then incubated at 37 ° C. in a humidified 5% CO2 atmosphere in 96 well plates with flat bottoms treated for cell culture, in the presence of increasing concentrations. rituximab (MabThera®) and cytotoxic effector cells (CD16-expressing T cells, 30,000 cells per well) in RPMI-1640 supplemented with 5% FCS. After 4 hours of incubation, the percentage of specific lysis is measured according to the corresponding method (release of 51 Cr or of nanoluciferase depending on whether the cells have been chromium-labeled or not): 25 μl of supernatant of the 51 Cr-labeled cells are recovered. and deposited on a Lumaplate® plate (Perkin Elmer). At the same time, for cells not labeled with 51 Cr, 25 μl of supernatant are recovered in each well, transferred to white 96 "half-well plates mixed with 25 μl of 1/50 NanoGlo® diluted substrate. in D-PBS, incubated for 3 minutes and read on a Mithras LB940 luminometer (Berthold Technologies). After a drying night, the radioactivity of the supernatants deposited in Lumaplate® is measured in a Microbeta-Jet counter (Perkin Elmer) and expressed in CCPM ("corrected count per minute") after normalization of the detectors. The percentage of specific lysis obtained for each concentration X is calculated in the same way for the two types of methods, from measurements of radioactivity (in CCPM) or of luminescence (in RLU) according to the formula. In both cases, the maximum and spontaneous conditions were obtained by incubation of the same cells in a medium containing 3000 ng / ml of trastuzumab instead of rituximab (spontaneous release) and 0.01% of triton X-100 (in the case maximum release only). The specific percent lysis percentages are shown in Fig. 9 in ordinates, each point representing the average of the nine determinations (3 replicates in 3 independent experiments) and the vertical bars representing the standard deviations. The data was modeled using a 4 parameter sigmoidal regression using the GraphPad Prism software. This modeling makes it possible to calculate four characteristic variables of the curve: the percentages of minimum lysis (Emin) and maximum (Emax), the slope of the linear part and the concentration of antibody necessary to obtain 50% of Emax (EC50 ). The ability of both methods to detect samples with slightly altered ADCC activity compared to a reference sample (which is the basis of a potency measurement) was assessed through the use of antibody ranges whose concentrations are shifted by 80% or 125% from the reference range (called "100%"). The potency of the diluted X% ranges is calculated according to the formula: (EC50 [100%]) / (EC50 [X%]) × 100. This approach also makes it possible to evaluate the accuracy of the method according to the criteria of ICH-Q2 (R1). The general results of these three experiments are illustrated in Figure 9, which shows the percentages of specific lysis obtained (mean ± standard deviation of the 3 experiments) and the resulting modelizations for the 100% range. They show a very good reproducibility of the two methods (the standard deviations for the same condition are low) and a very similar results profile between the two methods (the two curves are almost superimposable). To go further in the analysis, the dispersion of Emin and Emax calculated for each series of data, for the 3 ranges and in the 3 experiments was analyzed. The results are shown in Figure 10 (where each point corresponds to the measured value for an experiment, the horizontal line for each series of values representing the arithmetic average of the series considered). The dispersions are quite satisfactory, with mean values (Emin and Emax, ± standard deviation) of 1.0 ± 1.5% and 75.7 ± 6.0% for 51 Cr and 1.2 ± 1 , 2% and 73.6 ± 8.4% for nanoluciferase. These results show that Emin and Emax are constant and reproducible between tests, even when concentration ranges of 80% and 125% are used in addition to the standard range. In the same way, the analysis of the dispersion of [C50 of the ranges 100% [Figure 11: each point represents the value of EC50 obtained in an experiment using either the measurement of the liberated nanoluciferase (black circles) or that of the chromium -51 (white squares), the horizontal line representing the arithmetic mean of the 3 values] shows very satisfactory results for both methods and a slightly lower mean value for the luminescence method: 55.2 ± 15.6 ng / mL for 51Cr and 31.8 ± 7.3 ng / mL for nanoluciferase (means ± standard deviations), or coefficients of variation (CV) of 28.3% and 22.8%, respectively. Finally, the potency is calculated for each independent experiment as the ratio (expressed as percentages) between the EC50 of the reference sample (here the standard range 100%) and the EC50 of the sample to be tested (here the ranges 80 and 125%). The results are shown in Figure 12: the calculated potency is plotted on the ordinate for the two methods, nanoluciferase (black circles) and chromium-51 (white squares), the horizontal line (solid for the nanoluciferase method and thick dotted for the method 51Cr) represents the arithmetic mean of the 3 measurements. Dashed fine lines indicate the ideal theoretical potency of 80% and 125%). The results again show a comparable dispersion of measurements between the two methods and results in line with expectations. In fact, the mean values (± standard deviation) for the 80% and 125% ranges are 76.7 ± 10% (CV = 14.2%) and 131.9 ± 11.9% (CV = 25%). , 1%) for the 51Cr method and 83.6 ± 22% (CV = 26.4%) and 119.5 ± 22.7% (CV = 19.0%) for the nanoluciferase method. These results demonstrate the relevance of the method based on the release of nanoluciferase in the anti-CD20 ADCC model described here, whose performance is quite comparable to that of the 51Cr reference method.
[0027] Example 5: Using the same type of target cells as that of Example 4, the nanoluciferase method was compared to the 51 Cr method in a CDC anti-CD20 assay. The methodology applied was the same as that described in Example 4, the cytolytic system being constituted by a source of complement. Likewise, a standard range of antibodies (rituximab) was used, as well as ranges of concentrations modified at 80% and 125%. Three independent experiments were carried out, one of which was carried out by a second operator. Figure 13 shows the comparison of the sigmoid curves obtained from the average values from the 3 experiments. The standard deviations are satisfactory and the curves are quite similar in appearance, despite a slight EC50 shift. The fine analysis of Ern ,, and Emax (Figure 14) confirms the similarity of these parameters, with 3.0 ± 3.4% and 88.6 ± 7.8% for 51Cr and -0.3 ± 3 , 2% and 95.6 ± 7.0% for nanoluciferase, respectively (mean ± standard deviation) for all ranges 80, 100 and 125%. The analysis of the EC50 dispersion for the 100% range in the 3 experiments (FIG. 15) also gives satisfactory results with a mean value (± standard deviation) of 156.7 ± 30.1 ng / mL ( CV = 19.2%) for 51Cr and 73.1 ± 9.9 ng / mL (CV = 13.5%) for nanoluciferase. Finally, the calculation of the potencies (FIG. 16) shows similar performances between the two methods, with values (mean ± standard deviations) quite comparable for the 80% range (69.6 ± 12.3%). for 51Cr and 65.0 ± 12.9% for nanoluciferase, CV = 17.7% and 19.8% respectively) and better accuracy with the 125% range for the nanoluciferase method despite a fairness a little less good (105.8 ± 8.2% [CV = 7.7%], compared with 131.5 ± 16.4% [CV = 12.4%] for 51Cr). These results demonstrate the relevance of the method based on the release of nanoluciferase in the CDC anti-CD20 model described herein, whose performance is quite comparable to that of the 51Cr reference method. Example 6: The nanoluciferase method was compared to the 51 Cr method in an anti-HER2 ADCC assay. For this purpose, SKOV3 cells stably expressing nanoluciferase were or were not labeled with 51 Cr and used simultaneously in an ADCC assay under the same experimental conditions, according to the same principle as that described in Example 4. Target cells SKOV3 transformed to stably express the nanoluciferase were incubated or not 1 hour in a saline solution of Na2CrO4 (Perkin Elmer, France), equivalent to 100 u.Ci of 51 Cr per million cells, then washed 4 times with RPMI-1640 , 10% FCS and counted. The cells (3000 per well), labeled or not labeled with 51 Cr, were then incubated at 37 ° C. in a humidified 5% CO2 atmosphere in 96-well flat-bottomed plates treated for cell culture, in the presence of increasing concentrations ( 1, 5, 10, 25, 50, 100, 250, 500, 1000, and 5000 ng / mL, logarithms of abscissae) of trastuzumab antibody (Herceptin®) and cytotoxic effector cells (expressing T cells CD16, 30000 cells per well) in RPMI-1640 supplemented with 5% FCS. At the end of the 4 hours incubation, 25 μl of the supernatant of each condition are recovered, the cytolysis is measured and the percentage of specific lysis calculated in exactly the same manner as those described in Example 4.
[0028] A standard range (100%) of trastuzumab as well as variations at 80% and 125% of this range were used, and the experiment was repeated 3 times independently, including one performed by a second operator. The average specific lysis percentages obtained by the two methods in the three experiments as well as the associated modelizations are shown in Figure 17. Again, both methods give very similar results, with a dispersion of values that appears to be lower for the nanoluciferase method. These data are confirmed by the analysis of the minimum and maximum lyses (Figure 18: each point corresponds to the value measured for an experiment, the horizontal line for each series of values representing the arithmetic mean of the series considered.), Which shows values (means ± standard deviations) of Ern ,, and dEmax of 0.5 ± 2.2% and 41.3 ± 4.3% for 51 Cr and 0.3 ± 0.7% and 34, 1 ± 4.3% for nanoluciferase, respectively. The EC50 analysis (Figure 19: each point represents the EC50 value obtained in an experiment), the horizontal line representing the arithmetic mean of the 3 values) confirms the smallest dispersion obtained in this model thanks to the nanoluciferase method. Thus, the mean EC50 (± standard deviation) for the standard range is 181.6 ± 8.0 ng / mL in nanoluciferase (CV = 4.4%) while it is 402.5 ± 172. , 5 ng / mL for 51 Cr, a CV of 42.8%.
[0029] 3033335 Despite this higher dispersion of EC50s between experiments for the chromium method, the analysis of potencies (which are calculated experience by experiment by dividing the EC50 of the standard range by that of the modified range) shows no difference significant performance between the two methods (Figure 20). In this FIG. 20, the horizontal line (solid for the nanoluciferase method and thick dotted line for the 51 Cr method) represents the arithmetic mean of the three measurements. Dashed fine lines indicate the ideal theoretical potency of 80% and 125%. The mean potency (± standard deviation) calculated for the 80% and 125% ranges is 82.9 ± 13.0% (CV = 15.7%) and 154.2 ± 24.8% (CV = 16, 1%) for the 51 Cr method and 69.3 ± 7.9% (CV = 11.4%) and 136.9 ± 19.4% (CV = 14.2%) for the nanoluciferase method, respectively . All these results demonstrate the usefulness of the nanoluciferase method as a replacement for the 51 Cr method in this anti-HER2 ADCC model. Example 7: The method of measuring the release of nanoluciferase was compared with the chromium method in a third model, that of the anti-TNF-alpha adalimumab antibody (Humira®). This example illustrates its implementation in a test for measuring CDC activity. For this purpose, CHO cells previously transformed and selected to stably express human TNF-alpha in its membrane form have been transformed to stably express nanoluciferase. The methodology followed is then comparable to that described in Example 5. Briefly, these cells, labeled or not labeled with 51 Cr, were incubated in the presence of increasing concentrations of adalimumab (Humira®) and a source of complement ( Guinea pig (Sigma)). Specific lysis was then measured by the appropriate method (release of chromium or nanoluciferase). The percentage of specific lysis obtained for each concentration X is calculated in the same way for the two types of methods, from measurements of radioactivity (in CCPM) or of luminescence (in RLU) according to the formula. In both cases, the maximum and spontaneous conditions were obtained by incubating the cells in the same medium containing 8000 ng / mL of trastuzumab to replace adalimumab (spontaneous release) and 0.01% triton X-100 (in the case of the maximum release only). The percentages of specific lysis are represented on the ordinate, each point (black circle for nanoluciferase and white squares for 51 Cr) representing the average of the nine determinations (3 replicates in 3 independent experiments) and the vertical bars 3033335 representing standard deviations . The data was modeled using a 4 parameter sigmoidal regression using the GraphPad Prism software. The average results obtained for the 3 experiments are shown in FIG. 21. They again show very good comparability of the two methods, with curves of very similar aspects and characteristics (plateaux, slopes, EC50). The analysis of the dispersion of Ern ,, and Emax carried out confirms this similarity (FIG. 22) with mean values (± standard deviation, over all the ranges in the 3 experiments) of 3.2 ± 2.8, respectively. % and 50.4 ± 5.6% for 51Cr and 0.9 ± 1.5% and 45.7 ± 5.1% for nanoluciferase.
[0030] The analysis of the EC50 dispersion of the three CDC experiments described in FIG. 21 (FIG. 23) for the standard ranges here gives an almost perfect equality between the two methods, the mean values (± standard deviation, average represented by a horizontal line) of the 3 experiments being 150.8 ± 15 ng / mL for chromium (CV = 9.9%) and 156.9 ± 23.4 ng / mL for nanoluciferase (CV = 14.9 %).
[0031] Finally, the analysis of potencies across the 80% and 125% ranges also shows comparable performances between the two methods (Figure 24) with average potencies (± standard deviation) of 91.2 ± 23.6%. (CV = 25.8%) and 153.9 ± 29.2% (CV = 19.0%) for 51 Cr and 83.4 ± 10.1% (CV = 12.1%) and 131.7 ± 47 , 2% (CV = 35.8%) for nanoluciferase (respectively for the 80% and 125% ranges).
[0032] These results demonstrate that the method based on measuring the release of nanoluciferase is equivalent in terms of results and performance to the 51 Cr release method for measuring CDC activity in an anti-TNF-alpha model. Example 8: The nanoluciferase method was compared to the 51 Cr method in an anti-TNF-alpha ADCC assay. For this, the CHO cells described in Example 7, stably expressing TNF-alpha and nanoluciferase, were used, as well as a standard range of adalimumab and variations at 80% and 125% of this range. Three independent experiments were performed, following the same methodology as in the previous examples. From the EC50s generated in the sigmoidal regression models from the specific lysis data, the range of 80% and 125% were calculated as before and the results are presented in Figure 25. The results are comparable between both methods, with a slight advantage in terms of dispersion of values for the nanoluciferase method. In fact, the mean potencies 5 (± standard deviations) of the 80% and 125% ranges calculated are 91.4 ± 43.3% (CV = 47.4%) and 138.5 ± 43.9% (CV = 31.7%) for the 51Cr method and 75.5 ± 15.5% (CV = 20.6%) and 114.2 ± 22.8% (CV = 19.9%) for nanoluciferase. These results demonstrate the utility of the nanoluciferase method for measuring the ADCC activity of anti-TNF-alpha antibodies in place of the 51Cr method.
[0033] Example 9: This example illustrates the adequacy between the nanoluciferase method and the biological phenomena used in the ADCC and CDC mechanisms. For this, ADCC and CDC reactions were carried out in parallel and under the same experimental conditions using for the incubation either flat bottom plates or round bottom plates, the latter favoring contacts and interactions. thanks to the particular shape of the well. The results are shown in FIG. 26. They show that, in the case of the ADCC assay for which the interactions between effector cells and targets are essential for the cytotoxic effect, the shape of the well effectively influences the percentage of maximum lysis (38.1% on average for round funds versus 27.6% for flat bottoms), without this phenomenon significantly modifying the resulting EC50s (59.8 ng / mL against 65.3 ng / mL, respectively ). On the contrary, the shape of the well does not influence the reaction of CDC, with average Emax (47.0% in round bottom and 44.6% in flat bottom) and average EC50 (114.1 and 155.3 ng / mL, respectively) similar and independent of the well shape. Favoring the contacts between the cells by a well-adapted form thus makes it possible to improve the quantity of target cells lysed by ADCC and detected by the nanoluciferase method. Consistently, the CDC assay, in which the single cell population present is the target cell population and in which the effector system is soluble (the complement), is insensitive to the shape of the well.
[0034] EXAMPLE 10 In order to compare the performances of the nanoluciferase release method with those of the 51 Cr release method, the results of variability presented in Examples 4 to 8 were collated in Table 2 below. after.
[0035] 51Cr Model CV test on EC50 CV on Potency 80% CV on Potency 125% (and relative bias) (and relative bias) ADCC 28.3% 14.2% (-4.1%) 26.4% (+4, 5%) CD20 CDC 19.2% 17.7% (-13.0%) 12.4% (+ 5.2%) HER2 ADCC 42.8% 15.7% (+ 3.6%) 16, 1% (+ 23.4%) ADCC 19.9% 47.4% (+ 14.3%) 31.7% (+ 10.8%) TNF-alpha CDC 9.9% 25.8% (+ 14.0%) 19.0% (+ 23.1%) nanoluciferase CV on Potency 80% CV on Potency 125% Model CV test on EC50 (and relative bias) (and relative bias) ADCC 22.8% 9.1 % (+ 5.5%) 19.0% (-4.4%) CD20 CDC 13.5% 19.8% (-18.8%) 7.7% (-15.4%) HER2 ADCC 4 , 4% 11.4% (-13.4%) 14.2% (+ 9.5%) ADCC 14.5% 20.6% (-5.6%) 19.9% (-8.6%) %) TNF-alpha CDC 14.9% 12.1% (+ 4.3%) 35.8% (+ 5.4%) Table 2 All results are expressed as percentages. Three independent experiments were carried out in each of the models and tests. The coefficients of variation (CV) of the EC50s and potencies are calculated as the standard deviation divided by the average of the corresponding data. The relative bias expresses the difference between the expected value and that actually obtained, as a percentage of the expected value, according to the following formula (where P represents the potency): The purpose of Examples 11 and 12 below is to evaluate the ability of the present method to measure longer kinetic cell death phenomena than those involved in the mechanisms of ADCC or CDC, for example in the case of detecting the cytotoxic effect of polluting, toxic molecules or pro-apoptotic.
[0036] EXAMPLE 11 Physical Agent Raji cells transformed to stably express the nanoluciferase (50000 cells per well) are cultured for 48 hours at 37 ° C. (under a humidified atmosphere containing 5% CO 2) in RPMI-1640 medium containing 10% FCS. and in the presence or absence of 0.2% Triton X-100 (to determine the maximum release), after being exposed to ultraviolet (UV) radiation for a variable time (0, 10, 20, 30, 40, 50 or 60 seconds). After 48 hours, 25 μl of supernatant is recovered in each well, transferred to a white "half-well" plate, mixed with 25 μl of NanoGlo® substrate diluted 1/50 to D-PBS, incubated for 3 minutes and read on a Mithras luminometer LB940 (Berthold Technologies). The results of the reading are expressed in RLU ("relative luminescence unit"). The percentage of dead cells for each staurosporin concentration is determined according to the formula (RLU [medium without tritone]) / (RLU [0.2% triton]) × 100. After removal of the supernatant, the rest of the cells are resuspended, mixed extemporaneously with 0.25 μM final of the TO-PRO-3 vital marker (Life Technologies) and acquired on a C6 flow cytometer (BD / Accuri). The percentage of dead cells is determined when analyzing the cytometry data as the percentage of cells having integrated TO-PRO-3. Two identical independent experiments were carried out, the results of which are shown in graphs A and B of FIG. 27. The graph of the top (A) represents the percentages of dead cells determined according to the two methods (luminescence of nanoluciferase [" lumi. ", dotted line] or flow cytometry [" CMF ", solid line]) based on UV exposure time 3033335 for a representative test. The bottom graph (B) represents the percentage of dead cells determined by the luminescent method ("% lumen mortality") as a function of the corresponding percentage of dead cells measured by flow cytometry ("% CMF mortality") for the whole group. conditions in test 1 (solid line, solid circles) and in test 2 (dashed line, empty squares). A linear regression was calculated between these two parameters using the GraphPad Prism software and plotted on the graph (the equation of the line and the linear regression coefficient R2 are represented on the graph for each of the two tests). Example 12 Chemical Agent Raji cells transformed to stably express nanoluciferase (50000 cells per well) are grown for 48 hours at 37 ° C (under humidified atmosphere containing 5% CO2) in the presence of increasing concentrations of staurosporine, a pro-drug. -apoptotic, in RPMl-1640 medium containing 10% FCS, and in the presence or absence of 0.2% Triton X-100 (to determine the maximum release). At the end of the incubation, 25 μl of supernatant is collected in each well, transferred to a white "half-well" plate, mixed with 25 μl of NanoGlo® substrate diluted 1/50 to D-PBS, incubated 3 minutes and read on a Mithras LB940 luminometer. The results of the reading are expressed in RLU ("relative luminescence unit"). The percentage of dead cells for each staurosporine concentration is determined according to the formula (RLU [medium without tritone]) / (RLU [0.2% triton]) × 100.
[0037] After removal of the supernatant, the rest of the cells are resuspended, mixed extemporaneously with final 0.25 μM of the TO-PRO-3 vital marker (Life Technologies) and acquired on a C6 flow cytometer (BD / Accuri). The percentage of dead cells is determined when analyzing the cytometry data as the percentage of cells having integrated TO-PRO-3.
[0038] Two independent identical experiments were carried out, the results of which are shown in graphs A and B of FIG. 28. The top graph (A) represents percentages of dead cells determined according to the two methods (luminescence of nanoluciferase [" lumi. ", dashed line] or flow cytometry [" CMF ", solid line]) as a function of the staurosporine concentration in the presence of which the cells were incubated, for a representative test. The bottom graph (B) represents the percentage of dead cells determined by the luminescent method ("% lumen mortality") as a function of the corresponding percentage of dead cells measured by flow cytometry ("% CMF mortality") for 3033335 41 l set of conditions in test 1 (solid line, solid circles) and in test 2 (dotted line, empty squares). A linear regression was calculated between these two parameters using the GraphPad Prism software and plotted on the graph (the equation of the line and the linear regression coefficient R2 are represented on the graph for each of the two tests).
[0039] These results show, on the one hand, that there is a proportional relationship between staurosporine concentration and target cell mortality, irrespective of the method of measuring mortality. On the other hand, the mortality detected by the measurement of nanoluciferase is perfectly correlated with the analysis at the cellular level by flow cytometry. Indeed, the slope of the regression line is close to 1 (slope = 0.86), as is the linear regression coefficient (R2 = 0.9659). Similar results (not shown) were obtained with the SKOV3 and CHO-TNF-alpha target lines expressing nanoluciferase.

权利要求:
Claims (25)
[0001]
REVENDICATIONS1. Non-radioactive method for direct in vitro determination (control and quantification) of the cytolytic action of an agent active against target cells and / or a medium surrounding the target cells, comprising the following successive steps : i) Genetic transformation of target cells to express an exogenous enzyme to said target cells, ii) Exposure of said genetically transformed target cells to the active agent and / or said environment to be tested, which can lead to lysis of the target cells. at least a portion of the target cells by releasing said exogenous enzyme in the extracellular medium, iii) Measuring the activity of the exogenous enzyme released during the lysis of said target cells characterized in that said exogenous enzyme is a lower molecular weight enzyme or equal to 45 kDa, and whose activity is detectable by luminescence or fluorescence.
[0002]
2. Method according to claim 1, characterized in that the molar mass of the exogenous enzyme is less than or equal to 30 kDa, preferably less than or equal to 25 kDa, more preferably less than or equal to 20 kDa.
[0003]
3. Method according to any one of claims 1 or 2, characterized in that said exogenous enzyme is a luciferase.
[0004]
4. Method according to any one of claims 1 to 3, characterized in that said exogenous enzyme has a peptide sequence having at least 60% homology, preferably at least 80% homology, with the wild-type peptide sequence of the 19 kDa luciferase subunit produced by shrimp Oplophorus grocilirostris.
[0005]
5. Method according to claim 4, characterized in that said exogenous enzyme is in a form with optimized activity and stability, marketed by Promega under the name NanoLuc®. 25
[0006]
6. Method according to any one of the preceding claims, characterized in that the amount of exogenous enzyme released is measured by cleavage of a substrate specific for said enzyme, the substrate preferably being of the coelenterazine family, more particularly of the family of furimazine and its derivatives. 3033335 43
[0007]
7. Method according to any one of the preceding claims, characterized in that the test medium surrounding the target cells comprises a biological agent and / or a chemical agent and / or a physical agent potentially active (s) vis-à-vis the said target cells.
[0008]
8. Method according to claim 7, characterized in that the biological agent is an antibody, preferably selected from the therapeutic antibodies, including monoclonal antibodies for therapeutic purposes.
[0009]
9. Method according to claim 8, characterized in that the antibodies are antibodies directed against molecules expressed mainly by normal or pathological B lymphocytes, such as CD19, CD20 or IL-6R (CD126), preferably CD20. 10
[0010]
10. Method according to claim 8, characterized in that the antibodies are antibodies directed against molecules expressed mainly by normal or pathological T lymphocytes, such as CD3, CD25 or LPAM ("Peyer Lymphocyte Patch Adhesion Molecule" or alpha integrin [ 4] beta [7]).
[0011]
11. Method according to claim 8, characterized in that the antibodies are antibodies directed against molecules expressed mainly by cells of normal or pathological lymphoid origin, such as CTLA-4 (CD152), PD-1 (CD279) or Human CD30
[0012]
12. Method according to claim 8, characterized in that the antibodies are antibodies directed against molecules expressed mainly by cells of normal or pathological origin of lymphoid and myeloid origin, such as CD52, VLA4 (CD49d) or LFA-1 (CD11a). ) 20 human.
[0013]
13. The method of claim 8, characterized in that the antibodies are antibodies directed against molecules expressed mainly by carcinoid cells, such as EGFR ("Epidermal Growth Factor Receptor", also noted HER1 or ErbB1), EGFR2 (or HER2, or ErbB2), EGFR-3 (or HER3, or ErbB3) or EpCAM (CD326). 25
[0014]
14. The method of claim 8, characterized in that the antibody is directed against the TNF-alpha molecule.
[0015]
15. The method of claim 8, characterized in that the antibody is directed against the molecule VEGF ("Vascular Endothelial Growth Factor") or its VEGFR receptor. 3033335 44
[0016]
16. The method of claim 7, characterized in that the chemical agent and / or physical is selected from chemotherapeutic agents or anti-cancer molecules, preferably cytotoxic molecules or molecules of the family of protein kinase inhibitors . 5
[0017]
17. A method according to any one of the preceding claims wherein the target cells are genetically transformed to transiently or constitutively express said exogenous enzyme in a cytoplasmic and non-secreted form.
[0018]
18. A method according to claim 17, characterized in that the step of genetic transformation of said target cells comprises the introduction into said cells of an expression vector carrying the coding sequence of the exogenous enzyme and a promoter of constitutive type allowing its transcription in a cell, such as a eukaryotic cell.
[0019]
19. The method of claim 18, characterized in that the vector is a viral vector, or a plasmid vector, preferably a plasmid vector.
[0020]
20. Method according to one of claims 18 or 19, characterized in that the expression vector of the exogenous enzyme comprises an antibiotic resistance gene.
[0021]
21. Process according to claim 20, characterized in that the antibiotic resistance gene is a geneticin resistance gene (G418), puromycin, blasticidine, hygromycin B, mycophenolic acid or zeocin, preferably a puromycin resistance gene.
[0022]
22. Method according to any one of claims 19 to 21, characterized in that the introduction of said expression vector into said cells is carried out by infection with viral particles bearing the gene of the exogenous enzyme when the vector of expression is a viral vector, or by chemical or physical methods when the expression vector is a plasmid vector.
[0023]
23. The method of claim 22, characterized in that, when the vector is a plasmid vector, the introduction into the target cells is performed by electroporation.
[0024]
24. Use of the method according to any preceding claim for measuring antibody-dependent cellular cytotoxicity (ADCC), measuring complement-dependent cytotoxicity (CDC) and / or measuring apoptosis. 3033335 45
[0025]
25. Kit for carrying out the method according to any one of claims 1 to 16 or its use according to claim 24, characterized in that it comprises: - a target cell line expressing the exogenous enzyme, - effector cells cytotoxic agents for carrying out an ADCC test and / or a source of complement for carrying out a CDC assay; a substrate activating said exogenous enzyme to produce light emission, and any associated buffer solutions, - an explanatory note.

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同族专利:

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公开号 | 公开日

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PT3265579T|2019-03-21|

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FR3033335B1|2017-03-10|

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US20180051317A1|2018-02-22|

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ES2715557T3|2019-06-04|

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CA2977318A1|2016-09-09|

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EP3265579B1|2018-12-12|

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DK3265579T3|2019-04-08|

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CA2977318C|2021-08-24|

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FR3033334A1|2016-09-09|

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EP3265579A1|2018-01-10|

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CN108956979B|2018-08-02|2021-07-27|上海细胞治疗集团有限公司|Cytotoxicity detection reagent composition|

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CN112285083B|2020-10-28|2022-01-07|上海睿钰生物科技有限公司|Method for evaluating cell killing efficacy|

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优先权:

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申请号 | 申请日 | 专利标题

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FR1551835A|FR3033334A1|2015-03-05|2015-03-05|NON-RADIOACTIVE METHOD FOR DETERMINING THE CYTOLYTIC ACTION OF AN AGENT FOR TARGET CELLS, USE THEREOF AND KIT THEREOF|ES16714989T| ES2715557T3|2015-03-05|2016-03-01|Non-radioactive procedure for determining the cytolytic action of an agent against target cells, their use and their associated kit|

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EP16714989.7A| EP3265579B1|2015-03-05|2016-03-01|Non-radioactive method for determining the cytolytic activity of an agent with respect to target cells, use thereof and associated kit|

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US15/554,368| US20180051317A1|2015-03-05|2016-03-01|Non-radioactive method for determining the cytolitic activity of an agent with respect to target cells, use thereof and associated kit|

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CA2977318A| CA2977318C|2015-03-05|2016-03-01|Non-radioactive method for determining the cytolytic activity of an agent with respect to target cells, use thereof and associated kit|

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DK16714989.7T| DK3265579T3|2015-03-05|2016-03-01|NON-RADIOACTIVE PROCEDURE FOR DETERMINING THE CYTOLYTIC EFFECT OF A MEDICINE ON TARGET CELLS, APPLICATION THEREOF AND RELATED KITS|

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PT16714989T| PT3265579T|2015-03-05|2016-03-01|Non-radioactive method for determining the cytolytic activity of an agent with respect to target cells, use thereof and associated kit|

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PCT/FR2016/050458| WO2016139415A1|2015-03-05|2016-03-01|Non-radioactive method for determining the cytolytic activity of an agent with respect to target cells, use thereof and associated kit|