EP3870693A1 - Modified pdc line for secreting a cytokine - Google Patents
Modified pdc line for secreting a cytokineInfo
- Publication number
- EP3870693A1 EP3870693A1 EP19794953.0A EP19794953A EP3870693A1 EP 3870693 A1 EP3870693 A1 EP 3870693A1 EP 19794953 A EP19794953 A EP 19794953A EP 3870693 A1 EP3870693 A1 EP 3870693A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- genetically modified
- pdc
- cells
- line according
- antigens
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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- A61K2039/5156—Animal cells expressing foreign proteins
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
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- C12N2510/00—Genetically modified cells
Definitions
- the present invention relates to a genetically modified PDC (plasmacytoid dendritic cell) line for secreting a cytokine, and its use for increasing the expansion of antigen-specific cells in immunotherapy.
- PDC plasmacytoid dendritic cell
- Immunotherapy approaches seeking to promote the production of specific antigen cells are known.
- An approach using a plasmacytoid or PDC dendritic cell line (called “PDC * üne”) to induce antigen-specific cytotoxic cells from donor mononuclear cells (CMN), in particular peripheral blood mononuclear cells (PBMC) was developed by the inventors (WO 2009/138489).
- This approach consists in culturing CMNs in the presence of “PDC * üne” irradiated and loaded with an antigen, the mononuclear cells sharing at least one HLA molecule with “PDC * line” (HLA-A2, for example).
- the ability to induce the expansion of specific antigen cells is a determining factor in the implementation of these new therapeutic approaches. It is known, in particular from application WO 2009/138489, that the presence of cytokines is necessary for this expansion.
- the experimental protocol described consists, the first week, in a coculture of irradiated PDC * line loaded with an antigen with CMN carried out without cytokine, then, in the second week, in a new stimulation with the irradiated PDC * Nne loaded with an antigen and IL-2.
- IL-2 is produced mainly by activated T lymphocytes, while IL-15 and its associated receptor IL-15Ra (Dubois & al., 2002), is produced by monocytes or myeloid dendritic cells (MDC) (Jakobisiak & al., 201 1).
- MDC myeloid dendritic cells
- MDC and PDC are both antigen presenting cells, they are two different cell types capable of inducing different types of immune responses according to the origin of the pathogen or the antigen and the environmental context in general, differentiated by a certain number of characteristics: origin, tissue localization, expression of Toll-like receptor, production of cytokines, in particular. None of these articles on MDCs modified to express IL15 mentions the possibility of using PDCs, or even of envisaging an effect at least equivalent on PDCs with IL15 or even IL2.
- the invention therefore relates to a new line of PDC genetically modified to express a cytokine chosen from interleukin 15 (IL15) and interleukin 2 (IL2).
- IL15 interleukin 15
- IL2 interleukin 2
- the genetically modified PDC cells according to the invention are loaded with one or more antigens, in peptide form.
- the invention also relates to the use of the new genetically modified PDC line for the amplification of specific antigen cells, in particular mononuclear cells (CMN), more particularly peripheral blood mononuclear cells (PBMC).
- CMS mononuclear cells
- PBMC peripheral blood mononuclear cells
- the invention also relates to a combination product or kit, comprising on the one hand a line of PDC genetically modified according to the invention and on the other hand mononuclear cells (CMN), in particular for simultaneous use in the treatment of diseases by immunotherapy.
- CPN mononuclear cells
- the invention also relates to a vaccine composition
- a vaccine composition comprising a genetically modified PDC line according to the invention and a vehicle suitable for its administration.
- Figure 1 represents the measurement of the expression of the CD34 molecule on the PDC * Nne line not transduced (on the left) or transduced by the retroviral supernatant coding for I ⁇ I-2 or IL-15.
- Figure 2 represents the expression of IL2 or IL15 by the PDC * Nne line transduced by the retroviral supernatant IL2 or IL15.
- Cytokines are detected at the level of messenger RNA (A, normalized with respect to G6PDH ) or in the supernatant of transduced cells (B, in pg / ml).
- FIG. 3 represents the expansion of anti-Melan-A CD8 + T lymphocytes after 14-day coculture of the PDC line, unmodified or genetically modified by IL2 or IL15, loaded with the Melan-A antigen, and mononuclear cells from 3 healthy donors.
- Anti-Melan-A CD8 + T cells are detected by flow cytometry using HLA-A2 / Melan-A multimers.
- A a representative experience is shown; in B, the results of 3 experiments are shown (% of CD8 + multimer cells).
- Figure 4 shows the function of CD8 + cells from the 14-day coculture.
- the function of cytotoxic cells is objectified by the detection of intracytoplasmic IFNy after stimulation by the target line (T2) loaded with the peptide used derived from Melan-A.
- T2 target line
- A the illustration of intracytoplasmic detection of the cytokine on all specific and non-specific CD8 + cells, obtained with the different lines.
- B the percentage of CD8 + cells positive for IFN ⁇ in non-specific cells (Multimer-) and specific cells (Multimer +) generated with each line.
- the invention therefore relates to a new line of PDC genetically modified to express a cytokine chosen from IL15 and IL2.
- PDC lines useful according to the invention for being genetically modified are well known to those skilled in the art, in particular described in EP 1 572 989 and WO 2009/138489. These are in particular cells obtained from PDC leukemia cells. Mention will be made in particular of the GEN2.2 and GEN3 lines deposited under the numbers 2938 and 31 10 at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Dondel Roux, 75015Paris) and all lines derived from PDC leukemia cells .
- lines derived from PDC leukemic cells is meant according to the invention lines derived from tumor nodules following the injection of PDC leukemic cells into immunodeficient mice, these nodules being dilated to obtain a cell suspension which is cultured in a synthetic medium promoting the growth of said line.
- cytokine chosen from IL15 and IL2
- expression of a cytokine chosen from IL15 and IL2 is meant according to the invention the secretion of the cytokine by the genetically modified line.
- Cytokine sequences are well known to those of skill in the art.
- the protein sequence identified under the reference UniProtKB P60568 and the coding sequence identified under the reference Ensembl ENSG00000109471 and for the IL-15 the protein sequence identified under the reference UniProtKB P40933 and the coding sequence identified under the reference Ensembl ENSG00000164136.
- Variants or fragments of these cytokines which have the same activity as the above sequences are also part of the invention.
- the cytokines are the human cytokines identified above.
- genetically modified PDC line is meant according to the invention any line transduced by a viral particle allowing the integration of the gene into the genome of the line, this virus possibly being an adenovirus, a lentivirus or a retrovirus.
- this virus possibly being an adenovirus, a lentivirus or a retrovirus.
- a murine Moloney leukemia virus (Mo-MLV) type retrovirus will be used.
- These retroviral particles being produced by the HEK 293T encapsulation lines, express naturally or after transfection the retroviral gag / pol and env sequences.
- a viral particle comprising a coding sequence for the gene or genes of interest and regulatory elements, in particular the sequences used to allow the expression of cytokines in genetically modified mammalian cells.
- Methods of transducing PDC cells with a viral particle to obtain a new genetically modified PDC line to express a cytokine selected from IL15 and IL2 are also known to those skilled in the art. Mention will in particular be made of the use of polycationic agents, such as polybrene or DOGS (dioctadecylamidoglycyl spermine), or of agents promoting contacts between viruses and cells of interest, such as fragments of fibronectin (Retronectin). Preferably, the method using Retronectin will be used.
- the cytokine alone is expressed.
- the PDC line also expresses the cytokine receptor, in particular the IL15Ra receptor.
- the IL15Ra receptor is well known to those skilled in the art, in particular its protein sequence
- the PDC line modified according to the invention does not express said receptor
- the PDC line can also be modified to express said receptor, according to the usual methods of the technique.
- the PDC cells modified according to the invention are particularly useful for the amplification of specific antigen cells, in particular mononuclear cells (CMN), more particularly peripheral blood mononuclear cells (PBMC).
- CPN mononuclear cells
- PBMC peripheral blood mononuclear cells
- the PDC cells modified according to the invention are preferably associated with antigens, in particular antigens peptide.
- the term “antigen” defines a molecule or a part of the molecule recognized by cells of the immune system and capable of triggering a cell-mediated immune reaction.
- the antigens according to the present invention can be native or modified antigens, tumor or microbial (in particular bacterial or viral), such as peptides, proteins, glycopeptides, glycoproteins, phosphorylated proteins.
- antigen (s) to be associated with the modified PDC line according to the invention according to the disease to be treated.
- the antigens are peptides capable of being obtained from antigenic proteins of tumor or viral origin.
- These peptides are well known to those skilled in the art, described in particular in numerous patent or patent applications, in particular EP 1 485 719, EP 2 1 13 253, US 7 087 712, US 7 528 224, WO 94/020127 , WO 95/02553, WO 98/22589, WO 2000/003693, WO 2000/020445, WO 2000/052163, WO 2000/078806, WO 2004/067023, WO 2007/036638, WO 2007/039192, WO 2008/045286 , WO 201 1/012720, WO 2015/0965572 and WO 2016/179573.
- the peptides capable of being obtained from tumor antigens can be chosen from the peptides included in the sequence of the antigens CEA, NY-BR1, Her-2 / Neu, PSA, RAGE -1, PRAME, TRP-2, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C1, MAGE-C2, Multi-MAGE, MUC-1, p53 , hTERT, survivin, melan-A / MART-1, GP100, tyrosinase, CAMEL or NY-ES01, modified or not, alone or in combination.
- any antigen chosen from mutated proteins or proteins resulting from the transcription of a messenger RNA generated by a new reading frame for a nucleic sequence (neo-antigen).
- the peptides capable of being obtained from viral antigens can be chosen from the peptides included in the sequence of the env, nef, gp41, gp120, gag or pol antigens of the HIV virus , HBc or HBs from HBV, core, NS3 or NS5 virus of HCV virus, Flu M1 of influenza virus, CMVpp65 of CMV virus, BMLF1, LMP2, EBNA-2 or EBNA-3a of EBV virus, LT A or VOP1 of BKV virus, nucleoprotein of virus Hatan, NS3 of the Dengue virus, E6 and E7 of the HPV virus, protein E, NS3, or BS4b of the West Nile Virus, modified or not, alone or in combination.
- the genetically modified PDC cells are loaded with one or more antigens. It will also be said that they are pulsed, that is to say incubated with one or more antigens.
- the invention therefore relates to a genetically modified PDC line as defined above and in the examples, loaded with one of the antigens listed above.
- the cells are also genetically modified to express said antigens.
- the genetic modification tools are the same as those used to modify PDC cells to express cytokines.
- a person skilled in the art will also choose the genetic elements which make it possible to express the antigens at the cellular level so that they are transformed into epitopes and presented by the HLA molecules, in vitro or in vivo. Such elements are also well known to those skilled in the art (Hu, Immunological review 2011).
- the genetically modified PDC lines according to the invention are irradiated lines.
- the invention also relates to the use of the new genetically modified PDC line for the amplification of specific antigen cells, in particular mononuclear cells (CMN), more particularly peripheral blood mononuclear cells (PBMC).
- CMS mononuclear cells
- PBMC peripheral blood mononuclear cells
- the invention also relates to the new genetically modified PDC line for use in therapy, in particular for the treatment of diseases by immunotherapy.
- the invention also relates to a combination product or kit, comprising on the one hand a line of PDC genetically modified according to the invention and on the other hand mononuclear cells (CMN), in particular for simultaneous use in the treatment of diseases by immunotherapy.
- CPN mononuclear cells
- the disease treated will depend essentially on the antigens which will be associated with the line according to the invention, with for example the tumor antigens being used for the treatment of cancers and the viral antigens for the treatment of viral infections.
- the invention also relates to a vaccine composition comprising a genetically modified PDC line according to the invention and a vehicle suitable for its administration.
- the invention also relates to a method of preventing and / or treating cancers and / or infectious diseases, characterized in that a genetically modified irradiated and pulsed PDC line according to the invention is injected into a patient who needs treatment. , the specific cells of said patient's antigens and the PDCs sharing at least one allele of the major histocompatibility complex (MHC).
- MHC major histocompatibility complex
- the invention also relates to a method of prevention and / or treatment of cancers and / or infectious diseases characterized in that the specific effectors obtained by incubation of a line of PDC according to l are injected into a patient in need of treatment.
- invention with at least one antigen, the pulsed PDCs, possibly irradiated, then being brought into contact with cells specific for antigens of said patient and cultured, PDCs and cells specific for antigens sharing at least one allele of the major complex d 'histocompatibility (CMH).
- CSH major complex d 'histocompatibility
- specific effector is meant according to the invention the cells of the immunity capable of recognizing a specific antigen or a product derived from this antigen, in particular cytotoxic effectors and more particularly T lymphocytes specific for the antigen used, and including CD8 +.
- Example 1 Generation of genetically modified PDC * line lines to express IL-2 or IL15.
- the sequences of the genes of interest coding for IL2 (SEQ ID NO 1) and IL15 (SEQ ID NO 2) were synthesized by ThermoFischer (https://www.thermofisher.com/fr/fr/home/life- science / cloning / gene-synthesis / geneart-gene-synthesis.html) and supplied in a plasmid allowing the amplification of the plasmid after transformation of DH5alpha bacteria (NEB).
- the sequence of interest (IL2 or IL15) was then subcloned into the plasmid SFGACD34 (Quintarelli, Blood 2007, generously supplied by Dr M. Pule) between the restriction sites Sal-I and Mul-I at the using Gibson Assembly technology (NEB).
- a truncated CD34 sequence in the plasmid SFGACD34 allows the selection of cells expressing the gene of interest.
- a retroviral suspension was then obtained by triple transfection of the HEK-293T line with the plasmid SFGACD34-IL2 or SFGACD34-IL15 and the expression plasmids MoMLV gag-pol pEQ-PAM3 (-E) and RD114 env (generously provided by Dr M. Pule and Dr M. Collins (Cosset, J Virol 1995)) in the presence of GeneJuice (VWR).
- the cells of the PDC line were subsequently transduced with the supernatant retroviral corresponding to IL2 or IL15 in the presence of retronectin (Takara) and the expression of CD34 was measured, reflecting the transduction efficiency.
- This line is derived from the cells of a patient with PDC leukemia (Chaperot, Blood 2001), patient from which the GEN2.2 and GEN3 lines are derived.
- CD34 is correlated with the expression of the IL2 or IL15 genes.
- analysis by RT-qPCR shows a relative expression, relative to the G6PDH gene, high for IL2 or IL15, by a factor greater than 20 or 30 respectively ( Figure 2A).
- CBA BD
- R&D ELISA
- Example 2 Expansion of CD8 + lymphocytes after co-culture of CMN with transduced lines loaded with a tumor peptide.
- the capacity of the modified lines was then evaluated using a coculture with mononuclear cells (CMN) from a healthy donor HLA-A2 +. Briefly, the cells of the PDC line, genetically modified or not, were loaded with the peptide Melan-A26i_-35, irradiated then cultured with CMN in a 24-well plate for 14 days. Different amounts of PDC line cells were added to the 2 million CMN (10/1 or 20/1) per well. On D7, the cells are restimulated with the PDC line loaded with Melan-A in the presence of soluble IL-2.
- CPN mononuclear cells
- the expansion of anti-Melan-A CD8 + lymphocytes is measured using dextramer Melan-A A2 (Multimer) labeled with a fluorochrome (Immudex) and anti-CD3 and CD8 antibodies (Beckman Coulter).
- Example 3 Functionality of the effectors generated with the PDC line modified or not by IL2 or IL15: Intracytoplasmic expression of IFNy.
- FIG. 4A represents the phenotyping obtained representing the information of the IFNy positive cells as a function of the cells labeled with the multimer.
- Figure 4B shows that only specific Melan-A cells (multimer +) produce of IFNy when stimulated by the T2 / Melan-A line.
- the results show that Melan-A specific lymphocytes amplified in the presence of the PDC line modified to express IL2 or IL15 secrete 1.8 to 2.1 times more IFN ⁇ than Melan-A specific lymphocytes amplified with the unmodified line.
Abstract
The present invention relates to a genetically modified PDC (plasmacytoid dendritic cell) line for secreting a cytokine and to its use for increasing the expansion of antigen-specific cells in immunotherapies.
Description
LIGNÉE PDC MODIFIÉE POUR SECRÉTER UNE CYTOKINE MODIFIED PDC LINE TO SECRET A CYTOKINE
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
La présente invention concerne une lignée PDC (cellules dendritiques plasmacytoïdes) modifiée génétiquement pour secréter une cytokine, et son utilisation pour augmenter l’expansion de cellules spécifiques d’antigènes dans des immunothérapies. The present invention relates to a genetically modified PDC (plasmacytoid dendritic cell) line for secreting a cytokine, and its use for increasing the expansion of antigen-specific cells in immunotherapy.
ETAT DE LA TECHNIQUE STATE OF THE ART
Des approches d’immunothérapie cherchant à favoriser la production de cellules spécifiques d’antigènes sont connues. Une approche utilisant une lignée de cellules dendritiques plasmacytoïdes ou PDC (appelée « PDC*üne ») pour induire des cellules cytotoxiques spécifiques d’antigènes à partir de cellules mononucléées de donneur (CMN), en particulier des cellules mononuclées du sang périphérique (PBMC), a été développée par les inventeurs (WO 2009/138489). Cette approche consiste à mettre en culture des CMN en présence de « PDC*üne » irradiée et chargée avec un antigène, les cellules mononuclées partageant au moins une molécule HLA avec « PDC*line » (HLA-A2, par exemple). Immunotherapy approaches seeking to promote the production of specific antigen cells are known. An approach using a plasmacytoid or PDC dendritic cell line (called “PDC * üne”) to induce antigen-specific cytotoxic cells from donor mononuclear cells (CMN), in particular peripheral blood mononuclear cells (PBMC) , was developed by the inventors (WO 2009/138489). This approach consists in culturing CMNs in the presence of “PDC * üne” irradiated and loaded with an antigen, the mononuclear cells sharing at least one HLA molecule with “PDC * line” (HLA-A2, for example).
Les travaux menés par les demandeurs ont montré l’intérêt de cette approche thérapeutique et son potentiel important dans le traitement par immunothérapie de cancers tels que le mélanome ou les cancers du poumon (Aspord & al., 2012, données non publiées). The work carried out by the applicants has shown the interest of this therapeutic approach and its significant potential in the immunotherapy treatment of cancers such as melanoma or lung cancers (Aspord & al., 2012, unpublished data).
La capacité d’induire l’expansion des cellules spécifiques d’antigènes est un facteur déterminant de la mise en oeuvre de ces nouvelles approches thérapeutiques. Il est connu, notamment de la demande WO 2009/138489 que la présence de cytokines est nécessaire à cette expansion. Le protocole expérimental décrit consiste, la première semaine, en une coculture de PDC*line irradiée et chargée avec un antigène avec des CMN réalisée sans cytokine, puis, la deuxième semaine, en une nouvelle stimulation avec la PDC*Nne irradiée chargée avec un antigène et de l’IL-2. The ability to induce the expansion of specific antigen cells is a determining factor in the implementation of these new therapeutic approaches. It is known, in particular from application WO 2009/138489, that the presence of cytokines is necessary for this expansion. The experimental protocol described consists, the first week, in a coculture of irradiated PDC * line loaded with an antigen with CMN carried out without cytokine, then, in the second week, in a new stimulation with the irradiated PDC * Nne loaded with an antigen and IL-2.
L’importance des cytokines dans les mécanismes de développement des différentes réponses immunitaires est bien connue de l’homme du métier, notamment pour G IL-2 et l’IL15 (Waldmann, Nat Rev 2006). L’IL-2 est produite principalement par des lymphocytes T activés, tandis que l’IL-15 et son récepteur associé IL-15Ra (Dubois & al., 2002), est produite par des monocytes ou des cellules dendritiques myéloïdes (MDC) (Jakobisiak & al., 201 1 ). La modification transitoire ou permanente de MDC générées in vitro pour produire de l’IL15 a été décrite dans le but d’augmenter la fonction de cellules (van den Bergh & al., 2015, Steel & al (2010) ou Zhang & al. (2014)) dans l’induction de réponse antitumorale (NK, anticorps ou T spécifique d’antigène). The importance of cytokines in the development mechanisms of different immune responses is well known to those skilled in the art, especially for G IL-2 and IL15 (Waldmann, Nat Rev 2006). IL-2 is produced mainly by activated T lymphocytes, while IL-15 and its associated receptor IL-15Ra (Dubois & al., 2002), is produced by monocytes or myeloid dendritic cells (MDC) (Jakobisiak & al., 201 1). The transient or permanent modification of MDCs generated in vitro to produce IL15 has been described with the aim of increasing cell function (van den Bergh & al., 2015, Steel & al (2010) or Zhang & al. (2014)) in the induction of antitumor response (NK, antibody or specific T of antigen).
Même si les MDC et les PDC sont toutes les deux des cellules présentatrices d’antigène, ce sont deux types cellulaire différents capables d’induire différents types de
réponses immunitaires selon l’origine du pathogène ou de l’antigène et du contexte environnemental en général, différenciées par un certain nombre de caractéristiques : origine, localisation tissulaire, expression de Toll-like Récepteur, production de cytokines, notamment. Aucun de ces articles sur les MDC modifiées pour exprimer IL15 ne mentionne la possibilité d’employer des PDC, ni même d’envisager un effet au moins équivalent sur des PDC avec de l’IL15 ou même de l’IL2. Although MDC and PDC are both antigen presenting cells, they are two different cell types capable of inducing different types of immune responses according to the origin of the pathogen or the antigen and the environmental context in general, differentiated by a certain number of characteristics: origin, tissue localization, expression of Toll-like receptor, production of cytokines, in particular. None of these articles on MDCs modified to express IL15 mentions the possibility of using PDCs, or even of envisaging an effect at least equivalent on PDCs with IL15 or even IL2.
Alors que la méthode d’activation et d’expansion classique décrite dans WO 2009/138489 permet déjà d’obtenir des niveaux d’expansion particulièrement élevés (Aspord & al., 2010), il reste Intéressant d’améliorer cette nouvelle approche thérapeutique au niveau de l’expansion des cellules spécifiques d’antigènes. While the classic activation and expansion method described in WO 2009/138489 already makes it possible to obtain particularly high levels of expansion (Aspord & al., 2010), it remains interesting to improve this new therapeutic approach to level of expansion of specific antigen cells.
EXPOSE DE L'INVENTION STATEMENT OF THE INVENTION
L’invention concerne donc une nouvelle lignée de PDC génétiquement modifiée pour exprimer une cytokine choisie parmi l’interleukine 15 (IL15) et l’interleukine 2 (IL2). The invention therefore relates to a new line of PDC genetically modified to express a cytokine chosen from interleukin 15 (IL15) and interleukin 2 (IL2).
En particulier, les cellules PDC génétiquement modifiées selon l’invention sont chargées avec un ou plusieurs antigènes, sous forme peptidique. In particular, the genetically modified PDC cells according to the invention are loaded with one or more antigens, in peptide form.
L’invention concerne aussi l’utilisation de la nouvelle lignée PDC génétiquement modifiée pour l’amplification de cellules spécifiques d’antigènes, en particulier des cellules mononucléées (CMN), plus particulièrement des cellules mononuclées du sang périphérique (PBMC). The invention also relates to the use of the new genetically modified PDC line for the amplification of specific antigen cells, in particular mononuclear cells (CMN), more particularly peripheral blood mononuclear cells (PBMC).
L’invention concerne aussi un produit de combinaison ou kit, comprenant d’une part une lignée de PDC génétiquement modifiée selon l’invention et d’autre part des cellules mononucléées (CMN), en particulier pour une utilisation simultanée dans le traitement de maladies par immunothérapie. The invention also relates to a combination product or kit, comprising on the one hand a line of PDC genetically modified according to the invention and on the other hand mononuclear cells (CMN), in particular for simultaneous use in the treatment of diseases by immunotherapy.
L’invention concerne également une composition vaccinale comprenant une lignée PDC génétiquement modifiée selon l’invention et un véhicule approprié pour son administration. The invention also relates to a vaccine composition comprising a genetically modified PDC line according to the invention and a vehicle suitable for its administration.
DESCRIPTION DES FIGURES DESCRIPTION OF THE FIGURES
La Figure 1 représente la mesure de l’expression de la molécule CD34 sur la lignée PDC*Nne non-transduite (à gauche) ou transduite par le surnageant rétroviral codant pour IΊI-2 ou l’IL-15. Figure 1 represents the measurement of the expression of the CD34 molecule on the PDC * Nne line not transduced (on the left) or transduced by the retroviral supernatant coding for IΊI-2 or IL-15.
La Figure 2 représente l’expression de l’IL2 ou de l’IL15 par la lignée PDC*Nne transduite par le surnageant rétroviral IL2 ou IL15.Les cytokines sont détectées au niveau de l’ARN messager (A, normalisée par rapport au G6PDH) ou dans le surnageant des cellules transduites (B, en pg/ml). Figure 2 represents the expression of IL2 or IL15 by the PDC * Nne line transduced by the retroviral supernatant IL2 or IL15. Cytokines are detected at the level of messenger RNA (A, normalized with respect to G6PDH ) or in the supernatant of transduced cells (B, in pg / ml).
La Figure 3 représente l’expansion de lymphocytes T CD8+ anti-Melan-A après coculture de 14 jours de la lignée PDC, non modifiée ou modifiée génétiquement par l’IL2 ou l’IL15, chargée avec l’antigène Melan-A, et des cellules mononucléées de 3 donneurs sains.
Les lymphocytes T CD8+ anti-Melan-A sont détectés par cytométrie en flux en utilisant des multimères HLA-A2/Melan-A. En A, une expérience représentative est montrée; en B, les résultats de 3 expériences sont représentés (% de cellules CD8+ multimères). FIG. 3 represents the expansion of anti-Melan-A CD8 + T lymphocytes after 14-day coculture of the PDC line, unmodified or genetically modified by IL2 or IL15, loaded with the Melan-A antigen, and mononuclear cells from 3 healthy donors. Anti-Melan-A CD8 + T cells are detected by flow cytometry using HLA-A2 / Melan-A multimers. In A, a representative experience is shown; in B, the results of 3 experiments are shown (% of CD8 + multimer cells).
La Figure 4 représente la fonction des cellules CD8+ issues de la coculture de 14 jours. La fonction des cellules cytotoxiques est objectivée par la détection d’IFNy intracytoplasmique après stimulation par la lignée cible (T2) chargée avec le peptide utilisé dérivé de Melan-A. En A, l'illustration de la détection intracytoplasmique de la cytokine sur l’ensemble des cellules CD8+ spécifiques et non-spécifiques, obtenues avec les différentes lignées. En B, le pourcentage de cellules CD8+ positives pour l’IFNy dans les cellules non- spécifiques (Multimer-) et les cellules spécifiques (Multimer+) générées avec chaque lignée. Figure 4 shows the function of CD8 + cells from the 14-day coculture. The function of cytotoxic cells is objectified by the detection of intracytoplasmic IFNy after stimulation by the target line (T2) loaded with the peptide used derived from Melan-A. In A, the illustration of intracytoplasmic detection of the cytokine on all specific and non-specific CD8 + cells, obtained with the different lines. In B, the percentage of CD8 + cells positive for IFNγ in non-specific cells (Multimer-) and specific cells (Multimer +) generated with each line.
DESCRIPTION DETAILLEE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION
L’invention concerne donc une nouvelle lignée de PDC génétiquement modifiée pour exprimer une cytokine choisie parmi IL15 et IL2. The invention therefore relates to a new line of PDC genetically modified to express a cytokine chosen from IL15 and IL2.
Les lignées PDC utiles selon l’invention pour être génétiquement modifiées, également appelées lignées PDC initiales, sont bien connues de l’homme du métier, notamment décrites dans EP 1 572 989 et WO 2009/138489. Il s’agit en particulier de cellules obtenues à partir de cellules de leucémie à PDC. On citera en particulier les lignées GEN2.2 et GEN3 déposées sous les numéros 2938 et 31 10 à la CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75015Paris) et toutes lignées issues de cellules leucémiques à PDC. The PDC lines useful according to the invention for being genetically modified, also called initial PDC lines, are well known to those skilled in the art, in particular described in EP 1 572 989 and WO 2009/138489. These are in particular cells obtained from PDC leukemia cells. Mention will be made in particular of the GEN2.2 and GEN3 lines deposited under the numbers 2938 and 31 10 at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75015Paris) and all lines derived from PDC leukemia cells .
Par lignées issues de cellules leucémiques à PDC, on entend selon l’invention des lignées dérivées de nodules tumoraux consécutif à l’injection de cellules leucémiques à PDC à des souris immunodéficients, ces nodules étant dilacérés pour obtenir une suspension cellulaire qui est mise en culture dans un milieu synthétique favorisant la croissance de ladite lignée. By lines derived from PDC leukemic cells is meant according to the invention lines derived from tumor nodules following the injection of PDC leukemic cells into immunodeficient mice, these nodules being dilated to obtain a cell suspension which is cultured in a synthetic medium promoting the growth of said line.
Par expression d’une cytokine choisie parmi IL15 et IL2, on entend selon l’invention la sécrétion de la cytokine par la lignée modifiée génétiquement. By expression of a cytokine chosen from IL15 and IL2, is meant according to the invention the secretion of the cytokine by the genetically modified line.
Les séquences des cytokines sont bien connues de l’homme du métier. On citera en particulier, pour l’IL2 la séquence protéique identifiée sous la référence UniProtKB P60568 et la séquence codante identifiée sous la référence Ensembl ENSG00000109471 et pour l’IL-15 la séquence protéique identifiée sous la référence UniProtKB P40933 et la séquence codante identifiée sous la référence Ensembl ENSG00000164136. Les variants ou fragments de ces cytokines qui ont la même activité que les séquences ci-dessus sont également partie de l’invention. De préférence les cytokines sont les cytokines humaines identifiées ci-dessus. Cytokine sequences are well known to those of skill in the art. In particular, the protein sequence identified under the reference UniProtKB P60568 and the coding sequence identified under the reference Ensembl ENSG00000109471 and for the IL-15 the protein sequence identified under the reference UniProtKB P40933 and the coding sequence identified under the reference Ensembl ENSG00000164136. Variants or fragments of these cytokines which have the same activity as the above sequences are also part of the invention. Preferably the cytokines are the human cytokines identified above.
Par lignée PDC génétiquement modifiée, on entend selon l’invention toute lignée
transduite par une particule virale permettant l’intégration du gène dans le génome de la lignée, ce virus pouvant être un adénovirus, un lentivirus ou un rétrovirus. De préférence on utilisera un rétrovirus de type Moloney murine leukemia virus (Mo-MLV). Ces particules rétrovirales étant produites par la lignées d’encapsulation HEK 293T, expriment naturellement ou après transfection les séquences gag/pol et env rétrovirales. By genetically modified PDC line is meant according to the invention any line transduced by a viral particle allowing the integration of the gene into the genome of the line, this virus possibly being an adenovirus, a lentivirus or a retrovirus. Preferably, a murine Moloney leukemia virus (Mo-MLV) type retrovirus will be used. These retroviral particles being produced by the HEK 293T encapsulation lines, express naturally or after transfection the retroviral gag / pol and env sequences.
L’homme du métier saura préparer une particule virale comprenant une séquence codante pour le ou les gènes d’intérêt et les éléments de régulations, en particulier les séquences employées pour permettre l’expression des cytokines dans des cellules de mammifères génétiquement modifiées. On citera notamment les séquences d’enveloppe virale, permettant l’entrée du virus dans la cellule, telles que 4070A, MK-G, HA, LCMV, RD1 14, HIV, VSV, MLV, GALV, BAEV), les séquences promotrices permettant l'initiation de la transcription du gène d’intérêt, telles que les séquences de type LTR (Long Terminal Repeat) modifiées ou non, PGK, EF-1 , CMV, SFFV, RSV, ou SV40, les séquences augmentant l’efficacité de transduction telle que cPPT, les séquences facilitant ou initiant la traduction, telle que la séquence 1RES (Internai Ribosome Entry Site) (Pelletier, Nature 1988), ou la séquence Kozak (Kozak, Nucl acid Res 1991 ), des séquences favorisant l’expression de la protéine, telle que la séquence WPRE (Donello et al., J. Virol 1998). De préférence on emploiera les séquences LTR, 1RES, Kozak et WPRE. Those skilled in the art will know how to prepare a viral particle comprising a coding sequence for the gene or genes of interest and regulatory elements, in particular the sequences used to allow the expression of cytokines in genetically modified mammalian cells. Mention will in particular be made of the viral envelope sequences allowing the entry of the virus into the cell, such as 4070A, MK-G, HA, LCMV, RD1 14, HIV, VSV, MLV, GALV, BAEV), the promoter sequences allowing the initiation of the transcription of the gene of interest, such as the LTR (Long Terminal Repeat) type sequences modified or not, PGK, EF-1, CMV, SFFV, RSV, or SV40, the sequences increasing the efficiency of transduction such as cPPT, sequences facilitating or initiating translation, such as the sequence 1RES (Internai Ribosome Entry Site) (Pelletier, Nature 1988), or the sequence Kozak (Kozak, Nucl acid Res 1991), sequences promoting expression protein, such as the WPRE sequence (Donello et al., J. Virol 1998). Preferably, the sequences LTR, 1RES, Kozak and WPRE will be used.
Les méthodes de transduction des cellules de PDC avec une particule virale pour obtenir une nouvelle lignée de PDC génétiquement modifiée pour exprimer une cytokine choisie parmi IL15 et IL2 sont également connues de l’homme du métier. On citera en particulier l’utilisation d’agents polycationiques, tels que le polybrene ou le DOGS (dioctadecylamidoglycyl spermine), ou d’agents favorisant les contacts entre virus et cellules d’intérêt, tels que des fragments de fibronectine (Rétronectin). De préférence on emploiera la méthode utilisant la Rétronectine. Methods of transducing PDC cells with a viral particle to obtain a new genetically modified PDC line to express a cytokine selected from IL15 and IL2 are also known to those skilled in the art. Mention will in particular be made of the use of polycationic agents, such as polybrene or DOGS (dioctadecylamidoglycyl spermine), or of agents promoting contacts between viruses and cells of interest, such as fragments of fibronectin (Retronectin). Preferably, the method using Retronectin will be used.
De préférence, la cytokine seule est exprimée. Dans certains cas, notamment pour IL15, la lignée PDC exprime également le récepteur de la cytokine, en particulier le récepteur IL15Ra. Le récepteur IL15Ra est bien connu de l’homme du métier, en particulier sa séquence protéique Preferably, the cytokine alone is expressed. In certain cases, in particular for IL15, the PDC line also expresses the cytokine receptor, in particular the IL15Ra receptor. The IL15Ra receptor is well known to those skilled in the art, in particular its protein sequence
Dans le cas où la lignée PDC modifiée selon l’invention n’exprime pas ledit récepteur, la lignée PDC pourra également être modifiée pour exprimer ledit récepteur, selon les méthodes usuelles de la technique. In the case where the PDC line modified according to the invention does not express said receptor, the PDC line can also be modified to express said receptor, according to the usual methods of the technique.
Les cellules PDC modifiées selon l’invention sont particulièrement utiles pour l’amplification de cellules spécifiques d’antigènes, en particulier des cellules mononucléées (CMN), plus particulièrement des cellules mononuclées du sang périphérique (PBMC). The PDC cells modified according to the invention are particularly useful for the amplification of specific antigen cells, in particular mononuclear cells (CMN), more particularly peripheral blood mononuclear cells (PBMC).
Bien qu’elles puissent être employées directement, les cellules PDC modifiées selon l’invention sont de préférence associées à des antigènes, en particulier des antigènes
peptidiques. Although they can be used directly, the PDC cells modified according to the invention are preferably associated with antigens, in particular antigens peptide.
Dans le cadre de la présente invention le terme « antigène » définit une molécule ou une partie de la molécule reconnue par des cellules du système immunitaire et capable de déclencher une réaction immunitaire à médiation cellulaire. Les antigènes selon la présente invention peuvent être des antigènes natifs ou modifiés, tumoraux ou microbiens (notamment bactériens ou viraux), tels que des peptides, protéines, glycopeptides, glycoprotéines, protéines phosphorylées. In the context of the present invention, the term “antigen” defines a molecule or a part of the molecule recognized by cells of the immune system and capable of triggering a cell-mediated immune reaction. The antigens according to the present invention can be native or modified antigens, tumor or microbial (in particular bacterial or viral), such as peptides, proteins, glycopeptides, glycoproteins, phosphorylated proteins.
L’homme du métier choisira le ou les antigènes à associer à la lignée PDC modifiée selon l’invention en fonction de la maladie à traiter. Those skilled in the art will choose the antigen (s) to be associated with the modified PDC line according to the invention according to the disease to be treated.
Dans une mise en oeuvre préférée de l'invention les antigènes sont des peptides susceptibles d'être obtenus à partir de protéines antigéniques d'origine tumorale ou virale. Ces peptides sont bien connus de l’homme du métier, décrits notamment dans de nombreuses demandes de brevets ou brevets, en particulier EP 1 485 719, EP 2 1 13 253, US 7 087 712, US 7 528 224, WO 94/020127, WO 95/02553, WO 98/22589, WO 2000/003693, WO 2000/020445, WO 2000/052163, WO 2000/078806, WO 2004/067023, WO 2007/036638, WO 2007/039192, WO 2008/045286, WO 201 1/012720, WO 2015/0965572 et WO 2016/179573. In a preferred implementation of the invention, the antigens are peptides capable of being obtained from antigenic proteins of tumor or viral origin. These peptides are well known to those skilled in the art, described in particular in numerous patent or patent applications, in particular EP 1 485 719, EP 2 1 13 253, US 7 087 712, US 7 528 224, WO 94/020127 , WO 95/02553, WO 98/22589, WO 2000/003693, WO 2000/020445, WO 2000/052163, WO 2000/078806, WO 2004/067023, WO 2007/036638, WO 2007/039192, WO 2008/045286 , WO 201 1/012720, WO 2015/0965572 and WO 2016/179573.
De nombreux antigènes tumoraux susceptibles d’être employés sont également décrits dans des bases de données disponibles en ligne, comme par exemple aux adresses http://cvc.dfci.harvard.edu/tadb/ ou https://docs.google.eom/spreadsheets/u/1/d/1 BFn5_mu7ogUh35NsLUozj9EB2rbLtj7XjMyt7 BoYNxg/pubhtml?widget=true&headers=false#gid=1609210712 ou encore dans des publications telles que Djureinovic & al. (JCI Insight. 2016;1 (10):e86837). Many tumor antigens that may be used are also described in databases available online, such as at http://cvc.dfci.harvard.edu/tadb/ or https: //docs.google.eom / spreadsheets / u / 1 / d / 1 BFn5_mu7ogUh35NsLUozj9EB2rbLtj7XjMyt7 BoYNxg / pubhtml? widget = true & headers = false # gid = 1609210712 or in publications such as Djureinovic & al. (JCI Insight. 2016; 1 (10): e86837).
De même de nombreux antigènes viraux susceptibles d’être employés sont également décrits dans des bases de données disponibles en ligne comme par exemple aux adresses https://www.iedb.org/ ou http://crdd.osdd.net/raghava/antigendb/. Likewise, numerous viral antigens which may be used are also described in databases available online, for example at the addresses https://www.iedb.org/ or http://crdd.osdd.net/raghava/ antigendb /.
Selon un mode particulier de réalisation de l'invention les peptides susceptibles d'être obtenus à partir d'antigènes tumoraux peuvent être choisis parmi les peptides compris dans la séquence des antigènes CEA, NY-BR1 , Her-2/Neu, PSA, RAGE-1 , PRAME, TRP-2, MAGE-A1 , MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C1 , MAGE-C2, Multi-MAGE, MUC-1 , p53, hTERT, survivin, melan-A/MART-1 , GP100, tyrosinase, CAMEL ou NY-ES01 , modifiés ou non, seuls ou en association. De même tout antigène choisi parmi des protéines mutées ou des protéines issues de la transcription d’un ARN messager généré par un nouveau cadre de lecture d’une séquence nucléique (néo-antigène). According to a particular embodiment of the invention, the peptides capable of being obtained from tumor antigens can be chosen from the peptides included in the sequence of the antigens CEA, NY-BR1, Her-2 / Neu, PSA, RAGE -1, PRAME, TRP-2, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C1, MAGE-C2, Multi-MAGE, MUC-1, p53 , hTERT, survivin, melan-A / MART-1, GP100, tyrosinase, CAMEL or NY-ES01, modified or not, alone or in combination. Similarly, any antigen chosen from mutated proteins or proteins resulting from the transcription of a messenger RNA generated by a new reading frame for a nucleic sequence (neo-antigen).
Selon un autre mode de réalisation de l’invention, les peptides susceptibles d'être obtenus à partir d'antigènes viraux peuvent être choisis parmi les peptides compris dans la séquence des antigènes env, nef, gp41 , gp120, gag ou pol du virus HIV, HBc ou HBs du
virus HBV, core, NS3 ou NS5 du virus HCV, Flu M1 du virus influenza, CMVpp65 du virus CMV, BMLF1 , LMP2, EBNA-2 ou EBNA-3a du virus EBV, LT A ou VOP1 du virus BKV, la nucléoprotéine du virus Hatan, NS3 du virus de la Dengue, E6 et E7 du virus HPV, la proteine E, NS3, ou BS4b du West Nile Virus, modifiés ou non, seuls ou en association. According to another embodiment of the invention, the peptides capable of being obtained from viral antigens can be chosen from the peptides included in the sequence of the env, nef, gp41, gp120, gag or pol antigens of the HIV virus , HBc or HBs from HBV, core, NS3 or NS5 virus of HCV virus, Flu M1 of influenza virus, CMVpp65 of CMV virus, BMLF1, LMP2, EBNA-2 or EBNA-3a of EBV virus, LT A or VOP1 of BKV virus, nucleoprotein of virus Hatan, NS3 of the Dengue virus, E6 and E7 of the HPV virus, protein E, NS3, or BS4b of the West Nile Virus, modified or not, alone or in combination.
De manière préférentielle, on citera Melan-A, gp100, Tyrosinase, MAGE-A3, MAGE- A4, MAGE-A10, Multi-MAGE, CTAG2, CTAG1 , Survivin, Her-2/Neu, hTERT et MUC1. Preferably, mention will be made of Melan-A, gp100, Tyrosinase, MAGE-A3, MAGE-A4, MAGE-A10, Multi-MAGE, CTAG2, CTAG1, Survivin, Her-2 / Neu, hTERT and MUC1.
Selon un premier mode préféré de réalisation de l’invention, les cellules PDC génétiquement modifiées sont chargées avec un ou plusieurs antigènes. On dira également qu’elles sont pulsées, c’est à dire incubées avec un ou plusieurs antigènes. According to a first preferred embodiment of the invention, the genetically modified PDC cells are loaded with one or more antigens. It will also be said that they are pulsed, that is to say incubated with one or more antigens.
L’invention concerne donc une lignée PDC génétiquement modifiée telle que définie ci- dessus et dans les exemples, chargée avec un des antigènes listés précédemment. The invention therefore relates to a genetically modified PDC line as defined above and in the examples, loaded with one of the antigens listed above.
Selon un autre mode de réalisation de l’invention, les cellules sont également génétiquement modifiées pour exprimer lesdits antigènes. Les outils de modification génétiques sont les mêmes que ceux employés pour modifier les cellules PDC pour exprimer les cytokines. Dans le cas des antigènes, l’homme du métier choisira également les éléments génétiques qui permettent d’exprimer les antigènes au niveau cellulaire de façon à ce qu’ils soient transformés en épitopes et présentés par les molécules HLA, in vitro ou in vivo. De tels éléments sont également bien connus de l’homme du métier (Hu, Immunological review 2011 ). According to another embodiment of the invention, the cells are also genetically modified to express said antigens. The genetic modification tools are the same as those used to modify PDC cells to express cytokines. In the case of antigens, a person skilled in the art will also choose the genetic elements which make it possible to express the antigens at the cellular level so that they are transformed into epitopes and presented by the HLA molecules, in vitro or in vivo. Such elements are also well known to those skilled in the art (Hu, Immunological review 2011).
Pour un usage thérapeutique, les lignées PDC génétiquement modifiées selon l’invention, définies précédemment et ci-après, sont des lignées irradiées. For therapeutic use, the genetically modified PDC lines according to the invention, defined above and below, are irradiated lines.
Les méthodes et conditions d’irradiation pour inhiber la croissance et les capacités de multiplication des lignées PDC transformées selon l’invention sont bien connues de l’homme du métier, notamment décrites dans WO 2009/138489. The irradiation methods and conditions for inhibiting the growth and the multiplication capacities of the PDC lines transformed according to the invention are well known to those skilled in the art, in particular described in WO 2009/138489.
L’invention concerne aussi l’utilisation de la nouvelle lignée PDC génétiquement modifiée pour l’amplification de cellules spécifiques d’antigènes, en particulier des cellules mononucléées (CMN), plus particulièrement des cellules mononuclées du sang périphérique (PBMC). The invention also relates to the use of the new genetically modified PDC line for the amplification of specific antigen cells, in particular mononuclear cells (CMN), more particularly peripheral blood mononuclear cells (PBMC).
L’invention concerne aussi la nouvelle lignée PDC génétiquement modifiée pour son utilisation en thérapie, en particulier pour le traitement de maladies par immunothérapie. The invention also relates to the new genetically modified PDC line for use in therapy, in particular for the treatment of diseases by immunotherapy.
L’invention concerne aussi un produit de combinaison ou kit, comprenant d’une part une lignée de PDC génétiquement modifiée selon l’invention et d’autre part des cellules mononucléées (CMN), en particulier pour une utilisation simultanée dans le traitement de maladies par immunothérapie. The invention also relates to a combination product or kit, comprising on the one hand a line of PDC genetically modified according to the invention and on the other hand mononuclear cells (CMN), in particular for simultaneous use in the treatment of diseases by immunotherapy.
La maladie traitée dépendra essentiellement des antigènes qui seront associée à la lignée selon l’invention, avec par exemple les antigènes tumoraux étant employés pour le traitement des cancers et les antigènes viraux pour le traitement des infections virales.
L’invention concerne également une composition vaccinale comprenant une lignée PDC génétiquement modifiée selon l’invention et un véhicule approprié pour son administration. The disease treated will depend essentially on the antigens which will be associated with the line according to the invention, with for example the tumor antigens being used for the treatment of cancers and the viral antigens for the treatment of viral infections. The invention also relates to a vaccine composition comprising a genetically modified PDC line according to the invention and a vehicle suitable for its administration.
L’invention concerne également une méthode de prévention et/ou de traitement des cancers et/ou maladies infectieuses caractérisée en ce qu'on injecte une lignée de PDC génétiquement modifiée irradiée et pulsée selon l’invention dans chez un patient qui a besoin du traitement, les cellules spécifiques d’antigènes dudit patient et les PDC partageant au moins un allèle du complexe majeur d'histocompatibilité (CMH). The invention also relates to a method of preventing and / or treating cancers and / or infectious diseases, characterized in that a genetically modified irradiated and pulsed PDC line according to the invention is injected into a patient who needs treatment. , the specific cells of said patient's antigens and the PDCs sharing at least one allele of the major histocompatibility complex (MHC).
L’invention concerne aussi une méthode de prévention et/ou de traitement des cancers et/ou maladies infectieuses caractérisée en ce qu'on injecte chez un patient qui a besoin du traitement les effecteurs spécifiques obtenus par incubation d'une lignée de PDC selon l’invention avec au moins un antigène, les PDC pulsées, éventuellement irradiées, étant ensuite mises en contact avec des cellules spécifiques d’antigènes dudit patient et cultivées, les PDC et les cellules spécifiques d’antigènes partageant au moins un allèle du complexe majeur d'histocompatibilité (CMH). The invention also relates to a method of prevention and / or treatment of cancers and / or infectious diseases characterized in that the specific effectors obtained by incubation of a line of PDC according to l are injected into a patient in need of treatment. invention with at least one antigen, the pulsed PDCs, possibly irradiated, then being brought into contact with cells specific for antigens of said patient and cultured, PDCs and cells specific for antigens sharing at least one allele of the major complex d 'histocompatibility (CMH).
Par « effecteur spécifique » on entend selon l’invention les cellules de l'immunité capables de reconnaître un antigène spécifique ou un produit issu de cet antigène, en particulier des effecteurs cytotoxiques et plus particulièrement des lymphocytes T spécifiques de l'antigène utilisé, et notamment CD8+. By “specific effector” is meant according to the invention the cells of the immunity capable of recognizing a specific antigen or a product derived from this antigen, in particular cytotoxic effectors and more particularly T lymphocytes specific for the antigen used, and including CD8 +.
Ces méthodes sont notamment décrites dans la demande WO 2009/138489. These methods are in particular described in application WO 2009/138489.
EXEMPLES EXAMPLES
Exemple 1 : Génération des lignées PDC*line modifiées génétiquement pour exprimer IL-2 ou IL15. Example 1: Generation of genetically modified PDC * line lines to express IL-2 or IL15.
Les séquences des gènes d’intérêt codant pour IL2 (SEQ ID NO 1 ) et IL15 (SEQ ID NO 2) ont été synthétisées par ThermoFischer (https://www.thermofisher.com/fr/fr/home/life- science/cloning/gene-synthesis/geneart-gene-synthesis.html) et fournis dans un plasmide permettant l’amplification du plasmide après transformation des bactéries DH5alpha (NEB). La séquence d’intérêt (IL2 ou IL15) a ensuite été sous-clonée dans le plasmide SFGACD34 (Quintarelli, Blood 2007, généreusement fourni par le Dr M. Pule) entre les sites de restriction Sal-I et Mul-I à l’aide de la technologie Gibson Assembly (NEB). La présence d’une séquence tronquée de CD34 dans le plasmide SFGACD34 (sans domaine intracytoplasmique) permet la sélection des cellules exprimant le gène d’intérêt. Une suspension rétrovirale a ensuite été obtenue par triple transfection de la lignée HEK-293T avec le plasmide SFGACD34-IL2 ou SFGACD34-IL15 et les plasmides d’expression MoMLV gag-pol pEQ-PAM3(-E) et RD114 env (généreusement fournis par les Dr M. Pule et Dr M. Collins (Cosset, J Virol 1995)) en présence de GeneJuice (VWR). The sequences of the genes of interest coding for IL2 (SEQ ID NO 1) and IL15 (SEQ ID NO 2) were synthesized by ThermoFischer (https://www.thermofisher.com/fr/fr/home/life- science / cloning / gene-synthesis / geneart-gene-synthesis.html) and supplied in a plasmid allowing the amplification of the plasmid after transformation of DH5alpha bacteria (NEB). The sequence of interest (IL2 or IL15) was then subcloned into the plasmid SFGACD34 (Quintarelli, Blood 2007, generously supplied by Dr M. Pule) between the restriction sites Sal-I and Mul-I at the using Gibson Assembly technology (NEB). The presence of a truncated CD34 sequence in the plasmid SFGACD34 (without intracytoplasmic domain) allows the selection of cells expressing the gene of interest. A retroviral suspension was then obtained by triple transfection of the HEK-293T line with the plasmid SFGACD34-IL2 or SFGACD34-IL15 and the expression plasmids MoMLV gag-pol pEQ-PAM3 (-E) and RD114 env (generously provided by Dr M. Pule and Dr M. Collins (Cosset, J Virol 1995)) in the presence of GeneJuice (VWR).
Les cellules de la lignée PDC ont été par la suite transduites avec le surnageant
rétroviral correspondant à l’IL2 ou l’IL15 en présence de rétronectine (Takara) et l’expression de CD34 a été mesurée, reflétant l’efficacité de transduction. Cette lignée est dérivée des cellules d’un patient ayant une leucémie PDC (Chaperot, Blood 2001 ), patient dont sont issues les lignées GEN2.2 et GEN3. The cells of the PDC line were subsequently transduced with the supernatant retroviral corresponding to IL2 or IL15 in the presence of retronectin (Takara) and the expression of CD34 was measured, reflecting the transduction efficiency. This line is derived from the cells of a patient with PDC leukemia (Chaperot, Blood 2001), patient from which the GEN2.2 and GEN3 lines are derived.
Comme le montre la figure 1 , plus de 90% de PDC*line ont été transduites. As shown in Figure 1, more than 90% of PDC * line has been transduced.
Cette expression de CD34 est corrélée à l’expression des gènes IL2 ou IL15. En effet, l’analyse par RT-qPCR (technique TaqMan, Roche) montre une expression relative, par rapport au gène de la G6PDH, élevée pour l’IL2 ou l’IL15, d’un facteur supérieur à 20 ou 30 respectivement (Figure 2A). Par ailleurs, en utilisant la technique CBA (BD) ou ELISA (R&D), les deux cytokines sont retrouvées produites sous forme soluble dans le surnageant des lignées modifiées génétiquement correspondantes à la concentration de 20 000 pg/ml et de 2 500 pg/ml pour l’IL2 et pour l’IL15 respectivement (Figure 2B). This expression of CD34 is correlated with the expression of the IL2 or IL15 genes. Indeed, analysis by RT-qPCR (TaqMan technique, Roche) shows a relative expression, relative to the G6PDH gene, high for IL2 or IL15, by a factor greater than 20 or 30 respectively ( Figure 2A). Furthermore, using the CBA (BD) or ELISA (R&D) technique, the two cytokines are found produced in soluble form in the supernatant of genetically modified lines corresponding to the concentration of 20,000 pg / ml and 2,500 pg / ml for IL2 and for IL15 respectively (Figure 2B).
Exemple 2 : Expansion de lymphocytes CD8+ après co-culture de CMN avec les lignées transduites chargées avec un peptide tumoral. Example 2: Expansion of CD8 + lymphocytes after co-culture of CMN with transduced lines loaded with a tumor peptide.
La capacité des lignées modifiées a ensuite été évaluée à l’aide d’une coculture avec des cellules mononuclées (CMN) de donneur sain HLA-A2+. Brièvement, les cellules de la lignée PDC modifiée génétiquement ou non ont été chargées avec le peptide Melan-A26i_-35, irradiées puis mises en culture avec des CMN en plaque 24 puits pendant 14 jours. Différentes quantités de cellules de la lignée PDC ont été ajoutées aux 2 million de CMN (10/1 ou 20/1 ) par puits. A J7, les cellules sont restimulées avec la lignée PDC chargées avec Melan-A en présence d’IL-2 soluble. A D14, l’expansion des lymphocytes CD8+ anti- Melan-A est mesurée à l’aide de dextramer Melan-A A2 (Multimer) marqué à un fluorochrome (Immudex) et des anticorps anti-CD3 et CD8 (Beckman Coulter). The capacity of the modified lines was then evaluated using a coculture with mononuclear cells (CMN) from a healthy donor HLA-A2 +. Briefly, the cells of the PDC line, genetically modified or not, were loaded with the peptide Melan-A26i_-35, irradiated then cultured with CMN in a 24-well plate for 14 days. Different amounts of PDC line cells were added to the 2 million CMN (10/1 or 20/1) per well. On D7, the cells are restimulated with the PDC line loaded with Melan-A in the presence of soluble IL-2. At D14, the expansion of anti-Melan-A CD8 + lymphocytes is measured using dextramer Melan-A A2 (Multimer) labeled with a fluorochrome (Immudex) and anti-CD3 and CD8 antibodies (Beckman Coulter).
Les résultats présentés dans la figure 3 montrent que l’expression de l’IL2 ou de l’IL15 par la lignée PDC permet une expansion plus forte que la lignée non modifiée. Cette augmentation est en moyenne d’une facteur supérieur ou égal à 3,7. The results presented in FIG. 3 show that the expression of IL2 or IL15 by the PDC line allows a stronger expansion than the unmodified line. This increase is on average by a factor greater than or equal to 3.7.
Exemple 3 : Fonctionnalité des effecteurs générés avec la lignée PDC modifiée ou non par l’IL2 ou l’IL15 : Expression intracytoplasmique d’IFNy. Example 3: Functionality of the effectors generated with the PDC line modified or not by IL2 or IL15: Intracytoplasmic expression of IFNy.
Les lymphocytes T amplifiés après une coculture de 14 jours en présence de la lignée PDC chargée avec Melan-A, transduite ou non par l’IL-2 ou l’IL15, ont été marqués avec le dextramer Melan-A A2 (Multimer) et restimulés par la lignée cible T2 chargée avec Melan-A en présence d’un inhibiteur de dégranulation, le GolgiSTOP (Beckton Dickinson) pendant 4h. Les cellules ont ensuite été marquées avec un anticorps anti-IFNy (Becton Dickinson) et des anticorps anti-CD3 et CD8. La figure 4A représente le phénotypage obtenu représentant l’information des cellules IFNy positives en fonction des cellules marquées par le multimer. La figure 4B montre que seules les cellules spécifiques de Melan-A (multimer+) produisent
de l’IFNy quand elles sont stimulées par la lignée T2/Melan-A. Les résultats montrent que les lymphocytes spécifiques de Melan-A amplifiés en présence de la lignée PDC modifiée pour exprimer l’IL2 ou l’IL15 secrétent 1 ,8 à 2,1 fois plus d’IFNy, que les lymphocytes spécifiques de Melan-A amplifiés avec la lignée non modifiée.
The T lymphocytes amplified after a 14-day coculture in the presence of the PDC line loaded with Melan-A, transduced or not by IL-2 or IL15, were labeled with the dextramer Melan-A A2 (Multimer) and restimulated by the T2 target line loaded with Melan-A in the presence of a degranulation inhibitor, GolgiSTOP (Beckton Dickinson) for 4 h. The cells were then labeled with an anti-IFNγ antibody (Becton Dickinson) and anti-CD3 and CD8 antibodies. FIG. 4A represents the phenotyping obtained representing the information of the IFNy positive cells as a function of the cells labeled with the multimer. Figure 4B shows that only specific Melan-A cells (multimer +) produce of IFNy when stimulated by the T2 / Melan-A line. The results show that Melan-A specific lymphocytes amplified in the presence of the PDC line modified to express IL2 or IL15 secrete 1.8 to 2.1 times more IFNγ than Melan-A specific lymphocytes amplified with the unmodified line.
REFERENCES REFERENCES
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• https: //docs.google.eom/spreadsheets/u/1/d/1 BFn5_mu7ogUh35NsLUozj9EB2rbLtj7XjM yt7BoYNxg / pubhtml? Widget = true & headers = false # gid = 1609210712
Claims
1. Lignée de PDC génétiquement modifiée pour exprimer une cytokine choisie parmi l’interleukine 15 (I L15) et l’intrtlrukine 2 (IL2). 1. PDC line genetically modified to express a cytokine chosen from interleukin 15 (I L15) and intrtlrukine 2 (IL2).
2. Lignée génétiquement modifiées selon la revendication 1 , caractérisée en ce que la lignée PDC initiale est obtenue à partir de cellules dendritiques plasmacytoides leucémiques. 2. Genetically modified line according to claim 1, characterized in that the initial PDC line is obtained from leukemic plasmacytoid dendritic cells.
3. Lignée génétiquement modifiée selon l’une des revendications 1 ou 2, caractérisée en ce que la modification est réalisée à l’aide de particules virales 3. Genetically modified line according to one of claims 1 or 2, characterized in that the modification is carried out using viral particles
4. Lignée génétiquement modifiée selon l’une des revendications 1 à 3, caractérisée en ce que la cytokine seule est exprimée. 4. Genetically modified line according to one of claims 1 to 3, characterized in that the cytokine alone is expressed.
5. Lignée génétiquement modifiée selon l’une des revendications 1 à 4, caractérisée en ce qu’elle est chargée avec un ou plusieurs antigènes. 5. Genetically modified line according to one of claims 1 to 4, characterized in that it is loaded with one or more antigens.
6. Lignée génétiquement modifiée selon l’une des revendications 1 à 4, caractérisée en ce qu’elle est également génétiquement modifiée pour exprimer un ou plusieurs antigènes. 6. Genetically modified line according to one of claims 1 to 4, characterized in that it is also genetically modified to express one or more antigens.
7. Lignée génétiquement modifiée selon l’une des revendications 5 ou 6, caractérisée en ce que les antigènes sont des antigènes peptidiques. 7. Genetically modified line according to one of claims 5 or 6, characterized in that the antigens are peptide antigens.
8. Lignée génétiquement modifiée selon l’une des revendications 1 à 7, caractérisée en ce qu’elle est irradiée. 8. Genetically modified line according to one of claims 1 to 7, characterized in that it is irradiated.
9. Utilisation d’une lignée génétiquement modifiée selon l’une des revendications 1 à 8 pour l’amplification in vitro de cellules spécifiques d’antigènes. 9. Use of a genetically modified line according to one of claims 1 to 8 for the in vitro amplification of specific antigen cells.
10. Lignée génétiquement modifiée selon l’une des revendications 1 à 8 pour son utilisation en thérapie. 10. Genetically modified line according to one of claims 1 to 8 for use in therapy.
1 1. Produit de combinaison ou kit, comprenant d’une part une lignée de PDC génétiquement modifiée selon l’une des revendications 1 à 8 et d’autre part des cellules spécifiques d’antigènes. 1 1. Combination product or kit, comprising on the one hand a genetically modified PDC line according to one of claims 1 to 8 and on the other hand specific cells of antigens.
12. Produit de combinaison selon la revendication 1 1 , pour une utilisation simultanée dans le traitement de maladies par immunothérapie. 12. Combination product according to claim 1 1, for simultaneous use in the treatment of diseases by immunotherapy.
13. Composition vaccinale comprenant une lignée PDC génétiquement modifiée selon l’une des revendications 1 à 8 et un véhicule approprié pour son administration.
13. A vaccine composition comprising a genetically modified PDC line according to one of claims 1 to 8 and a vehicle suitable for its administration.
Applications Claiming Priority (2)
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| FR1859773A FR3087448B1 (en) | 2018-10-23 | 2018-10-23 | PDC LINE MODIFIED TO SECRET A CYTOKINE |
| PCT/EP2019/078845 WO2020083974A1 (en) | 2018-10-23 | 2019-10-23 | Modified pdc line for secreting a cytokine |
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| BR9302851A (en) | 1993-07-14 | 1995-04-25 | Pereira Antonio Martins | Integrated unit for bottle caps / stoppers treatment |
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| AU4990999A (en) | 1998-07-14 | 2000-02-07 | Jenner Biotherapies, Inc. | Survivin, and peptides thereof, as an anti-cancer vaccine |
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| US7157091B1 (en) | 1999-06-18 | 2007-01-02 | Ludwig Institute For Cancer Research | MAGE-A1 peptides presented by HLA class II molecules |
| US7892559B2 (en) | 2002-01-30 | 2011-02-22 | Survac Aps | Survivin-derived peptides and use thereof |
| FR2837837B1 (en) | 2002-03-28 | 2006-09-29 | Roussy Inst Gustave | PEPTIDE EPITOPES COMMON TO ANTIGENS OF THE SAME MULTIGENIC FAMILY |
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| ES2603418T3 (en) | 2006-10-04 | 2017-02-27 | Janssen Pharmaceutica Nv | Preparation of inactivated artificial antigen presenting cells and their use in cell therapies |
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| CA2767767A1 (en) | 2009-07-30 | 2011-02-03 | Helmholtz-Zentrum Fuer Infektionsforschung Gmbh | Compositions for generating an antigen specific immune response |
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| CN113302286A (en) | 2021-08-24 |
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