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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
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  • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

• the invention relates to obtaining regulatory CD4 + T lymphocytes which can be used in the prevention and treatment of autoimmune and inflammatory diseases.
• Autoimmune diseases are caused by a deregulation of the immune system, which results in an undesirable immune response by an organism to its own antigens.
• CD4 + T lymphocytes also called helper T lymphocytes.
• Thl lymphocytes involved in the development of the cellular immune response, produce pro-inflammatory cyto ines such as interleukin-2 (IL-2) and interferon ⁇ ( IFN ⁇ ) and exhibit activating effects of macrophages; Th2 lymphocytes, which produce cytokines such as interleukins IL-4, IL- ⁇ , IL-10 and IL-13, promote the secretion of antibodies.
• IL-2 interleukin-2
• IFN ⁇ interferon ⁇
• Tri cells When re-stimulated by the antigen used for their induction, the Tri cells proliferate only slightly, produce very high quantities of IL-10, large quantities of TGF- ⁇ (tumor growth factor ⁇ ), very small amounts of IL-2, and do not produce IL-4.
• activated Tri cells When activated Tri cells are grown in the presence of other CD4 + T cells they suppress proliferation of these in response to an antigen; this effect results from the secretion of cytokines, and in particular of IL-10, by the Tr lymphocytes, and not from a direct action of these on the CD4 + T cells; it can therefore be obtained without having to know the antigen responsible for the proliferation of these cells. This has an important advantage in the case of autoimmune diseases, the treatment of which can thus be envisaged without the need to know the exact antigen against which the pathogenic cells are directed.
• Tri inhibitory cells are capable not only of preventing, but also to cure these different pathologies. They also observed that the Tri cells migrated in a specific and intense way in the various inflammatory sites. This property of Tri cells represents an additional advantage in the use of these cells as a vector for anti-inflammatory molecules in sites of intense inflammation. Tri cells obtained from the T cells of a patient can therefore potentially be used in the context of cell therapy to regulate the immune response in this patient.
• Tri lymphocytes can thus be used in particular to prevent or treat, not only the autoimmune and inflammatory diseases mentioned above, but also any other pathology characterized by an aberrant inflammatory response, such as diabetes, psoriasis, atherosclerosis, rheumatoid arthritis or asthma; they can also be used in the treatment of transplant rejection or of the graft versus host reaction.
• the method for obtaining Tri lymphocytes which is described in the publication by GROUX et al. cited above, requires repeated stimulation of T cells with the antigen in the presence of IL-10. This method is cumbersome to implement, and does not make it possible to quickly obtain, from a patient, a population of Tri lymphocytes which can be used in the context of a therapeutic treatment.
• BULLENS et al. (Int. Immunol. 13, 181-191, 2001) report that co-stimulation by CD58 induces the production of IL-10 and IFN- ⁇ by T cells stimulated by an anti-CD3 antibody.
• DONG et al. (Nat. Med., 5, 1365, 1999) have described a co-stimulation molecule related to the B-7 family, and named B7-H1, which preferentially induces the secretion of IL-10.
• VAN GOOL et al. (Eur. J. Immunol., 29, 2367,
• CHABOT et al. report that the production of IL-10, induced by the interaction of microglial cells with T lymphocytes is reduced by blocking CD40 / CD40L, B7 / CTLA-4 or B7 / CD28, or CD23.
• the inventors have now developed a new method for rapidly obtaining antigen-specific Tri lymphocytes, in a quantity much greater than that obtained by the activation of CD4 + T cells in the presence of IL-10. They indeed found that the culture of CD4 + T cells in the presence of an inducing antigen, and of antigen presenting cells expressing a class II HLA molecule and the human LFA-3 molecule (CD58), but not expressing none of the co-stimulating molecules B7-1 (CD80), B7-2 (CD86), B7-H1, CD40, CD23 and ICAM-1 (CD54), induced the differentiation of Tri cells specific for said antigen.
• the LFA-3 molecule is the ligand of the CD2 receptor for T lymphocytes.
• LFA-3 Two forms have been described: a transmembrane form (WALNER et al., J. Exp. Med. 166, 923-932, 1987; PCT application WO 88/09826) and a form, called "Pl-linked LFA3" which is anchored to the cell membrane via a glycolipid containing phosphatidylinositol (PCT application WO 90/02181).
• the present invention relates to the use of artificial antigen presenting cells, expressing a molecule of the HLA class II system and a human LFA-3 molecule, and not expressing any of the co-stimulating molecules B7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1
• regulatory lymphocytes Tri specific for antigen, cells having the following characteristics, after re-stimulation by said antigen:
• IL-10 IL-10
• IL-2 an amount of IL-2 equal to or less than 50 pg / 10 6 cells; the ratio IL-10 / IL-2 produced by said cells is at least equal to 50/1, generally from 100/1 to 500/1;
• IL-4 an amount of IL-4 equal to or less than 50 pg / 10 6 cells; the IL-10 / IL-4 ratio produced by said cells is at least equal to 300/1, generally from 500/1 to 10000/1;
• TCD4 + cells stimulated by an antigen in the presence of Tri cells is reduced by at least 2 times, generally by at least 2 to 100 times.
• the subject of the present invention is in particular a method for preparing regulatory lymphocytes Tri specific for antigen from the lymphocytes of a patient, characterized in that it comprises: - In vitro activation of said lymphocytes in the presence of the chosen antigen, presented by artificial antigen presenting cells as defined above; the recovery, from said lymphocytes, of a population of activated CD4 + T lymphocytes comprising at least 10%, preferably at least 50%, and very preferably, at least 80% of Tri lymphocytes specific for said antigen.
• Artificial antigen presenting cells which can be used for the implementation of the present invention can advantageously be obtained by co-transfection of animal cells expressing none of the co-stimulation molecules indicated above with an acid sequence nucleic acid coding for the ⁇ chain of a class II HLA molecule, a nucleic acid sequence coding for the ⁇ chain of a class II HLA molecule and a nucleic acid sequence coding for any of the 2 forms of the LFA-3. If desired, these cells can also be transfected with a nucleic acid sequence coding for the antigen against which it is desired to induce the specificity of the Tri lymphocytes. These nucleic acid sequences can be carried by different nucleic acid molecules, or 2 or more of them can be carried by the same nucleic acid molecule.
• Animal cells which can be used for obtaining said artificial antigen presenting cells can be cells of human origin, autologous or heterologous, or cells of xenogenic origin, in particular mammalian cells. Recently isolated primary crops can be used; generally one will prefer to use established cell lines, which are more homogeneous, and capable of proliferating for several generations. They can be, for example, fibroblasts, keratinocytes, kidney tubule cells, Schwann cells, myoblasts, endothelial cells, etc.
• the MHC class II molecule expressed by the cells presenting the artificial antigen will be chosen from the various human HLA-2 molecules available, depending on the HLA-class II type of the patient from which CD4 + T lymphocytes are taken, and of the intended use for the Tri cells.
• an HLA-2 molecule also expressed by the patient; however, it is also possible to choose an HLA-2 molecule different from those expressed by said patient; for example, if it is desired to produce Tri cells which can be used in the context of the prevention of transplant rejection, an HLA-2 molecule expressed by the graft cells; in this case, the HLA-2 molecule constitutes the antigen against which the Tri cells obtained will be directed.
• the antigen used for the implementation of the process according to the invention will be chosen according to the use to which the specific cells of the antigen which are to be produced are intended. It may be an antigen associated with an inflammatory or autoimmune pathology; it can also be an antigen unrelated to a particular pathology, but which can be administered if one wishes to activate the Tri cells to fight against an undesirable immune response.
• the loading of the antigen on the cells presenting the artificial antigen can be carried out in a conventional manner, by co-incubation of the cells and of said antigen.
• This can be in native form, and be prepared by said antigen presenting cells; it can also be in the form of one or more antigenic peptides, which can be fixed directly by the HLA-2 molecules expressed by said cells.
• the antigen can be expressed by the artificial antigen presenting cells, in the case where these have been previously transfected with a nucleic acid sequence coding for it.
• the in vitro activation of the lymphocytes can be carried out directly on PBMCs (mononuclear cells of the peripheral blood) taken from said patient. It can also be performed on CD4 + T lymphocytes previously purified from these PBMCs.
• the activation of the lymphocytes is carried out by co-culture of these, for 2 to 10 days, preferably for 6 to 8 days, in the presence of the artificial antigen presenting cells described above, loaded with the selected antigen.
• the activated CD4 + T lymphocytes are selected on the basis of the expression of the CD4 + marker, and of one or more activation markers such as CD25, CD69, CD45RO, etc.
• Tri lymphocytes represent at least 10% and generally between 50 and 80% of the activated antigen-specific T lymphocytes.
• the stimulation can be repeated under the conditions defined above.
• Tri lymphocyte clones can also be isolated from this population. These clones can easily be identified on the basis of the characteristic cytokine production profile of lymphocytes
• the expansion of the clones can be carried out in a nutrient medium containing non-specific growth factors such as IL-4 and IL-2.
• a preferred embodiment is to use as stimulating agent beads coupled with anti-CD3 and CD28 antibodies.
• the sorting cells obtained by the process according to the invention have all the characteristics of Tri cells obtained by activation of CD4 + T lymphocytes in the presence of IL-10, and therefore have the same uses as these for the regulation of the immune response.
• the present invention also relates to a process for the preparation of a pharmaceutical composition intended for the treatment of inflammatory diseases and / or autoimmune diseases, characterized in that it comprises the preparation of Tri lymphocytes specific for antigen by a process in accordance with the invention, and the packaging of said Tri lymphocytes in a formulation suitable for their administration to a patient.
• PBMC Peripheral mononuclear cells
• CD4 + T cells are obtained by eliminating non-CD4 + cells, using anti-CD8 antibodies
• Mouse L fibroblast cells (ATCC CCL-1) were co-transfected with nucleic acid sequences encoding LFA-3 and an HLA class II molecule. Obtaining a cDNA encoding LFA-3
• a cDNA encoding LFA-3 was prepared from a total cDNA library of human peripheral mononuclear cells, by polymerase chain reaction (PCR) using the primer called 5'LFA-3, which comprises the nucleotide sequence 13 to 33 of the sequence coding for human LFA-3 (GENBANK access number X06296), flanked at (5 ') by a Kpnl site, and the primer called 3'LFA-3, which comprises the sequence of nucleotides 708 to 728 of the sequence coding for human LFA-3, flanked at (5 ') by a NotI site.
• 5'LFA-3 which comprises the nucleotide sequence 13 to 33 of the sequence coding for human LFA-3 (GENBANK access number X06296), flanked at (5 ') by a Kpnl site
• 3'LFA-3 which comprises the sequence of nucleotides 708 to 728 of the sequence coding for human LFA-3, flanked at (5 ') by a NotI
• a cDNA coding for the alpha chain of DR1 was obtained from a total cDNA library of human peripheral mononuclear cells from a donor DR1 +, by polymerase chain reaction (PCR) using the primers called DR1A5 'and DR1A3 ', which respectively comprise the sequence of nucleotides 151 to 176, and the sequence of nucleotides 769 to 792 of the sequence coding for the alpha chain of DR1 (accession number GENBANK K01171).
• a cDNA coding for the beta chain of DR1 was obtained in the same way using the primers called DR1B5 'and DR1B3' which comprise the sequence of nucleotides 52 to 74 and 850 to 923 of the sequence coding for the beta chain of DR1 respectively. (GENBANK access number NM002124).
• Each of the cDNAs obtained was cloned into a pcDNA 3.1 / hygro vector (INVITROGEN), at the TA site.
• the vectors obtained were used to co-transfect L cells by electroporation.
• the stable transfectants are sorted by flow cytofluorometry (FACSVantage SE, BECTON DICKINSON) by labeling the cells with an anti-LFA-3 antibody (1C3) and an anti-DR antibody (L243).
• FACSVantage SE BECTON DICKINSON
• LFA-3 antibody 1C3
• L243 anti-DR antibody
• Cells expressing both LFA-3 and DR are retained, and cultured on F12 medium (LIFE TECHNOLOGIES) supplemented with 10% fetal calf serum (BOEHRINGER), and added penicillin and streptomycin.
• Antigen presenting cells co-expressing LFA-3 and DR1 were also prepared, as described above, by co-transfection of P815 cells (ATCC-TIB64).
• non-transfected L or P815 cells or cells obtained by transfection of L or P815 cells, and expressing only the DR1 molecule were also used.
• CD4 + T cells isolated as described above, from PBMCs of a non-DR1 donor are suspended, at the rate of 2 ⁇ 10 6 cells / ml, in YSSEL medium (YSSEL et al., J. Immunol. Methods, 72, 219-227, 1984).
• the cell suspension is distributed in a 24-well culture plate, at a rate of 1 ml per well.
• L-DR1-LFA3 transfected cells or L-DR1 transfected cells, obtained as described above, or as a control, B-EBV DR1 + lymphoblastoid cells are irradiated (60 Gy) and added to each well, at the right rate of 5 x 10 5 cells / ml.
• CD4 + CD25 + cells are sorted by flow cytofluorometry, and are cloned at the rate of 1 cell / well in a 96-well plate, in YSSEL medium. Clonal expansion is carried out in the presence of IL-2 and IL-4 by the technique described by SPITS and YSSEL (J. Immunol. Methods, 9, 416-421, 1996). After clonal expansion, the different clones are re-stimulated by B-EBV DR1 + lymphoblastoid cells.
• the CD4 + T cells isolated from PBMCs of a DRl + donor are mixed, as described above, with cells presenting the antigen expressing the DRl molecule and the LFA- molecule 3.
• An inducing antigen (peptide HA 307-319, corresponding to a fragment of the capsid antigen of the Haemophilus influenzae virus) is added to the mixture of cells, at a rate of 50 ⁇ g / ml.
• CD4 + CD25 + are sorted by flow cytofluorometry, and are cloned as described above. After clonal expansion, the different clones are re-stimulated by B-EBV lymphoblastoid cells loaded with the HA peptide (10 ⁇ M). 48 hours after stimulation, the cell supernatant is recovered and the production profile of cytokines IL-2, IL-4, IL-10, and IFN- ⁇ in response to this stimulation was determined by assaying these cytokines by ELISA according to the protocol described by ABRAMS and collaborators.
• Isolated CD4 + CD25 + show the cytokine production profile of Tri cells.
• Clones of Tri cells having the same cytokine production profile were also obtained by using P815-DR1-LFA-3 cells as antigen presenting cells, which shows that obtaining Tri cells is not linked to the properties of the cell line from which the antigen presenting cells are obtained.
• the immunoregulatory properties of the Tri specific antigen cells were tested as follows:
• DR1 + (1 x 10 6 / ml) are stimulated by irradiated syngeneic monocytes (1 x 10 6 / ml (60 GY), in the presence of the peptide HA 307-319 (50 ⁇ M) and of tetanus toxoid
• the same experiment is carried out by adding cells (2 ⁇ 10 5 / ml) of a specific Tri HA clone obtained as described above, alone or in the presence of an antibody directed against the IL-10 receptor.
• anti-IL-lOR 10 ⁇ g / ml
• an anti-TGF- ⁇ antibody 20 ⁇ g / ml
• a mixture of these 2 antibodies is carried out by adding cells (2 ⁇ 10 5 / ml) of a specific Tri HA clone obtained as described above, alone or in the presence of an antibody directed against the IL-10 receptor.
• CD4 + T cell proliferation is determined after 5 days by measuring the incorporation of tritiated thymidine.
• TT + HA peptide CD4 + T cells stimulated by the HA 307-319 peptide and tetanus toxoid; + Trl HA specifies: CD4 + T cells stimulated by the peptide HA 307-319 and tetanus toxoid, in the presence of Tri cells specific for the HA antigen;
• CD4 + anti IL-10R CD4 + T cells stimulated by the peptide HA 307-319 and tetanus toxoid, in the presence of Tri cells specific for the HA antigen, and anti-IL-10R antibodies;

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