Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2000-10-02
2003-07-22
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C435S372000
Reexamination Certificate
active
06596275
ABSTRACT:
The invention relates to suppressive monocyte derived cells, a process for their preparation, and their uses in pharmaceutical compositions.
It is known, that macrophages, or other cells derived from monocytes or from their precursors, with their strong capacity for endocytosis, digestion, and surface antigen presentation, are capable of controlling the immune response.
Monocytes derived cells (MDCs) are immune cells such as obtained by culture of blood mononuclear cells in non adherent gas permeable plastic or Teflon bags for 5 to 10 days at 37° C. in O
2
/CO
2
atmosphere. Their culture medium (RPMI, IMDM, AIM5 (Gibco) or X-VIVO (Biowhittaker)) contains eventually cytokines or ligands as defined in patents PCT/EP93/01232, WO94/26875 or EP 97/02703 or in the articles mentioned below:
“Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages”. (Lopez M., Martinache Ch., Canepa S., Chokri M., Scotto F., Bartholeyns J.; J. of Immunological Methods, 159:29-38, 1993);
“Immune therapy with macrophages: Present status and critical requirements for implementation” (Bartholeyns J., Romet-Lemonne J-L., Chokri M., Lopez M.; Immunobiol., 195:550-562, 1996);
“Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion” Finkelman, Lees, Birnbaum et al., J. Immunology, 157:1406-1414, 1996;
“Dendritic cells as adjuvants for immune-mediated resistance to tumors” (Schuler G. and Steinman R. M.; J. Exp. Med., 186:1183-1187, 1997). All these patents applications and articles are included herein for references.
They can be centrifuged to be concentrated and purified before resuspension in isotonic solution.
Monocytes derived cells (MDCs) can either be macrophages, phagocytozing cells, growth factors and cytokine releasing cells, or dendritic cells according to their conditions of differentiation. Dendritic cells can for example be obtained as described in “Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion” Finkelman, Lees, Birnbaum et al., J. Immunology, 157:1406-1414, 1966 and “Dendritic cells as adjuvants for immune-mediated resistance to tumors” (Schuler G. and Steinman R. M.; J. Exp. Med. , 186:1183-1187, 1997), and EP 97/02703.
In physiology, monocyte derived cells are called initially to induce an immune response.
In a normal situation, this immune response has to be stopped in order to avoid a pathological enhanced response, and this control is mediated, in the body, by monocyte derived cells which have not yet been completely identified and are not yet mastered in ex vivo conditions.
One of the aims of the invention is to provide suppressed monocyte derived cells which present the properties of controlling the immune response, when compared to normal monocyte derived cells described until now.
Another aim of the invention is provide a process for the preparation of said suppressive monocyte derived cells.
Another aim of the invention is to provide new pharmaceutical compositions containing said suppressive monocyte derived cells.
Another aim of the invention is to provide new methods for inducing inmmunotolerance.
Another aim of the invention is to provide new methods for treating autoimmune diseases.
Another aim of the invention is to provide new methods for the treatment of chronic inflammations.
Another aim of the invention is to provide new methods for the treatment of allogenic graft rejection.
Another aim of the invention is to provide new methods for gene therapy.
The invention relates to suppressive monocyte derived cells presenting the following characteristics:
1) increased release, with respect to normal monocyte derived cells, of at least one of the following compounds:
PGE-2
IL-10
IL-4
and decreased level of expression and secretion of inflammatory and immunostimulating cytokines such as IL-1, IL-12, IFN&ggr;, with respect to normal monocytes derived cells,
and decreased presence, on their membrane, with respect to normal monocyte derived cells, of at least one of the following activation or accessory molecules such as CD80, CD86, and CD40,
and/or
2) presence in their nucleus of at least one exogenous nucleic acid which has been integrated in the absence of the monocyte derived cell division.
The expression “normal monocyte derived cells” corresponds to monocytes cultured in defined media or in the presence of cytokines which present MHCI and MHCII molecules at their surfaces, release cytokines and growth factors, induce proliferation of lymphocytes in mixed lymphocyte reaction assays.
Normal monocyte derived cells can be obtained for instance from blood derived monocytes purified and cultured in the presence of GM-CSF and other cytokines.
Monocyte derived cells properly stimulated can trigger the immune system leading to T-cell activation and production of antibodies. In contrast, monocyte derived cells which do not present costimulatory signals on their membranes and release suppressive cytokines (e.g. TGF-&bgr;) or cytokines inducing a TH2 response (e.g. IL-4, IL-10) or suppressive factors (e.g. PGE2) do inhibit the immune system. If such monocyte derived cells have interiorized and processed antigens of interest, they can specifically induce peripheral tolerance, with durable antigen specific unresponsiveness in the absence of generalized inununosuppression.
According to an advantageous embodiment of the invention, the increased release, with respect to normal monocyte derived cells, of at least one of the following compounds:
prostaglandins such as PGE-2
arachidonic acid metabolites
TGF-&bgr;
IL-10
IL-4
is in an amount higher than 0.1 pg/cell/hr.
This can be measured by ELISA method.
According to an advantageous embodiment of the invention, decreased level, with respect to normal monocyte derived cells, of expression and of secretion of inflammatory and immunostimulating cytokines such as IL-1, IL-12, IFN&ggr;, is below 0.01 pg/cell/hr.
This can be measured by ELISA methods.
According to another embodiment of the invention, the decreased presence, with respect to normal monocyte derived cells, on their membrane of at least one of the following activation or accessory molecules such as CD80, CD86, CD1a and adhesins such as CD40 or ICAM, MHCI and MHCII molecules is in an amount of less than about 10
3
molecules/cell, as measured by flow cytometry.
According to another embodiment of the invention, the decreased phagocytosis capacity, with respect to normal monocyte derived cells is in the average of less than 5 particles of yeast phagocytosed in one hour.
In a particular embodiment of the invention, the monocyte derived cells as described above, contain exogenous compounds in their cytoplasm such as drugs, protein, growth factors of interest.
In another embodiment, the monocyte derived cells as described above contain in their cytoplasm exogenous DNA coding for a protein of interest.
It should be made clear that depending upon the conditions in which the monocyte derived cells are preferred and more particularly depending upon the nature of the physical stress to which the monocyte derived cells of the invention are submitted, as explained hereafter, either the DNA contained in the cytoplasm of said monocyte derived cells remain in the cytoplasm after the physical stress, or there is an uptake of said exogenous DNA by their nucleus which is made possible by the physical stress.
The invention also relates to suppressive monocyte derived cells, which present the following characteristics:
increased release, with respect to normal monocyte derived cells, of at least one of the following compounds:
prostaglandins such as PGE-2
arachidonic acid metabolites
TGE-&bgr;
V-EGF
IL-10
IL-4
and decreased level, with respect to normal monocyte derived cells, of expression and secretion of inflammatory and immunostimulating cytokines such as IL-1, IL-12, IFN&ggr;,
and decreased presence, on their membrane, with respect to normal monocyte derived cells of at least of the following activation or accessory molecules such as CD80, CD86, CD1a and
Bartholeyns Jacques
Chokri Mohamed
Romet-Lemonne Jean-Loup
Chan Christina
Huynh Phuong N.
I.D.M. Immuno-Designed Molecules
Young & Thompson
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