Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-10-02
2002-06-04
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S375000
Reexamination Certificate
active
06399372
ABSTRACT:
The invention relates to stimulated monocyte derived cells, processes for their preparation, and pharmaceutical compositions containing the same.
It has long been established that macrophages have a primary role in wound and tissue repair (see for example Wong and Wahl, Inflammation and repair, in Handbook of Exp Pharmacol, 95:509-548, 1990). They are inducers and regulators of healing; they favor angiogenesis and recruit cells which will complete wound repair. After a tissue has been injured (burn, ulcers, wounds, trauma, mucosal damage, infarcts or even reconstructive surgery), macrophages called locally clean the wound by elimination of the necrotic debris formed by dead cells (by phagocytosis and proteolysis). At the same time macrophages, activated locally by this phagocytosis, actively release growth factors, monokines and chemokines. These autologous factors stimulate surrounding cells to multiply and to migrate towards the wound and to replace the dead cells.
Macrophages play a major role in the antitumoral response, and they are able to be activated by immunological activators against cancer cells (Adams D. and Hamilton T.: “Activation of macrophages for tumor cell kill: effector mechanism and regulation”; in Heppner & Fulton (eds), Macrophages and cancer. CRC Press, 1988, p. 27; Fidler M. Macrophages and metastases. A biological approach to cancer therapy. Cancer Res. 45: 4714, 1985).
Furthermore, 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 inducing a specific immune response. In this way, they represent good candidates for the preparation of vaccines, and more specifically cellular autologous vaccines.
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 no PCT/EP93101232, no 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);
“In vitro generation of CD83
+
human blood dendritic cells for active tumor immunotherapy” (Thurnher M., Papesh C., Ramoner R., Gastlt G. and al.; Experimental Hematology, 25:232-237, 1997);
“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 patent applications and articles are included herein for references.
They can be activated by IFN-&ggr; at the end of culture to obtain in particular cytotoxic macrophages. They can be entrifuged to be concentrated and purified before resuspension in isotonic solution.
Monocyte derived cells (MDCs) can either be killer macrophages, phagocytozing cells, growth factors and cytokines releasing cells, or dendritic cells according to their conditions of differentiation. Dendritic cells can for example be obtained as described in “In vitro generation of CD83+ human blood dendritic cells for active tumor immunotherapy” (Thurnher M., Papesh C., Ramoner R., Gastlt G. and al.; Experimental Hematology, 25:232-237, 1997) 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 97/092703.
Mature dendritic cells are very potent antigen presenting cells to initiate an immune response. The dendritic cells can be characterized by the induction of T cell proliferation and by their phenotype (presence of CD80, CD86, CD83, MHC-I, MHC-II on their membranes).
The dendritic cells play an important role, but are difficult to obtain in large quantities, necessary for therapeutic purpose in particular because of the required presence of multiple cytokines. Moreover, the dendritic cells obtained according to standard procedures are not stimulated and therefore do not present direct anti-tumoral or tissue repair properties.
One of the aim of the invention, is to provide stimulated monocyte derived cells having enhanced biological activities as described above, when compared to monocyte derived cells described until now.
Another aim of the invention is to provide processes for the preparation of said stimulated monocyte derived cells.
Another aim of the invention is to provide new pharmaceutical compositions containing said stimulated monocyte derived cells.
Another aim of the invention is to provide new methods for the treatment of tissue injuries.
Another aim of the invention is to provide new methods for the treatment or vaccination against tumors or infectious (bacterial or viral) diseases.
Another aim of the invention is to provide new methods for gene therapy.
The invention relates to stimulated monocyte derived cells presenting the following characteristics:
1)—increased release, with respect to normal monocyte derived cells, of at least one of the following polypeptides, proteins or compounds:
PDGF
(platelet derived growth factor)
IGF1
(insulin growth factor)
MDGF
(macrophage derived growth factor)
bFGF
(basic fibroblast growth factor)
GM-CSF
(granulocyte macrophage - colony stimulating factor)
heat shock or stress proteins,
chemokines and monokines such as IL12 and IFN&ggr;
enzymes or enzyme inhibitors,
complement components,
transfer proteins,
peroxides, NO (nitrous oxide),
bioactive lipids,
hormones,
and
increased presence, on their membranes, with respect to normal monocyte derived cells, of at least one of the following activation markers: CD1&agr;, CD11a, CD80, CD83, CD86, MHC class I and MHC class II molecules, adhesions, or accessory molecules for immunostimulation such as ICAM, or 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 correspond to monocytes cultured in defined media or in the presence of cytokines which have not been specifically stressed and therefore which do not release increased levels of immunostimnulatory proteins or compounds and simultaneously do not express markedly increased levels of MHC and accessory molecules on their membranes.
NO is usually not released due to NO synthase suppression, but, in the case of the invention, NO synthase is uninhibited which causes release of NO.
Monocytes derived cells can be obtained for instance from blood derived monocytes purified and cultured in the presence of GM-CSF and another cytokine, such as IL-4 or IL-13.
The invention relates more particularly to stimulated monocyte derived cells as described above, wherein the released polypeptides, proteins and compounds are those listed on Table 1.
According to an advantageous embodiment, tie activation markers are present in an amount of at least about 1000 molecules/cells.
This can be measured by flow cytometry.
In a particular embodiment of the invention, the monocyte derived cells as described above contain exogenous compounds in their cytoplasm such as drugs, protein and 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 nature of the physical stress, 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 physically
Bartholeyns Jacques
Chokri Mohamed
Latour Nathalie
Horlick Kenneth R.
I.D.M. Immuno-Designed
Strezecka Teresa
Young & Thompson
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