Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2000-12-28
2004-03-09
Falk, Anne-Marie (Department: 1636)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S325000, C435S366000, C435S372000
Reexamination Certificate
active
06703016
ABSTRACT:
The present invention relates to new apoptotic bodies, monocyte derived cells containing the same, a process for their preparation and their uses as vaccines.
In advanced cancer, tumor cells are notoriously poor immunogens in vivo. In vitro, the immune system can recognize and kill tumor cells. Tumor cells have antigens for T cells, as reported for melanoma
1
, but lack immunogenicity in vivo. Probably, tumor antigens are either masked, not presented by specialized cells or suppressed by the tumor milieu. The use of vaccines consisting of peptides
2
or single transfected
3
, antigen presented by professional antigen presenting cells (APC)
4
, caused limited benefits in antitumoral therapies
5,6
because of the narrowness of specificity and immune escape. An alternative strategy is to use unfractionated or fractionated tumor cells
7,8,9
limited to immunogenic tumors.
Programmed Cell Death (PCD) is the most common form of eukaryotic cell death. The term of apoptosis, which is often equated with PCD, refers to morphological alterations exhibited by dying cells. In contrast to necrosis, apoptosis is an active process. Apoptosis occurs under normal and pathological physiological conditions, the cell being an active participant of its own “cellular suicide”, and cells undergoing apoptosis show characteristic morphological and biochemical features.
Apoptotic bodies express surface markers
14
that cause them to be specifically and rapidly phagocytosed by macrophages. Thus, the important consequence of this process is the disappearance of the apoptotic bodies without release of the cytoplasmic contents into the intercellular space. This avoids both inflammation and autoimmunization with intracellular cell constituents
15
.
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, AIMV (Gibco) or X-VIVO (Biowhittaker)) contains eventually cytokines or ligands as defined in patents no PCT/EP93/01232, 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.; Immnunobiol., 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 INF-&ggr; at the end of culture to obtain in particular cytotoxic macrophages. They can be centrifuged to be concentrated and purified before resuspension in isotonic solution.
Monocytes derived cells (MDCs) can either be kifler 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 no 97/02703.
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).
One of the aims of the invention is to provide new apoptotic bodies which can be used as anti-tumor vaccines.
Another aim of the invention is to provide new monocyte derived cells, which can be used as anti-tumor vaccines after integration of apoptotic bodies.
One of the aims of the invention is to provide potential anti-tumor vaccines, with improved immunogenicity.
Another aim of the invention is to provide potential and-tumor vaccines which are specific for a given patient and thus more efficient.
The invention relates to apoptotic bodies isolated from human tumor cells recovered from a patient's tumor biopsy and induced to apoptosis, said apoptotic bodies having the following characteristics:
they maintain plasma membrane integrity,
they are vesicles above about 0,1 &mgr;m, particularly above about 0,5 &mgr;m,
they contain intact mitochondria and cleaved nuclear DNA originating from the tumor cells,
they present unmasked tumor antigens,
they present specific tumor and MHC antigens from the patient.
The invention relates to apoptotic bodies isolated from human tumor cells recovered from a patient's tumor biopsy and induced to apoptosis, said apoptotic bodies having the following characteristics:
they maintain plasma membrane integrity,
they are vesicles of about 0,5 to about 5 &mgr;m,
they contain intact mitochondria and cleaved nuclear DNA originating from the tumor cells,
they present unmasked tumor antigens on their membranes,
they present specific tumor and MHC antigens from the patient.
Apoptotic tumor cells provide effective antigens recognised by the immune system. In the present invention, it is shown that apoptotic cells are more immunogenic than necrotic tumor extracts. Apoptotic bodies derived cells are phagocytosed by monocyte derived antigen presenting cells and potentiate the effective tumor antigenic presentation to the immune system (FIG.
1
).
The processing and presentation of antigenic molecules unmasked in apoptotic bodies derived from cancer cells offer new opportunities in anti-cancer immunotherapy to enhance the specific cellular and humoral immune responses against tumors.
The expression “apoptotic bodies” designates cell fragmentation bodies containing in an intact membrane, mitochondria and fragmented nuclear DNA originating from tumor induced to apoptosis.
Apoptotic cells can be distinguished from necrotic cells (other dead cells) according to characteristic morphological and biochemical features. Apoptosis is characterized by shrinkage of the cell, dramatic reorganization of the cell nucleus, cell membrane and cell metabolism, active membrane blebbing, and ultimate fragmentation of the cell into membrane-enclosed vesicles (apoptotic bodies)
13
. The nuclear events of apoptosis begin with collapse of the chromatin against the nuclear periphery and into one or a few large clumps within the nucleus. Nuclear features include chromatin aggregation followed by DNA fragmentation (a specific marker of apoptotic process) after activation of endonucleases resulting in multiples subunits of DNA of an approximately 180 bp. The cellular events include cytoplasmic condensation and partition of the cytoplasm
Bartholeyns Jacques
Gregoire Marc
Falk Anne-Marie
INSERM Institut National de la Sante et de la Recherche Medicale
Qian Celine
Young & Thompson
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