Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing
Reexamination Certificate
2001-11-27
2004-05-18
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
C424S093700, C435S325000, C435S366000, C435S373000, C435S372000, C435S391000, C435S405000
Reexamination Certificate
active
06737051
ABSTRACT:
The invention relates to a cell composition containing macrophages, presenting anti-infectious and hematopoietic properties, and a process for preparing the same.
Peripheral hematopoietic apheresis instead of bone marrow puncture and purification are widely used to collect hematopoietic progenitor cells. These cells are used in allogeneic or autologous transplantations for the treatment of genetic diseases and mainly of neoplasic diseases to support high dose chemotherapy or radiotherapy.
Positive selection of the apheresis products for cells bearing the CD34 antigen (CD34
+
stem cells) and cryopreservation until use has become a recognized method.
However, it offers several drawbacks including cost (the process of selection of cells on antibody coated beads), limited amounts of progenitor cells recovered, and delays in immune reconstitution (resulting in infectious complications in the post transplant period).
The number of progenitor cells present in blood can be increased by the preapheresis conditioning of the patient treated with GM-CSF and/or G-CSF colony stimulating factors and eventually with chemotherapy drug such as a cyclophosphamide.
However, cancer patients often relapse due to the presence of residual tumor cells resistant to the chemotherapy regimen (Bhatia M et al.: Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med
. 186: 619-624, 1997, Bonnet D et al.: Cytokine treatment or accessory cells are required to initiate engrafment of purified primitive human hematopoietic cells transplanted at limiting doses into NOD/SCID mice,
Bone Marrow Transplant
. 23: 203-209, 1999, Breems D A et al.: Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells.
Blood
. 91: 111-117, 1998, Civin et al.: Highhly purified CD34-positive cells reconstitute hematopoiesis.
J Clin Oncol
. 14: 2224-2233, 1996, Morrison S J et al.: The biology of hematopoietic stem cells.
Annu Rec Cell Dev Biol
. 11: 35-71, 1995, Traycoff C M et al.: Proliferation-induced decline of primitive hematopoietic progenitor cell activity is coupled with an increased in apoptosis of ex vivo expanded CD34+ cells.
Exp Hematol
. 26: 53-62, 1998).
An aim of the invention is to provide a new cell composition containing macrophages, presenting interesting properties in cancer immunotherapy and in stem cell transplantation.
Another aim of the present invention is to provide a new cell processing method allowing under improved standardised procedures the expansion of progenitor and stem cells from peripheral blood without costly purification of a defined cell population.
The aims of the invention are achieved by a cell composition containing macrophages, myeloid cells and progenitor cells, with said progenitor cells being preferably present in a mean ratio of at least about 1%, preferably about 0.1 to 20%, with said myeloid cells being preferably present in an amount of about 10% to about 30%, with said macrophages being preferably in an amount of about 10 to about 70%, these percentages being expressed with respect to the total number of cells.
Macrophages, myeloid cells and progenitor cells are defined as CD14
+
and CD64
+
cells (macrophages), CD33
+
cells (myeloid cells) and CD34
+
cells and/or GM-CFU (progenitor cells). GM-CFU are cells able to form colonies of granulocyte and macrophage in cytokine containing semi-solid culture medium after 14 days of culture.
GEMM-CFU are myeloid stem cells, and are able to give BFU-E, CFU-GM, CFU-M, CFU-EO, and CFU-B. BFU-E are progenitor cells able to differentiate into erythrocytes.
According to an advantageous embodiment, the cell composition of the invention contains T lymphocytes, preferably in a ratio of about 10 to 60%, expressed with respect to the total number of cells.
According to an advantageous embodiment of the invention, the progenitor cells contain from about 0.1 to about 20% of stem cells, expressed with respect to the total number of progenitor cells.
Stem cells are defined as expressing CD34 molecules and/or by their ability to form colonies in cytokine containing semi-solid culture medium.
According to an advantageous embodiment, the progenitor cells are generated from and possibly included in peripheral blood mononuclear cells, and in particular are chosen among:
myelo-erythroid progenitor cells, myeloid progenitor cells, lymphoid progenitor cells or a mixture thereof.
The expression “included in” means that the progenitor cells are present in the cell composition.
The expression “generated from” means that the progenitor cells are differentiated from stem cells originally present in the cell composition.
In the cell composition of the invention, the macrophages, myeloid cells and the lymphocytes if present, are included in/or generated from blood mononuclear cells.
The cell composition of the invention has gained a new combination of activities useful in cancer immunotherapy and in stem cell transplantation. These properties include:
1) purge by macrophages and cytotoxic T/NK cells of the tumor cells eventually present in the graft,
2) eradication of residual cancer disease in the patient by macrophages and/or antigen presenting cells (MAC-DCs dendritic cells) present in the autologous or in the allogeneic graft,
3) avoiding most infectious episodes after injection at the beginning of the aplasia period following therapy in the patient, thanks to the potent anti-viral, anti-bacterial and anti-parasite properties of macrophages and eventually of contaminating polynuclear cells and their precursors present in the product,
4) facilitating engraftment by the enhanced amount of stem cells, of hematopoietic cells, progenitors of myeloid cells, erythroid and lymphoid as well as of cells at intermediate states of differentiation present in the graft,
5) decrease significantly of the aplasia period (correlated with patient's fever and infections) by markedly increasing the recovery rate of the different blood populations.
The invention also relates to a process for the preparation of a cell composition containing macrophages, myeloid cells and progenitor cells, with said progenitor cells being preferably present in an amount of about 0.1% to about 10%, with said macrophages being preferably in an amount of about 10 to about 60%, these percentages being expressed with respect to the total number of cells, comprising the step of mobilization of the progenitor cells in the blood of a patient, for instance by premeditation of said patient with G-CSF and/or GM-CSF, or G-CSF and cyclophophosphamide, thus increasing the amount of progenitor cells in peripheral blood.
The term “mobilization” means stimulation of bone marrow cells to release increased amount of progenitor cells in the blood.
The process of the invention can comprise an additional step of coculture of the blood mononuclear cells and progenitors, after washing off the platelets, the granulocytes and erythrocytes, for about 4 to about 10 days, in a medium allowing differentiation of monocytes into macrophages and myeloid progenitors into polynuclear cells.
According to an advantageous embodiment of the process, the coculture of the blood mononuclear cells and progenitors is carried out in the presence of cytokines or growth factors, for example: IL3, IL6, stem cell factor, EPO, thrombopoietin, GM-CSF, G-CSF, Flat-3 ligand, c-kit ligand or their agonists.
The process of the invention can also comprise an additional step of macrophage activation, at the end of the coculture, for instance by addition of &ggr;-interferon or muramyl peptides.
The aim of activation macrophages is to gain more anti-infectious and anti-tumoral activity.
The process of the invention can comprise an additional step of concentration of the cells obtained at the end of the coculture, and resuspension in a vehicle suitable for administration to a patient.
The process of the invention can comprise after the resuspension of the coculture, a ste
Bartholeyns Jacques
Klein Bernard
Lu Zhao Yang
Belyavskyi Michail A
Chan Christina
I.D.M. Immuno-Designed Molecules
Young & Thompson
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