Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1996-04-24
2001-04-03
Priebe, Scott D. (Department: 1633)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S069100, C435S320100, C435S455000, C424S093210
Reexamination Certificate
active
06210963
ABSTRACT:
This application claims priority under 35 U.S.C. § 119 to French patent application number 93/10222, filed Aug. 25, 1993.
The present invention relates to cell compositions, to their preparation, to pharmaceutical compositions containing them and to their use in therapy. More especially, it relates to the isolation, culture and activation of cells of the mononuclear phagocytic system, and to their use in cell therapy, for example in adoptive immunotherapy.
The cells of the mononuclear phagocytic system comprise peripheral blood monocytes, their bone marrow or blood precursors and tissue macrophages. Monocytes are formed in the bone marrow, which they leave after maturation, passing from the peripheral blood to the tissues. Human monocytes circulating in the blood have a half-life of approximately 3 days. When it reaches the tissues, the monocyte is called a macrophage. The total number of tissue macrophages greatly exceeds the number of circulating monocytes, by a factor of approximately 400. Macrophages are found everywhere in the body, but are especially numerous in the liver (Kupffer cells), in the lymph nodes, in the lungs, in the peritoneum and in the skin (Langerhans' cells). The exact half-life of tissue macrophages is not known, but appears to be counted in months rather than in days. Lastly, the passage of monocytes from the general circulation to the tissues is irreversible.
Monocytes and macrophages are known to have numerous and important functions, including induction of immune responses in acute phases (Anonymous, Lancet ii (1985) 536-537), regulation of haematopoiesis (Sieff C. A. J. Clin. Invest. 79 (1987) 1549-1557), activation of the immune system (Unanue E. R. Annu. Rev. Immunol. 2 (1984) 395-428) and of coagulation (Prydz H., Allison A. C. Thromb. Haemost. 39 (1978) 582-591), destruction of organisms and of tumour cells (Sharma S. D., Remington J. S. Lymphokines 3 (1981) 181-212 and Carswell E. A. et al. Proc. Natl. Acad. Sci. USA 72 (1975) 3666-3670) and tissue repair and cicatrization (Korn J. H. et al., J. Clin. Invest. 65 (1980) 543-54).
Throughout the following text, a monocyte-macrophage line is defined as a line of the mononuclear system comprising peripheral blood monocytes, their bone marrow or blood precursors and tissue macrophages. It also comprises the monocytes obtained by culturing precursor cells as well as the macrophages obtained after culturing monocytic cells, under the conditions detailed in the examples (see also Andressen R. et al., Cancer Res. 50:7450; Bartholeyns J et al., Anticancer Res. 11 (1991) 1201-1204; Lopez M. et al., Journal of Immunological Methods 159 (1993) 29-38). The precursors of monocytes-macrophages comprise, in particular, pluripotent stem cells, myeloid stem cells (CFU-GEMM), myelomonocytic stem cells (CFU-GM), CFU-M, monoblasts and promonocytes.
At the present time, monocytes-macrophages are used in adoptive immunotherapy for the treatment of some types of cancer in man. These cells are purified from the circulating blood of patients, cultured ex vivo and activated with interferon &ggr; to induce their differentiation and increase their tumoricidal power, then reinjected into the patients. However, this treatment is rather arduous for the patient, since the monocytes-macrophages have to be withdrawn regularly and frequently, the ex vivo activation requires the expenditure of considerable periods of time and interferon &ggr; is still very expensive. For this reason, it is important to have treatments at one's disposal which are more effective, less demanding for the patient and less expensive. The present invention provides an advantageous approach to this problem. The Applicant has, in effect, shown that it is possible, using suitable vectors, to transfer genes ex vivo into monocyte-macrophage cells, thereby enabling them to be endowed with superior properties both of cytotoxicity and of stimulation of the immune system.
A first subject of the invention hence lies in a cell composition comprising cells of the monocyte-macrophage line containing a recombinant nucleic acid comprising one or more therapeutic genes under the control of regulatory elements permitting its expression.
The present invention thus makes it possible to obtain simply and effectively healthy and active monocytes-macrophages which are usable in adoptive immunotherapy for the treatment of certain pathologies, such as cancers. The present invention also makes it possible to endow monocytes-macrophages with novel or enhanced therapeutic properties compared to those of the body's monocytes-macrophages, in particular in the field of defence against infectious agents and against tumour cells or in activation of the immune system. Such cells are advantageously usable for the curative or preventive treatment of infectious (in particular viral) diseases, autoimmune diseases and immune deficiencies, or alternatively for the purpose of vaccination.
For the purposes of the invention, the term therapeutic gene denotes any gene whose transcription and, where appropriate, translation in the cell generates a product having a therapeutic effect. Such genes can comprise, in particular, those for all or part of a therapeutic protein (interleukin, interferon, tumour necrosis factor, colony stimulating factors, and the like), or for an antigenic peptide for production of a vaccine or stimulation of the immune system, or can alternatively comprise an antisense RNA capable of regulating the expression of a specific protein such as, for example, a protein of viral origin, or alternatively of interfering with the infection and/or replication cycle of a virus.
Advantageously, the gene codes for all or part of a protein capable of endowing the cells of the said line with novel or enhanced anti-infectious, anticancer or immunostimulatory properties.
More preferably, the gene is chosen from the genes coding for interferons (preferably gamma), tumour necrosis factors (preferably alpha), interleukins (IL-1 to -12) and colony stimulating factors (G-CSF, M-CSF, GM-CSF, and the like), the MDR (multi-drug resistance) gene and a gene coding for an antigen of an infectious particle or specific to a tumour (for example a surface protein of a virus, namely gp160 protein of the HIV virus in particular, or Muc-1 antigen characteristic of epithelial tumours).
In a particular mode, the subject of the present invention is cell compositions as defined above, in which the recombinant nucleic acid is carried by a vector, preferably a viral vector. The use of a vector according to the invention makes it possible, in particular, to improve the administration of the nucleic acid in the cells, and also to increase its stability in the said cells, thereby enabling a lasting effect to be obtained. Furthermore, it is possible to introduce several genes into the same vector, thereby also increasing the efficacy of the treatment.
The vector used is preferably of viral origin and, in particular, it may be chosen from adenoviruses, adeno-associated viruses (AAV), herpesvirus, vaccinia virus, cytomegalovirus (CMV), and the like.
Advantageously, the virus used is a defective virus. The term “defective virus” denotes a virus incapable of replicating in the target cell. Generally, the genome of the defective viruses used in the context of the present invention hence lacks at least the sequences needed for replication of the said virus in the infected cell. These regions may be either removed (wholly or partially), or rendered non-functional, or replaced by other sequences, and in particular by the recombinant nucleic acid. Preferably, the defective virus nevertheless retains the sequences of its genome which are needed for encapsidation of the viral particle.
An especially advantageous vector for the preparation of the cell compositions according to the invention is an adenoviral vector. The Applicant showed, in effect, that adenoviruses were capable of very effectively infecting cells of the monocyte-macrophage line, of being maintained stably therein and of expressing a therapeutic gene
Haddada Hedi
Lopez Manuel
Perricaudet Michel
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut National de la Sante et de la Recherche Medicale
Nguyen Dave Trong
Priebe Scott D.
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