Process for preparing macrophages, kits, and compositions...

Details Process for preparing macrophages, kits, and compositions... Process for preparing macrophages, kits, and compositions...

1999-09-23

2001-04-24

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Maintaining blood or sperm in a physiologically active state...

C435S325000, C435S326000, C435S372000, C435S374000

Reexamination Certificate

active

06221576

ABSTRACT:

The present invention relates to a process for preparing macrophages, specifically human macrophages, in particular activated macrophages (also designated as cytotoxic macrophages) or macrophages (or other cells derived from monocytes or from their precursors) for the presentation of antigens, as well as kits and compositions which may be used for this process.
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 I.: 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. Macrophages, or other cells derived from monocytes, presenting antigens on their surface specifically capable of inducing a specific immune response, are designated in the text which follows as MD-APCs (Monocyte-Derived Antigen Presenting Cells).
The MD-APCs are obtained by culture of mononucleated cells (monocytes or their precursors), taken from a patient or healthy individual, in the presence of one (or several) antigen(s) which are desired to be expressed as fragments on the surface of the cells obtained at the end of the culture period.
The antigens which may be cultured with the above mentioned mononucleated cells are, for example, antigens expressed by tumor cells (in particular fragments of killed tumor cells) or by pathogens (in particular bacterial or viral protein fragments).
The MD-APCs thus obtained are used for the preparation of vaccines directed against the pathology associated with the antigen co-cultivated with the mononucleated cells (in particular vaccines against tumors or viral diseases) and more particularly for the preparation of autologous cellular vaccines which are advantageously administered to the patient from whom the starting mononucleated cells were taken.
MD-APCs are also able to be obtained by transfection of the above mentioned cells placed in culture, or of differentiated cells, in particular macrophages, obtained after culture, with DNA sequences coding for antigen fragments such as those defined above.
Macrophages presenting cytotoxic activity which is particularly significant (also designated as activated macrophages or MAK (registered trade mark)) as well as a culture medium and process for obtaining said macrophages were the subject matter of international patent application no. PCT/EP 93/01232, filed on May 18, 1993.
The process for preparing macrophages defined in the above mentioned international patent application is carried out with a composition rich in blood cells obtained by apheresis from a healthy individual, and includes a stage of monocyte culture in a culture medium containing vitamin D3 1.25-dihydroxyl and GM-CSF (Granulocyte-Macrophage colony stimulating factor). Advantageously, the monocytes are cultivated in the presence of lymphocytes for about 6 to 7 days, and the differentiated macrophages thus obtained are activated in the culture medium by the addition of interferon &lgr; (IFN-&lgr;).
This culture stage is preceded, as in all procedures for obtaining macrophages described up to today, by a stage of separation of, in one part, mononuclear cells, and, in another part, red cells, granulocytes, and part of the platelets contained in the composition derived from blood obtained by apheresis; and by a stage of elimination, by washing part of the blood platelets and anticoagulants remaining after the preceding separation stage.
The above mentioned stage of separation of red cells and granulocytes is generally carried out by centrifugation of the medium containing the monocytes on a density gradient, particularly in a solution having a specific weight of about 1.0 to about 1.3 g/ml, such as Ficoll Paque solution (Pharmacia) with a specific weight of 1.077 g/ml.
In the above mentioned international patent application, the composition derived from the blood is obtained by apheresis containing approximately 7 to 9×10
9
leukocytes in a volume of approximately 200 ml. After the stage of separation of the red cells and granulocytes by centrifugation on a density gradient, the composition of cellular suspension placed in culture thus contains, in a volume of approximately 500 ml to 1000 ml:
Hematocrit (Red cells)
<0.1% of the number of white cells
present in the composition
Platelets
<10
10
White cells
3 to 5 × 10
9
Lymphocytes
50-70% of the number of white cells
present in the composition
Monocytes
30-50% of the number of white cells
present in the composition
Polynuclear cells (or granulocytes)
<5% of the number of white cells
present in the composition
The elimination of red cells and granulocytes, which is done beforehand in the culture stage, has always been considered as an indispensable stage, due particularly to the fact that the granulocytes are liable to totally or partially inhibit the differentiation of monocytes into macrophages during the culture stage.
This inhibition would principally be due to the fact that red cells, granulocytes and platelets use the constituents of the culture medium for their own metabolism, thus exhausting the reserves necessary for the development of the monocytes, and leading to acidification of the culture medium in proportions such that the culture stage cannot be completed (usually requiring about 6 to 7 days).
Furthermore, the granulocytes and platelets would be particularly liable to secrete suppresser factors, such as TGF (transforming growth factor), and prostaglandins, against the functionality of macrophages.
Therefore, it is currently acknowledged that the granulocytes and as many of the platelets as possible should be separated from the monocytes before being placed in culture with the latter, even though this separation leads to the elimination of about 20% to about 50%, in particular about 30%, of the mononucleated cells present in the medium before separation.
The present invention aims at providing a new process for obtaining macrophages or MD-APCs, optionally activated, which is the most simple procedure described up to today and presents the best results of all the processes for obtaining macrophages described up to today.
The invention also aims at providing kits (or cases) for carrying out this process.
The invention also aims at providing for the user of said kits, compositions which can be used as internal controls in order to control the different stages of the process, particularly to control the contents of monocytes, and optionally of other constituents of the starting composition of the process of the invention, and to control the contents of macrophages or MD-APCs, optionally activated, in the final composition being sought.


REFERENCES:
patent: 4434237 (1984-02-01), Dinarello
patent: 4912045 (1990-03-01), Kung et al.
patent: 5286482 (1994-02-01), Jones
patent: 5374549 (1994-12-01), Leung
patent: 5662899 (1997-09-01), Chokri et al.
patent: 5804442 (1998-09-01), Romet-Lemonne et al.
patent: 01232 (1994-11-01), None
patent: WO 94/26875 (1994-11-01), None
E. Helsinki et al., “Tumor-Cytolytic Human Macrophages Cultured As Nonadherent Cells: Potential for the Adoptive Immunotherapy of Cancer”,Cancer Detection and Prevention, 1990, pp. 471-481.
R. Andreesen et al., “Primary Cultures of Human Blood-Born Macrophages Grown on Hydrophobic Teflon Membranes”,Journal of Immunological Methods, vol. 56, 1983, pp. 295-304.
R. Andreesen et al., “Activation of Human Monocyte-Derived Macrophages Cultured on Teflon: Response to Interferon-y during Terminal Maturation in vitro”, Immunobiol., vol. 177, 1988, pp. 186-198.
Chok

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