Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
1999-05-04
2004-11-23
Harris, Alana M. (Department: 1642)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C424S093210, C424S093100, C424S093700, C424S093710, C424S133100, C424S135100, C424S138100, C424S141100, C424S144100, C424S153100, C424S154100, C424S155100, C435S325000, C435S375000, C435S377000, C435S384000, C435S385000, C530S387300
Reexamination Certificate
active
06821516
ABSTRACT:
TECHNICAL FIELD
The invention relates to new macrophages, to a process for preparing the same and to their use as active substances of pharmaceutical compositions.
BACKGROUND ART
Cytotoxic leukocytes (NK, T lymphocytes, monocytes-macrophages) are potent effectors in the host defense against tumors. In vitro expansion and enhancement of the tumoricidal potential of autologous effector cells and their subsequent reinfusion into the host are currently being used in local or systemic adoptive immunotherapy (Rosenberg et al.: Progress report on the treatment of 157 patients with advanced cancer using LAK cells and IL2 or IL3 alone. N. Engl. J. Med. 316: 889, 1990; Rosenberg et al.: Use of tumor infiltrating lymphocytes and IL2 in the immunotherapy of patients with metastatic melanoma. N. Engl. J. Med. 319: 1676, 1990; Lacerna et al.: Adoptive cancer immunotherapy utilizing lymphokine activated killer cells and IFN-&ggr; activated killer lymphocytes. Pharm. Ther. 38: 453, 1988).
Macrophages play a central role in the antitumoral response and can be activated against neoplastic cells by immunopotentiators (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).
In murine models, it has been demonstrated that activated macrophages given locally in the tumor inhibited tumor growth and decreased metastatic development (Bartholeyns et al.: Immunotherapy of cancer: experimental approach with activated macrophages proliferating in culture. Cancer Detect. & Prev. 12: 413, 1988; Chokri et al.: Antitumoral effect of LPS, TNF, IFN and activated macrophages: synergism and tissue distribution. Anticancer Res. 9: 1185, 1989). Human monocytes isolated from blood could be differentiated in vitro into macrophages by a 7 day culture in hydrophobic bags (Chokri et al.: Antitumoral effects of LPS, TNF, IFN, and activated macrophages: synergism and tissue distribution. Anticancer Res. 9: 1185, 1989). After incubation in the presence of IFN-&ggr;, these macrophages became activated and tumoricidal, (Macrophages activated killer=MAK) for a number of human tumors in vitro or for tumors engrafted in nude mice (Andreesen et al.: Surface phenotype analysis of human monocytes to macrophages differentiation. J. Leuk. Biol. 47; 490, 1990; Dumont et al.: Control of the antitumoral activity of human macrophages produced in large amount in view of adoptive transfer. Eur. J. Cancer 24: 1691, 1988).
Adoptive transfer of MAK has undergone phase I clinical trials by different investigators in Strasbourg, Freiburg and Paris for patients with metastatic cancer infused systematically or intraperitoneally with 10
8
to 2×10
9
autologous MAK (Andreesen et al.: Adoptive transfer of tumor cytotoxic macrophages generated in vitro from circulating monocytes: a new approach to cancer immunotherapy. Cancer Res. 50: 7450, 1990; Bartholeyns et al. Adoptive immunotherapy of solid tumors with activated macrophages: experimental and clinical results. Anticancer Res. 11: 1201, 1991; Lopez et al.: One step separation by elutriation of monocytes from leukapheresis products of cancer patients for production of IFN-&ggr; activated macrophages in view of adoptive immunotherapy. J. Immunotherapy, 11: 209, 1992; Faradji et al.: Phase I trial of IV infusion of ex-vivo activated blood derived macrophages in patient with non small cell lung cancer: toxicity and immunomodulatory effects. Cancer Immunol. Immunother. 33: 319, 1991).
The clinical tolerance was excellent with minor side effects such as low grade fever and chills. The maximal tolerated dose could not be attained due to the limited number of MAK generated from one cytapheresis.
The recovery of larger quantities of MAK is therefore a need for optimal antitumoral efficacy in clinical trials and in therapy.
The macrophages prepared thus far in the conventional culture medium are obtained in a yield which is less than about 40%.
However, the need for macrophages presenting a higher cytotoxic activity is present in order to increase the efficacy in vivo and, subsequently, to delay the next macrophage administration.
DISCLOSURE OF THE INVENTION
One of the aims of the invention is to provide macrophages having a higher cytotoxic activity over standard macrophages.
Another aim of the invention is to provide macrophages in which the kinetics of the deactivation of the cytotoxic activity are slower with respect to standard macrophages.
Another aim of the invention is to provide a new culture medium which enables to prepare macrophages having an improved cytotoxic activity and slower deactivation kinetics of the cytotoxic activity with respect to standard macrophages.
The aims of the present invention are achieved by macrophages which have at least one of the following properties:
their cytotoxic activity without activation with IFN-&ggr; is increased by about 20 to 30% with respect to standard macrophages, and is preferably of about 70%;
their cytotoxic activity further to activation with IFN-&ggr; is increased by about 20 to about 40% with respect to standard macrophages, and is preferably of about 93%;
the extension of the deactivation time of the cytotoxic activity in reply to an activation of IFN-&ggr; is in a ratio such that after 60 h of activation with IFN-&ggr;, the cytotoxic activity is higher than or equal to 30%, preferably of about 55%, compared to the maximum cytotoxic activity presented by the macrophages further to an activation with IFN-&ggr;, with said cytotoxic activity being measured as the percentage of inhibition of 3-H thymidine incorporation by target tumoral cells, particularly U 937 cells.
The expression “standard macrophages” corresponds to the ones obtained by the culture of monocytes in a standard medium, such as defined in the Example section.
The “cytotoxic activity” corresponds to the one measured as the percentage of inhibition of 3-H thymidine incorporation by target tumoral cells, particularly U937 cells, and a test for this measure is hereafter given:
Differentiated macrophages were seeded into 96-well flat microtiter plates at 10
4
macrophages/well in 0.2 ml. After 2 h incubation at 37° C. in 5% CO
2
humidified atmosphere, the medium was removed and replaced by IMDM+5% hu AB serum containing or not 250 U/ml of rhu IFN&ggr;; plates were further incubated overnight. After washing, 10
4
U937 tumor cells in 0.2 ml fresh medium (IMDM+10% FCS) were added to each well. After 24 h contact, 0.1 &mgr;Ci of tritiated thymidine was added to each well for a further 24 h incubation.
The cells were collected and the determination of incorporated radioactivity on the fiber glass filters was carried out by &bgr; counting. The percentage inhibition of thymidine incorporation by tumor cells could be calculated according to the formula:
(
cpmT
-
cpmTM
)
cpmT
×
100
where
cpmT=radioactivity in control tumor cells.
cpmTM=radioactivity in tumor cells+macrophages mixed culture.
It is to be noted that such method enables to determine the three main types of antitumoral activity of the macrophages described in the invention, i.e.: cytolysis, cytostasis and phagocytosis.
The macrophages originate from human subjects, who can be either healthy or patients suffering from various diseases.
By way of example, standard macrophages originating from culture of monocytes from healthy subjects present a spontaneous cytotoxic activity (i.e. without activation with IFN-&ggr;) of about 45%, versus about 70%, in the case of the macrophages of the invention; and further to IFN-&ggr; activation, standard macrophages present a cytotoxic activity of about 76%, versus about 93%, in the case of the macrophages of the invention.
Similar enhancement of cytotoxic activity is obtained with macrophages issued from patients.
As to the kinetics of the deactivation time, table I hereafter gathers the results relative to the measu
Bartholeyns Jacques
Chokri Mohamed
Harris Alana M.
Holleran Anne L.
I.D.M. Immuno-Designed Molecules
Young & Thompson
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