Tumoricidal T lymphocytes

Details Tumoricidal T lymphocytes Tumoricidal T lymphocytes

1995-12-04

2003-06-10

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Primate cell, per se

C435S372000, C435S372200, C435S375000, C435S377000, C435S347000, C424S053000, C424S520000

Reexamination Certificate

active

06576466

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention concerns a mammalian cell line and its active fragments which when it is co-cultured with lymphocytes during which allogenic stimulation is avoided, activate lymphocytes to form tumoricidal T cells a process for the production of tumoricidal T lymphocytes by co-culturing lymphocytes with such cell lines or with active fragments thereof, the tumoricidal T lymphocytes obtainable by this process as well as the use of these T lymphocytes for the production of a therapeutic agent which can be used in tumour therapy.
The cellular immune defence plays an important role in the elimination of pathologically changed endogenic cells such as e.g. cells infected by viruses or tumour cells. In this process cytotoxically active T lymphocytes recognize the changed endogenic cells on the basis of surface antigens. These surface antigens are usually protein fragments which are formed by the cells and are present on the cell surface bound to surface receptors of the so-called major-histocompatibility complex (MHC) (Zinkernagel et al., Nature 248 (1974), 701-702 and Babbit et al., Nature 317 (1985), 359-361). However, if these surface antigens of the tumour cells only differ slightly from the corresponding antigens of healthy cells, the immune system may possibly form no cytotoxically active T lymphocytes which could eliminate the tumour cells.
Therefore attempts have already been made to induce a cellular immune resistance against such tumour cells. For this it was firstly attempted to achieve an active immunization with unspecific immunostimulants such as Bacillus Calmette-Guerin (BCG), Corynebacterium parvum or vaccines from tumour cell extracts (Terry and Rosenberg eds., Immunotherapy of Human Cancer (1982), Elsevier North Holland). Better results were obtained using the concept of so-called adoptive immunotherapy. In this case lymphocytes of the patient are activated in vitro and then re-implanted. The in vitro activation to form such “promiscuous killer cells” (D. Thiele et al., Immunology Today 10 (1989), 375-381) is usually carried out by addition of interleukin 2. The cytotoxic lymphocytes obtained are then denoted lymphokine-activated killer cells (LAK cells) (Rosenberg, Immunology Today 9 (1988), 58-62). In contrast to cytotoxic T lymphocytes, the action of LAK cells against tumour cells does not depend on a correct expression of the MHC genes for the recognition of tumour antigens and in contrast to the natural killer cells of the immune system LAK cells are also effective against fresh tumour cells. It has even already been possible to achieve the first clinical successes using LAK cells. However, a disadvantage of this form of adoptive immunotherapy are side-effects of interleukin 2 which is required in relatively high doses over a longer time period. This results primarily in an increase in the permeability of the capillaries and concomitant functional disorders of the organs (Rosenberg, Immunology Today 9 (1988), 58-62, Rosenstein et al., Journal Immunology 137 (1986), 1735-1742 and Ettinghausen et al., Surg. Forum 37 (1987), 388-389). In addition such LAK cells are obtained which when stimulated with interleukin 2, are directed against healthy endogenous cells (B. Chen et al., Cell. Immunol. 118 (1989), 458-469).
In the search for more effective methods for adoptive immunotherapy the lymphocytes to be activated were also cultured in the presence of autologous tumour cells (mixed lymphocyte tumor cultures, G. Fossati et al., International Journal of Cancer 42 (1988), 239-245; G. Degiovanni et al., Eur. J. Immunol. 18 (1988), 671-676; Wölfel et al., J. Exp. Med. 170 (1989), 797-819; Darrow et al., J. Immunol. 142 (1989), 3329-3335 and Notter et al., Int. J. Cancer 45 (1990), 834-841). In addition a method for the proliferation of tumour-infiltrating lymphocytes (TIL) in vitro has also been described (Yron et al., J. Immunol. 125 (1980), 238-245) in contrast to LAK cells, these tumour-infiltrating lymphocytes have a high tumour specificity i.e. they are only active against the tumour from which they themselves were isolated. Such tumour-infiltrating lymphocytes are not even effective against the same type of tumours from other patients. This significantly limits their therapeutic applicability.
SUMMARY OF THE INVENTION
The object of the present invention was therefore to provide tumoricidal T lymphocytes which are more suitable for tumour therapy than the previously known in vitro activated T lymphocytes.
This object is achieved by a mammalian cell line or active subcellular fractions thereof which are characterized in that
a) when they are co-cultured with lymphocytes during which allogenic stimulation is avoided they activate lymphocytes to form tumoricidal T cells without the need to add mitogens or growth factors such as e.g. interleukin 2 and
b) the lymphocytes activated in this way proliferate in their presence without addition of interleukin 2.
Surprisingly it turned out that tumoricidal T lymphocytes with a broad tumoricidal activity without HLA restriction can be obtained from lymphocytes by co-culture with a cell line according to the present invention or active subcellular fractions/fragments thereof. In this connection tumoricidal activity is understood as a killing effect and in particular a lytic effect on the respective tumour cells as well as an inhibitory action on the proliferation of these tumour cells.
A cell line is understood as those cells which have the capability of unlimited proliferation such as is characteristic of HeLa cells (ATCC CCL 2) (James D. Watson et al., Molecular Biology of the Gene, 4th edition, The Benjamin/Cummings Publishing Co., Inc. (1987), p. 963). Such cell lines are for example obtained by immortalizing human blood lymphocytes. Immortalization is preferably carried out by fusion with cytoplasts from the mouse myeloma cell line Ag8.653 according to the method described in EP-B 0 093 436 or in EP-B 0 256 512 (the content of which is also subject matter of the present patent application). The immortalized lymphocyte lines thus obtained are then co-cultured with human donor lymphocytes.
Blood lymphocytes are preferably used as lymphocytes. However, it is also possible to use tumour-infiltrating lymphocytes (TIL) as well as lymphocytes from the spleen or lymphatic nodes. In this connection it is preferable to purify the lymphocyte preparation before use. When using blood lymphocytes it is particularly expedient to substantially remove the erythrocytes and to concentrate the mononuclear cells. It is also advantageous to deplete the number of cells which can be allogenically stimulated by the cell line according to the invention or active fragments thereof.
In order to avoid allogenic stimulation, lymphocytes which are susceptible to such stimulation are eliminated from the donor lymphocyte population before co-culture. For this monocytes, macrophages, natural killer cells and MHC-restricted cytotoxic T cells, also especially those directed against allogenic MHC of the activator cell line and their precursor cells are preferably eliminated by incubation with L-leucyl-L-leucine methyl ester according to Thiele and Lipsky (The Journal of Immunology, Vol. 136, No. 3 (1986), p. 1038-1048). Those immortalized lymphocyte lines are selected after the co-culture which cause an activation of the donor lymphocytes to form tumoricidal T lymphocytes during this co-culture. In this process those activating lymphocyte lines which are lysed by the donor lymphocytes which activate them during the co-culture are preferably examined further. For the further selection, these activating lymphocyte lines are cultured together with the donor lymphocytes which are activated by them and a series of different tumour cell lines. Finally those activating lymphocyte lines are selected which lead to activated donor lymphocytes with tumoricidal action against the examined tumour cell lines. In this case tumoricidal action is not only to be understood as the killing, in particular lysis, of the examined tumour cell lines bu

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