CA2270117A1

CA2270117A1 - Process for producing tumoricide t-lymphocytes

Info

Publication number: CA2270117A1
Application number: CA002270117A
Authority: CA
Inventors: Herbert Jungfer; Heinrich Barchet
Original assignee: Individual
Current assignee: Roche Diagnostics GmbH
Priority date: 1996-10-30
Filing date: 1997-10-29
Publication date: 1998-05-07
Also published as: AU6908898A; WO1998018907A1; EP0942961A1; JP2000504230A

Abstract

The disclosed process, which consists in submitting lymphocytes to a lysosomial pretreatment, including incubation in the presence of anti-CD43 antibodies, enables the production of tumoricide T-lymphocytes.

Description

Process for the production of tumoricidal T lymphocytes The invention concerns a process for activating, producing and multiplying tumoricidal T lymphocytes and a composition which is suitable as a medium for the production, activation and multiplication of tumoricidal T lymphocytes.
The cellular immune defence plays an important role in eliminating pathologically changed endogenous cells such as e.g. virus-infected cells or tumour cells. In this case cytotoxic T lymphocytes recognize these changed endogenous cells with the aid 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 differ only very slightly from the corresponding antigens of healthy cells, the immune system may not form cytotoxic T lymphocytes that could eliminate the tumour cells. Therefore attempts have already been made to also induce a cellular immune defence against such tumour cells. Firstly it was attempted to achieve an active immunization with non-specific immuno stimulants such as the 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 with the concept of so-called adoptive immunotherapy. In this case lymphocytes of the patient are activated in vitro and then reimplanted. In most cases they are activated in vitro to form promiscous killer cells (D. Thiele et al., Immunology Today 10 (1989), 375 - 381) by adding interleukin 2. The cytotoxic lymphocytes that are obtained are then referred to as lymphokine-activated killer cells (LAK cells) (Rosenberg, Immunology Today 9 (1988), 58 - 62). In contrast to cytotoxic T lymphocytes, LAK cells and their action on tumour cells is not dependent 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. First clinical successes have already been achieved with LAK
cells. However, a disadvantage of this form of adoptive immunotherapy is the side-effects of the interleukin 2 that is required over a long period at relatively high doses. This leads in particular to an increase of the permeability of the capillaries which results in organ malfunction (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). Moreover LAK cells are also obtained during stimulation with interleukin 2 which 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 tumour cultures, G. Fossati et al., International Journal of Cancer 42 (1988), 239 - 245; G.
Degiovanni et al., Eur. J. Immunol. 18 (l988), 671 - 676;
Wolfel 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 (l990), 834 - 841). In addition a method for multiplying tumour-infiltrating lymphocytes (TIL) in vitro has also been described (Yron et al., J.
Immunol. 125 (1980), 238 - 245). In contrast to LAK cells which can be obtained from peripheral blood cells, tumour tissue must be removed from the patient to obtain tumour-infiltrating lymphocytes. This considerably limits their therapeutic application.
Tumoricidal T lymphocytes and a process for their production are described in WO 94/23014. According to this tumoricidal T lymphocytes are produced by co-culture of lymphocytes with a mammalian cell line having certain properties. -It is known from Mentzer et al., J. Exp. Medicine 165 (1987) 1383 - 1392 that a sialoglycoprotein on the cell surface (later referred to as CD43) plays a role in the proliferation of T lymphocytes. However, the antibody described by Mentzer only stimulates T lymphocytes in the presence of monocytes.
Vargas-Cortes et al., describe in Scand. J. Immunol. 27 (1988) 661 - 671 the stimulation of peripheral blood lymphocytes (PBL) with anti-CD43 antibodies. In the described method a spontaneous cytotoxicity of human PBLs is potentiated, the effect being primarily directed towards the NK cells and the cytotoxicity of the cells is increased towards the tumour cell line K562.
The object of the invention was to provide an improved and simplified process for the specific production of specific tumoricidal T lymphocytes in which stimulation and proliferation of other T lymphocytes and in particular of NK cells is avoided.
This object is achieved by a process for the specific production of tumoricidal T lymphocytes which do not cytotoxically destroy cells of a chronic myelogenic leukemia cell line (K-562, ATCC CCL 243) but only inhibit their growth for a limited time, by culturing lysosomotropically pretreated lymphocytes with anti-CD 43 antibodies and isolating the tumoricidal T
lymphocytes.
It has surprisingly turned out that it is not, as described in WO 94/23014, necessary to co-culture white blood cells (lymphocytes) with a mammalian cell line but rather that surprisingly specific tumoricidal T
lymphocytes can be obtained when lysosomotropically pretreated lymphocytes are cultured and incubated with an anti-CD 43 antibody.
Tumoricidal T lymphocytes within the meaning of the invention are T cells as described in WO 94/23014 and which recognize tumour cells in an antigen-unspecific manner and kill them via apoptosis induction and/or cytolysis. Tumoricidal T lymphocytes are characterized in that a) they have a tumoricidal effect on the tumour cell lines MOLT-4, Jurkat, THP-1, HL-60, Hela, K-562, Malme3M and/or Y79 and b) no interleukin 2 is detectable at a detection limit of 0.5 IU/ml in the culture supernatant of these tumoricidal T lymphocytes during the proliferation of these cells in the presence of the cell line HB 654 or HB 617.
Tumoricidal killer T cells are not natural killer (NK) lymphocytes. Compared to tumour-infiltrating lymphocytes they are more suitable for a therapeutic application due to their broad tumoricidal activity. In contract to natural killer cells, the tumoricidal cells produced by the process according to the invention are T cells. A
further criterium which differentiates between NK cells and tumoricidal T cells is that NK cells recognize and cytotoxically destroy the tumour cell line K-562 whereas tumoricidal T cells only inhibit the growth of the cell line K-562 for a limited time.
An inhibition for a limited time within the sense of the invention means that the cells of the cell culture are not killed but only their proliferation is inhibited and preferably their proliferation is completely inhibited.
The proliferation inhibition is reversible and can be abolished by separating the tumoricidal T cells and the cells of the cell line K-562.
In this connection a tumoricidal effect does not only mean the killing (in particular lysis) of the examined tumour cell lines but also an inhibitory effect on proliferation. This tumoricidal effect can for example be detected by cytotoxicity tests that are familiar to a person skilled in the art for example by the fact that the tumour cell lines which can be distinguished morphologically from the tumoricidal T lymphocytes disappear from the culture or at least their growth is retarded when they are cultured for a long period with the tumoricidal T lymphocytes. The tumoricidal T
lymphocytes are preferably CD3+ and/or CD4+.

Lymphocytes within the sense of the invention are understood as preparations of white blood cells which can for example be obtained from whole blood. Such methods are known to a person skilled in the art. For example the fraction of mononuclear cells (PBMNC) which contains the blood lymphocytes can be obtained by gradient centrifugation (Ficoll gradient). The PBMNC
obtained in this manner must be pretreated in order to form specific tumoricidal T lymphocytes. The cells are pretreated with a lysosomotropic substance such as L-leucyl-leucine methyl ester or an analogous ester. Such substances are for example described by P.L. Triozzi et al., Immunopharmacology 28 (1994) 39 - 45; C.S.
Rosenfeld, Stemcells, Dayt 12 (1994) 198 - 204; N. Seo, Cancer Immunol. Immunother. 38 (1994) 277 - 280. The lysosomotropic pretreatment allows a fraction of surviving lymphocytes to be obtained enriched in the precursor cells of tumoricidal T lymphocytes.
An anti-CD 43 antibody is understood as an antibody to the cell surface molecule CD43 (DeSmet, W. et al., in:
Schlossman, S.F., et al. (Eds.] Leukocyte Typing V, pp.
1706 - 1707, Oxford, United Kingdom: Oxford University Press, 1995). CD43 is a sialoglycoprotein which is formed on cells of the haematopoietic system and - with the exception of dendritic cells and a subpopulation of B lymphocytes - is expressed on a11 cells of the immune system (Fukuda, M., Glycobiology 1 [1991], 347 - 356).
Such an antibody is preferably directed against that part of the CD43 molecule which is involved in the binding of CD43 to MHC class I molecules. The antibody can be monoclonal or polyclonal and be produced by immunization, synthesis or recombinant methods. Complete antibodies or antibody derivatives which mediate a characteristic binding can be used. However, an antibody or antibody derivative is preferably used which is able to cross-link CD43 molecules on cell surfaces in a similar manner to intact CD43 antibodies.
It has been shown that a co-stimulation via the surface marker CD43 (binding to MHC-I on the stimulator cell) and CD2 (binding to CD58 on the stimulator cell) is involved in the differentiation of precursor cells (CD3-positive, leucyl leucine methyl ester-resistant lymphocytes) to tumoricidal T lymphocytes. The mammalian cell line described in Wo 94/23014 which leads to the production of tumoricidal T lymphocytes when co-cultured with lymphocytes has been shown to cause a differentiation of precursor cells apparently via the said surface markers. Surprisingly it has turned out that an anti-CD43 antibody alone is able to achieve an analogous effect.
In a preferred embodiment one or several anti-CD2 antibodies are added to improve the yield of tumoricidal T lymphocytes and preferably a T-cell-stimulated pair (directed against two different epitopes on CD2) of anti-CD2 antibodies (Schwarz, M., et al., J. Immunol.
154 (1995), 5813 - 5820; Moingeon, P., et al., Immunol.
Rev. 111 (1989), 111 - 140).
The lymphocytes are expediently cultured with the anti-CD43 antibody in conventional culture media for several days (preferably 5 - 30 days). The culture medium is preferably serum-free. It is especially preferred to use an autologous serum for the culture if it is intended to prepare autologous tumoricidal T lymphocytes.
The tumoricidal T lymphocytes obtained in this manner - g -can be administered to a patient as a therapeutic agent optionally after purification.
It has surprisingly turned out that specific tumoricidal T lymphocytes with a broad tumoricidal activity without HLA restriction can be obtained from lymphocytes by culture with anti-CD43 antibodies. .In this connection a tumoricidal activity is understood as a killing, in particular apoptosis-inducing and/or lysing effect on the corresponding tumour cells as well as an inhibitory affect on the proliferation of these tumour cells.
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 lymph nodes. The lymphocyte preparation is preferably purified before use. When using blood lymphocytes it is expedient to remove the erythrocytes. The fraction of precursor cells of tumoricidal T lymphocytes is concentrated before culture with the anti-CD43 antibody.
For this monocytes, macrophages, NK cells, suppressor T
cells and allogen-reactive, cytotoxic T cells and precursor cells thereof are eliminated by lysosomotropic treatment preferably 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 ) .
In order to carry out the process according to the invention lymphocytes are firstly isolated by known methods e.g. by means of Ficoll density gradient centrifugation preferably from the blood or from tumours of a donor. Subsequently the lymphocytes that remain after lysosomotropic treatment are cultured in a conventional lymphocyte culture medium together with an anti-CD43 antibody. The antibody concentration is between 1 ng/ml - 100 ug/ml, preferably 10 ~g/ml culture medium. The culture is continued until the activation of tumoricidal T cells can be detected. A culture for about 8 days is usually required for this. The culture according to the invention activates tumoricidal T
lymphocytes without having to add growth factors or differentiation factors such as lymphokines and in particular interleukin 2. This is a particular advantage for a therapeutic application of the tumoricidal T
lymphocytes that are obtained since such factors can trigger side effects in the therapeutic application.
As already described human tumoricidal T lymphocytes can be obtained using the process according to the invention without having to add differentiation factors such as e.g. lymphokines or interleukin 2 which could lead to side effects in a therapy. Therefore a further subject matter of the invention is the use of the process according to the invention to produce a therapeutic agent which can be used in tumour therapy. For such a therapeutic use the tumoricidal T lymphocytes produced according to the invention are washed by methods familiar to a person skilled in the art (e. g. by centrifugation and resuspension of the pellet in physiological saline which is repeated several times e.g. three times), optionally isolated and taken up in a suitable medium for the administration (e. g. physiological saline).
In addition to this ex vivo activation of lymphocytes to tumoricidal T lymphocytes, lymphocytes can also be activated in vivo by application of an anti-CD43 antibody to tumoricidal T lymphocytes. For such a therapeutic application the antibody is taken up and administered in a suitable medium for the administration such as physiological saline.
The tumoricidal T lymphocytes can also be used to eliminate tumour cells in a cell preparation ex vivo.
This can be preferably used to eliminate (purge) tumour cells from stem cell isolates (e. g. bone marrow stem cells) by culture with tumoricidal T lymphocytes. The stem cells purified in this manner can for example be reimplanted in a patient after a radiotherapy or chemotherapy (autologous bone marrow transplantation).
Antibodies to the human CD43 molecule are described by DeSmet, W., et al. (In: Schlossman, S.F., et al. [Eds.]
Leukocyte Typing V, pp. 1706 - 1707 "Oxford, United Kingdom: Oxford University Press, 1995). The anti-CD43 antibody named "6F5" is for example suitable for the process according to the invention.
In a preferred embodiment the tumoricidal T lymphocytes obtained after being cultured with the anti-CD43 antibody are proliferated. This proliferation is preferably carried out by the method described in the International Application PCT/EP97/01924.
The cell line HB 654 was deposited on the 24.03.1993 at the "Deutsche Sammlung fur Zellkulturen and Mikroorganismen GmbH, Mascheroder Weg lb, D-38124 Braunschweig under the number DSM ACC 2122.
The cell line HB 617 was deposited on the 01.03.94 at the "Deutsche Sammlung fur Zellkulturen and Mikroorganismen GmbH, Mascheroder Weg lb, D-38124 Braunschweig under the number DSM ACC 2166.

The invention is elucidated by the following examples, publications and tables, the protective scope of which is derived from the patent claims. The described methods are to be understood as examples which also describe the subject matter of the invention even after modifications.
The antibodies mentioned in the examples have been characterized in the international Leucocyte Typing Workshops. Instead of the said antibodies it is also possible to use antibodies with an analogous binding specificity.
Example 1 Culture of lymphocytes with an anti-CD43 antibody Mononuclear cells from the peripheral blood (PBMNC) of human donors are separated from erythrocytes by means of gradient centrifugation (lymphocyte separation medium, Boehringer Mannheim GmbH (BM)), washed twice with phosphate-buffered saline (Boehringer Mannheim GmbH
(BM)) and incubated for 20 minutes at room temperature at a density of ca. 5 x 106 cells/ml RPMI-1640 medium (Boehringer Mannheim GmbH (BM)) with 250 ACM leucyl-leucine methyl ester (Boehringer Mannheim GmbH (BM)) according to Thiele and Lipsky (The Journal of Immunology, Vol. 136, No. 3 (1986), p. 1038 - l048).
After washing twice with RPMI-1640 medium, the surviving cells are cultured at 37~C in an 8 % C02 atmosphere at a density of ca. 2 x 106 cells/ml in X-Vivo-20 medium (Bio-Whittacker) to which the 6F5 anti-CD43 antibody had been added at a concentration of 10 ~Cg/ml. On the 5th day after the start of the culture, half of the medium is replenished and fresh 6F5 anti-CD43 antibody is added up to a final concentration of 10 ~g/ml. On the 10th day after starting the culture, the cells are used for example 3.
The phenotyping of the cells on day 10 of the culture show that > 95 % of the cells are CD3+. Cells with the markers CD14 or CD16 are not found.
Example 2 Culture of lymphocytes with anti-CD43 and anti-CD2 antibodies As described in example 1, lymphocytes are prepared from the blood of a human donor by gradient centrifugation and treatment with leucyl-leucine methyl ester. The lymphocytes are suspended at a density of 1 x 106 cells/m1 in Iscoves modified DMEM (Gibco) to which 10 0 heat-inactivated (56~C/30 minutes) autologous serum had been added and the anti-CD43 antibody 6F5 and the anti-CD2 antibodies TS2i18 and VIT13 (Schwarz, M., et al., J.
Immunol. 154 (1995), 5813 - 5820) were each added at a concentration of 10 ~.g/ml. On day 5 half of the culture medium with the antibodies contained therein was replenished. The cell count on day 10 after the start of the culture showed that there was a 2.2-fold proliferation of the lymphocytes.

Example 3 Effect of tumoricidal T lymphocytes The tumoricidal T lymphocytes obtained according to example 1 are added to cultures of human tumour lines (see Tables I and II). The tumoricidal effect on these tumour cells is monitored under a microscope. These various tumour cell lines are killed or their growth is inhibited by the tumoricidal T lymphocytes according to the invention.
Table 1 Human Tumour E/T11 Surviving Growth Source tumour cell tumour of line density cells (% tumour of cells3>
control~2>

MOLT4 1x10~/ml 5/1 0 no ATCC CRL l582 JURKAT 1x105/m1 5J1 0 no ATCC TIB 152 THP-1 5x104/ml 5/1 <5 yes5~ ATCC TIB 202 HL-60 5x104/ml 10/1 <15 yes~> ATCC CCL 240 HELA 1x103icm2 10/1 <5 yes7> ATCC CCL 2 K562 1x105/ml 5/1 ca. 404> yes8~ ATCC CCL 243 MALME3M 2x103/cm2 10/1 <5 no ATCC HTB 64 Y79 2x103/cm2 10/1 <5 no ATCC HTB 18 1) effector/tumour cell ratio 2) microscopic evaluation after 48 hours 3) after 14 days culture 4) growth inhibition 5) ca. 4 colonies per 5 x 104 THP-1 cells 6) ca. 15 colonies per 5 x 104 HL-60 cells 7) 6 colonies per 2.5 x 104 HELA cells 8) abolition of the growth inhibition after 3 days Table II
Human tumour line Tumour type MOLT 4 acute lymphoblastic leukemia Jurkat acute T cell leukemia THP-1 acute monocyte leukemia HL-60 promyelocyte leukemia HeLa cervix carcinoma K-562 chronic myelogenic leukemia Malme-3M malignant melanoma Y79 retinoblastoma List of references Babbit et al., Nature 317 (1985), 359-361 Chen, B., et al., Cell.Immunol. 118 (1989), 458-469 Darrow et al., J. Immunol. 142 (1989), 3329-3335 Degiovanni, G., et al., Eur. J.Immunol. 18 (1988), 671-676;
DeSmet, W., et al., In: Schlossman S.F. et al., [Eds.]
Leukocyte Typing V, pp. 1706-1707; Oxford, United Kingdom: Oxford University Press, 1995 Ettinghausen et al., Surg. Forum 37 (1987), 388-389 Fossati, G., et al., International Journal of Cancer 42 (1988), 239-245 Fukuda, M., Glycobiology 1 [1991], 347-356 Mentzer et al., J.Exp.Medicine 165 (1987) l383-1392 Moingeon, P., et al., Immunol. Rev. 111 (1989), 111-140 Notter et al., Int.J.Cancer 45 (1990), 834-841 Rosenberg, Immunology Today 9 (1988), 58-62 Rosenfeld, C.S., Stemcells, Dayt 12 (1994) 198-204 Rosenstein et al., Journal Immunology 137 (1986), 1735-1742 Schwarz, M., et al., J.Immunol. 154 (1995), 5813-5820 Seo, N., Cancer Immunol.Immunother. 38 (1994), 277-280 Terry and Rosenberg (eds. Immunotherapy of Human Cancer (1982), Elsevier North Holland) Thiele and Lipsky (The Journal of Immunology, Vo1.136, No.3 (1986), p. 1038-1048 Thiele, D., et al., Immunology Today 10 (1989), 375-381 Triozzi, P.L. et al., Immunopharmacology 28 (1994), 39-45 Vargas-Cortes et al., Scand. J. Immunol. 27 (1988) 661-67l Wolfel et al., J.Exp.Med.~170 (1989), 797-810 Yron et al., J. Immunol. 125 (1980) , 238-245 Zinkernagel et al., Nature 248 (1974), 701-702

Claims

1. Process for the specific production of tumoricidal T lymphocytes which do not cytotoxically destroy cells of a chronic myelogenic leukemia cell line K-562, (ATCC CCL 243) but only inhibit their growth for a limited time, by culturing lysosomotropically pretreated lymphocytes with anti-CD 43 antibodies and isolating the tumoricidal T lymphocytes.

2. Process as claimed in claim 1, wherein one or several anti-CD2 antibodies are added.

3. Process as claimed in claims 1 or 2, wherein an autologous serum is added to the culture.

4. Process as claimed in claims 1 - 3, wherein L-leucyl-leucine methyl ester is used for the lysosomotropic pretreatment.