Note: Descriptions are shown in the official language in which they were submitted.
CA 02270117 1999-04-27
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
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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
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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
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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
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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+.
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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
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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
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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
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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
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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.
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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
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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.
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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
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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
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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
PCT/EP97/01924
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
WO 94/23014
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
