Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02251889 1998-10-16
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Process for producing and multiplying lymphocytes
The invention concerns a process for producing and
multiplying lymphocytes as well as a composition which
is suitable as a culture medium for lymphocytes.
The production and multiplication of lymphocytes is
problematic. Some types of lymphocytes cannot be
cultured at all in vitro or are very difficult to
culture in vitro. Native B lymphocytes can for example
only be cultured for a short period and T lymphocytes
require difficult culture conditions for longer culture
such as a combination of growth factors) and "feeder
cells" ("nurse cells") or the use of unphysiological and
potentially dangerous substances such as tumour
promoters (phorbol esters) in combination with
ionophoric substances (e. g. ionomycin) or plant lectins
(e. g. phytohaemagglutin, PHA). This puts severe
constraints on possibilities for the long-term culture
and multiplication of T lymphocytes and other
lymphocytes, and the production of lymphocytes in
significant amounts for diagnostic or therapeutic
purposes is very limited or impossible.
However, there is manifold interest in the ability to
produce, culture and multiply lymphocytes in vitro e.g.
B lymphocytes as producers of specific antibodies or
cytotoxic T lymphocytes (CTL) to treat infections or
tumours, in addition regulator T lymphocytes (helper or
suppressor T lymphocytes) for the diagnosis and
treatment of autoimmune diseases or "natural killer (NK)
CA 02251889 1998-10-16
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lymphocytes for treating malignant growths.
Hybridoma cells which are formed from a fusion of B
lymphocytes or T lymphocytes with malignant, lympoid
cells (e.g. myeloma cells) can be cultured and
multiplied without difficulty. Such hybridoma cells have
the immunological function of the original lymphocytes
as well as the essentially unlimited ability to
proliferate of the malignant fusion partner. However,
the application of the hybridoma technique is limited to
a few animal species (mouse, rat) and essentially fails
in other mammalian species and also in particular in
humans.
A process for the production of proliferating CD4+
lymphocytes is described in W090/10059. According to
this peripheral mononuclear blood cells (PBNMC) are
treated with alkyl esters to remove monocytes and
granulocytes and subsequently cultured in a culture
medium which contains a T cell stimulant and/or IL-2.
Mitogens such as PHA are used as the T cell stimulant.
However, the use of IL-2 alone only leads to a low
proliferation of the cells. The addition of mitogenic
substances during the culture of cells which are
subsequently to be implanted into a patient is critical.
A process for the culture of T cells in the presence of
interleukin-2 is also described in EP-A 0 203 403. A
disadvantage of this process is also that the T cells
can only be proliferated to a slight extent by this
means.
A process for culturing and multiplying tumoricidal T
lymphocytes is described in W094/23014 by co-culturing
CA 02251889 1998-10-16
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lymphocytes with a cell line (stimulator cells) while
avoiding an allogenic stimulation and without addition
of interleukin-2. In this process resting T lymphocytes
are activated to effector cells which recognize and kill
tumour cells or inhibit their growth. A considerable
amount of fermentation is required to provide such
stimulator cells for the mass proliferation of for
example tumoricidal killer T cells. In addition the
killer T cells must be separated in a sterile manner
from these stimulator cells or their cell debris before
use (reinfusion into the patient).
V. Kutnik et al., Period. Biol. 92 (1990) 48 describe
that, in the allogenic mixed lymphocyte reaction of
mouse spleen lymphocytes, the addition of IL2 restores
the cyclosporin A-induced inhibition of the
proliferation of the responder cells and increases their
alloreactivity.
Pierson, B.A. et al., Blood 87 (1996) 180 - 189 describe
that isolated human NK cells (CD56+, CD3-) can be
multiplied to a slight extent in culture by adding IL2.
The addition of supernatants of irradiated mononuclear
cells from blood increases the multiplication of the NK
cells. Thrombospondin was identified as the active
principle of this effect which does not act directly but
rather indirectly by activation of latent TGF~. TGFa
activated in this manner inhibits the proliferation in
the early phase of the culture and increases the further
proliferation. A repeated addition of TGF~ when the
medium is changed inhibits the growth of the cells and
in this case suppresses the proliferation of the NK
cells.
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In Immunological Invest. 25 (1996) 129 - 151 I.A. Ayoub
investigates the effect of human TGFa on a bovine CD4+
lymphoblastoid T cell line (BLTC) which grows
autonomously in IL2-containing medium. The cell line is
arrested in medium without IL2. The addition of TGF~ to
the arrested BLTC drives them rapidly into apoptosis.
The simultaneous addition of IL2 abolishes the arrest
and prevents the induction of apoptosis. The addition of
TGFa to suboptimal concentrations of IL2 co-stimulates
the proliferation of the BLTC.
In Intern. Immunol. 6 (1994) 631 - 638 R. de Jong
describes the effect of TGFpl on the proliferation of
isolated subpopulations of human CD4+ T lymphocytes.
TGFal amplifies the proliferation of CD4 cells (CD45
RA+) by antibodies in the presence of IL2, but the
proliferation of pre-activated T cells (CD45 RO+) is
inhibited by the addition of TGF~1. However, it turned
out that the induced proliferation of the CD45 RA+ cells
is inhibited after five days by addition of TGF~1.
A. Cerwenka describes in J. Immunol. 156 (1996) 459 -
464 that the presence of TGFal during the primary
stimulation of human T lymphocytes increases their
ability to survive in secondary cultures and reduces
their susceptibility to apoptosis-inducing anti-Fas
antibodies. The addition of TGF~1 also reduces the
apoptosis susceptibility of primary activated T
lymphocytes to secondary activation. A survival of the T
cells over a long period is ensured in the presence of
IL2 and TGF~1.
T.H. Inne et al., J. Immunol. 148 (1992) 3847 - 3856
describe that the proliferation of CTLL-2 cells (murine
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CA 02251889 1998-10-16
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- 6, Kakalis, LT., et al., FEBS-Lett. 362 (1995) 55-8.
Surprisingly aurintricarboxylic acid (ATA) can also be
used to increase the proliferation of lymphocytes.
Aurintricarboxylic acid is a substance which inhibits
the interaction of proteins and nucleic acids [Gonzales,
R.G. et al.: Biochemistry 19: 4299 - 4303 (1980)] and is
a general inhibitor of nucleases.
The amount of aurintricarboxylic acid which is added in
the process according to the invention can also be
varied and is to a certain extent dependent on the
medium used and on the cell to be cultured. It has
turned out that for example when culturing tumour-
infiltrating lymphocytes (TIL) in serum-containing
medium it is advantageous to add 0.1 - 100 ~M ATA.
Lymphocytes within the sense of the invention are
understood as leucocytes which are derived from
lymphocyte progenitor cells in the haematopoietic system
and can be for example found in blood, in the lymph, in
the spleen, in lymph nodes, in tumours (tumour-
infiltrating lymphocytes, TIL) or inflamed tissue.
Important subgroups are T lymphocytes, NK lymphocytes
and B lymphocytes. B lymphocytes are antibody-producing
lymphocytes in their mature form.
T lymphocytes (T cells) are understood as lymphocytes
which are for example involved in cell-mediated
cytotoxicity, in allergy of the delayed type and in the
activation of B lymphocytes. There are numerous
different types (subtypes) of T cells which can be each
distinguished by their function and/or their cell
surface antigens (see e.g. Imm. Rev. (1993), 74 and
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(1992), 82; Advances in Immunology 58 (1995) 87). Such
surface antigens are for example referred to as CD
(Cluster of Differentiation) antigens. The expression of
the antigen-recognizing T cell receptor is typical for
all T cells. T cells develop from haematopoietic stem
cells and mature, with some exceptions, in the thymus.
Examples of T cells are cytotoxic T cells, helper T
cells, suppressor T cells, suppressor-inducer T cells
and killer T cells.
Natural killer cells (NK cells) are lymphoid cells which
develop from haematopoietic stem cells and differ from T
cells and B cells in that they express neither the T
cell receptor nor the B cell receptor and are CD3-.
The culture of lymphocytes in the process according to
the invention is carried out in a conventional basic
culture medium which additionally contains a lymphocyte
growth factor and cyclosporin and/or ascomycin. All media
are suitable as basic culture media which are usually
used to culture mammalian cells. Such culture media can
either contain serum or be serum-free and are known to
any person skilled in the art. Examples are RPMI 1640-
medium, Dulbecco's Modified Eagles Medium (DMEM), F12
medium or a mixture of the latter (DF medium) which can
be used in a serum-containing and also in a serum-free
form. If serum-free media are used, the medium must be
supplemented by critical components. Such critical
components are, as known to any person skilled in the
art, albumin, transferrin, selenite and insulin. Serum-
free media which already contain all critical supplements
such as e.g. the culture medium X-Vivo 20~ (Bio-
Whittaker, Serva) are also particularly suitable.
CA 02251889 1998-10-16
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The amounts of lymphocyte growth factor and cyclosporin
and/or ascomycin which are added in the process
according to the invention can vary and, to a certain
extent, depend on the medium used (serum-free or serum-
containing) and on the cell to be cultured. It has
turned out that in serum-free culture amounts of 0.1 -
x 10'9 mol/1 growth factor and 10'1 - 10'2 mol/1
cyclosporin and/or ascomycin are suitable. Consequently
in the culture of T cells and/or NK cells in serum-free
medium it is advantageous to for example add 10 -
ng/ml IL-2 or IL-15 and 5 - 20 ng/ml Cyclosporin-A~
and/or 1 - 10 ng/ml Ascomycin~.
The process according to the invention is especially
suitable for culturing and multiplying killer T cells
(KT cells) and tumoricidal killer T cells. Such
tumoricidal killer T cells can for example be produced
according to WO 94/23014 by co-culturing lymphocytes,
which for example have been isolated from blood, with
stimulator cells. In this process resting T lymphocytes
are activated to effector cells which recognize and kill
tumour cells and inhibit their growth. The activated T
lymphocytes are stimulated to proliferate in this co-
culture and can be further cultured and multiplied by
the process according to the invention.
The process according to the invention is also
advantageous for the production and multiplication of
pancytotoxic T cells. A combination of a lymphocyte
growth factor with aurintricarboxylic acid, and a
substance which binds to cyclophilin and inhibits
calcineurin in this complex, or an apoptosis antagonist
is suitable. In addition the process according to the
invention enables the sustained proliferation of blood
lymphocytes after treatment with leucyl-leucine-methyl
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ester. The phenotyping of such cells produced according
to the invention on the basis of surface markers shows
that the proliferating cells are uniformly T lymphocytes
(100 ~ CD3+). These cells exhibit a previously unknown
activity which is referred to as pancytotoxic activity
in the following. Pancytotoxic T cells are characterized
in that they indiscriminately kill normal cells such as
e.g. fibroblasts, keratinocytes or endothelial cells and
also tumour cells such as e.g. malignant melanoma, T
lymphoma or lung carcinoma. Surprisingly pancytotoxic T
cells can be produced from mononuclear cells by
treatment with a combination IL-2 and apoptosis
antagonists. Pancytotoxic T cells can be used
advantageously for the local treatment of tumours (e. g.
tumour metastases).
An apoptosis antagonist (apoptosis inhibitor) is to be
understood as a substance which is able to not allow the
genetically determined self-destruction program to
become effective in a cell that would lead to cell death
after activation, and which partially or completely
slows or prevents the lysis of a cell after activation
of a suicide signal. Suitable substances are for example
described in Kroemer, G., Advances in Immunology 58
(1995) 27.1 - 296. According to the invention substances
are suitable which are able to inhibit agents with an
apoptosis signal effect on lymphocytes (e. g. an antibody
to TNFa which prevents binding of this cytokine to its
receptor). Furthermore substances are suitable which
prevent reception of an apoptosis signal by the
lymphocytes (e.g antibody to the TNFa receptor which
inhibits binding of TNFa to this receptor). Substances
are also suitable which are able to prevent apoptosis by
interrupting the signal chain from the cell membrane
into the inside of a lymphocyte (e.g. an inhibitor of
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sphingomyelinase, inhibition of the formation of
ceramide). Finally an apoptosis inhibitor is also to be
understood as a substance which is able to activate the
anti-apoptosis program in a lymphocyte which is also
genetically determined (e.g. up-regulation of the bcl-2
expression). Cyclosporin and ascomycin are also suitable
as calcineurin inhibitors.
It has surprisingly turned out that when mononuclear
blood cells (PBMNC) are cultured without pre-treatment
with leucyl-leucine-methyl ester, lymphocytes usually
grow after a latency period of 14-28 days which can be
multiplied as desired. Phenotyping of these cells shows
that they represent a mixture of T cells (CD2+, CD3+)
and NK cells (CD2+, CD3-, CD16+). Functionally they are
also pancytotoxic and thus differ from KT cells. In this
case it is also preferable to use IL2 or IL15 as a
lymphocyte growth factor and Cyclosporin or Ascomycin.
The following examples, publications and the figures
further elucidate the invention, the scope of which
results from the patent claims. The described processes
are to be understood as examples but still describe the
subject matter of the invention even after
modifications.
Fig. 1 shows the growth behaviour of killer T cells in
serum-free DF medium on addition of IL-2 +
CsA(A) and CsA alone (B).
Fig. 2 shows the growth behaviour of killer T cells in
serum-free DF medium on addition of IL-15 and
CsA(A) and IL-15 alone (B).
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Fig. 3 shows the growth behaviour of killer T cells in
serum-free DF medium on addition of IL-2 and
Ascomycin (FK520) (A) and Ascomycin alone (B).
Fig. 4 shows the growth behaviour of killer T cells in
serum-free DF medium on addition of IL-2 and
aurintricarboxylic acid (A) and arintri-
carboxylic acid alone (B).
Example 1
Production of killer T cells
Mononuclear cells from peripheral blood (PBMNC) of human
donors are isolated by means of gradient centrifugation
(lymphocyte separation medium, BM), washed twice with
phosphate-buffered saline solution and incubated at a
density of 5 - 10 x 106 cells/ml DF medium according to
Thiele and Lipsky (J. Immunol. 136 (1986) 1038-1048)
with 250 ACM leucyl-leucine-methyl ester (BM) for 20
minutes at room temperature. After washing with DF
medium the cells are cultured at 37°C in 8 % C02
atmosphere at a density of 1 - 2 x 106 per ml DF medium
together with irradiated (2000 rad) HB654 cells or HB617
cells (stimulator cells; 2 - 5 x 105 per ml). On day 5 -
6 of the co-culture half to two thirds of the culture
medium is renewed and irradiated stimulator cells (2 - 5
x 105 per ml) are added again. From day 8 - 10 after
starting the co-culture, when all stimulator cells have
been destroyed by the cytotoxic activity of the killer T
cells, the killer T cells are used for the following
examples.
Phenotyping the cells on day 10 of the co-culture shows
that >95 % of the cells are CD3+, ca. 40 ~ CD4+ and ca.
CA 02251889 1998-10-16
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60 % CD8+. Cells with the markers CD19 or CD16 are not
found.
Example 2
Multiplication of killer T cells in serum-free medium
which contains interleukin-2 (IL-2) and Cyclosporin A
(CsA).
Killer T cells which have been produced according to
example 1 are washed once in DF medium and cultured at a
density of 5 x 105 per ml DF medium in two separate
preparations that are denoted A and B. Human recombinant
IL-2 (BM; 20 ng/ml) and CsA (Sandoz; 12.5 ng/ml) are
added to preparation A. Only CsA (12.5 ng/ml) is added
to preparation B. Half of the culture medium is renewed
every second day and the cell count is adjusted to 5 x
105 per ml.
As shown in Figure 1 the killer T cells multiply in
preparation A (IL-2 + CsA) with a doubling time of about
48 hours for at least ten doubling cycles (corresponds
to 1000-fold multiplication). In preparation B (only
CsA) the killer T cells do not multiply.
Example 3
Multiplication of killer T cells in serum-free medium
which contains interleukin-15 (IL-15) and Cyclosporin A
(CsA) .
Killer T cells which have been produced according to
example 1 are washed once in DF medium and cultured at a
density of 5 x 105 per ml DF medium in two separate
preparations that are denoted A and B. Human recombinant
IL-15 (R & D Systems; 15 ng/ml) and CsA (Sandoz;
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12.5 ng/ml) are added to preparation A. Only IL-15 (15
ng/ml) is added to preparation B. Half of the culture
medium is renewed every second day and the cell count is
adjusted to 5 x 105 per ml.
As shown in Figure 2 the killer T cells multiply in
preparation A (IL-15 + CsA) with a doubling time of
about 48 hours for at least 11 doubling cycles. In
preparation B (only IL-15) the proliferation stagnates
after 3 doubling cycles.
Example 4
Multiplication of killer T cells in serum-free medium
containing interleukin-2 (IL-2) and Ascomycin (FR 520).
Killer T cells which have been produced according to
example 1 are washed once in DF medium and cultured at a
density of 5 x 105 per ml DF medium in two separate
preparations that are denoted A and B. rh IL-2 (20 ng/ml)
and Ascomycin (Calbiochem; 2.5 ng/ml) are added to
preparation A. Only Ascomycin (2.5 ng/ml) is added to
preparation B. Half of the culture medium is renewed
every second day and the cell count is adjusted to 5 x 105
per ml.
As shown in Figure 3 the killer T cells multiply in
preparation A (IL-2 + Ascomycin) with a doubling time of
about 48 hours for at least ten doubling cycles. In
preparation B (only Ascomycin) no multiplication of
killer T cells is observed.
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Example 5
Multiplication of NR and T lymphocytes from mononuclear
cells of human blood in serum-free medium which contains
interleukin-2 (IL-2) and transforming growth factor-(31
( TGF-(31 ) .
Mononuclear cells from peripheral blood (PBMNC) of a
human donor are isolated by means of gradient
centrifugation (lymphocyte separation medium, Boehringer
Mannheim GmbH, Germany (BM)), washed twice with
phosphate-buffered saline solution and taken up in DF
medium at a density of 1 x 106 cells/ml and cultured in
three separate preparations which are denoted A, B and
C. Recombinant human IL-2 (BM, 20 ng/ml) and rh TGF~31
(BM, 4 ng/ml) are added to preparation A, only IL-2
(20 ng/ml) is added to preparation B and only TGF(31
(4 ng/ml) is added to preparation C. Half of the culture
medium (+cytokine(s)) is renewed every second day and
the cell count is adjusted to ca. 1 x 106 cells/ml
during the first 7 days and subsequently to ca. 0.5 x
105 cells/ml.
In preparation A (IL-2 + TGF(31) the number of non-
adherent lymphoid cells increases approximately from day
after the beginning of the culture, firstly with a
doubling time of ca. 96 hours, from day 20 when no more
colonies with adherent monocytic cells are detectable
with a doubling time of less than 48 hours. On day 50 of
the continuous culture the cell count has increased more
than 104-fold compared to the initial state. The
analysis of the cells with regard to their surface
markers yields the following result on day 50: the
population contains:
CA 02251889 1998-10-16
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* ca. 50 % NK cells (CD2+, CD3-, CD16+, CD56+) and
* ca. 50 % T cells (CD3+, CD4+, CD8+)
In preparation B (only IL-2) lymphoid cells only
multiply moderately (< 50-fold) over ca. 16 days, then
stagnate and die after ca. 20 further days.
In preparation B (only TGF(31) the cells do not multiply.
Example 6
Multiplication of killer T cells in serum-free medium
containing interleukin 2 (IL-2) and aurintricarboxylic
acid (ATA).
Killer T cells which have been produced according to
example 1 are washed once in DF medium and cultured at a
density of 5 x 105 per ml DF medium in two separate
preparations that are denoted A and B. IL-2 (20 ng/ml)
and aurintricarboxylic acid (Aldrich Chemie; 4.2 ~cg/ml)
are added to preparation A. Only aurintricarboxylic acid
(4.2 ~g/ml) is added to preparation B. Half of the
culture medium is renewed every third day and the cell
count is adjusted to 5 x 105 per ml.
As shown in Figure 4 the killer T cells multiply in
preparation A (IL-2 + ATA) with a doubling time of about
72 hours for at least il doubling cycles. In preparation
B (only ATA) the killer T cells do not multiply.
CA 02251889 1998-10-16
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Example 7
Multiplication of tumour-infiltrating lymphocytes in a
medium which contains interleukin-2 (IL-2) and
aurintricarboxylic acid (ATA).
The nodules of a human colon carcinoma which was removed
by an operation from the large intestine is freed of
connective tissue and normal parts of the intestine and
cut into ca. 2 x 2 x 3 mm pieces. The tumour fragments
are taken up in Iscove-modified DME medium (Gibco) which
contains 15 % FCS (BM) and divided equally into four
culture dishes which are named A, B, C and D. IL-2
(20 ng/ml) is added to preparation A, aurintricarboxylic
acid (4.2 ~g/ml) is added to preparation B and IL-2
(20 ng/ml) plus aurintricarboxylic acid (4.2 ~,g/ml) is
added to preparation C. Preparation D remains without
additions. Three quarters of the respective culture
media is removed each second day without changing the
tissue or cell content. Microscopic control of the
cultures shows that after 24 - 48 hours lymphocytic
cells migrated out of the tumour tissue fragments which
multiplied in the partial culture C which contains IL-2
plus ATA but not in the partial cultures A, B or D.
After a ten day culture the picture is as follows: In A
and B (only IL-2 or ATA) the number of emigrated
lymphocytes has remained constant compared to day 2, in
D (no additives) hardly any lymphocytes are detectable.
In the partial culture C the number of lymphocytes has
increased 20-fold compared to A or B. The lymphocytes
from C are isolated from the partial culture C and they
are analysed on the basis of their surface markers. The
population contains:
ca. 60 % T lymphocytes (CD3+, CD4+, CD8+, CD19-)
ca. 25 % NK cells (CD2+, CD3-, CD56+, CD19-) and
ca. 15 % B lymphocytes (CD19+, CD3-).
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