Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WQ91/~4317 PCT/US90/05051
-1- 2066~4~ :~
~ET~OD FOR T~ PROD~CTION
OF IN VITRO EXPAND~D LYMPHOID CELLS
FOR USE IN ADOPTIVE IMMUNOTHERAPY
This invention is a continuation-in-part of U.S.
application serial number 07/238,445, which was filed on
August 31, 1988, the subject matter of which is
incorporated herein by raference thereto.
BACKGROUND OF INVENTION
A recent and highly pro~ising development in the
therapeutic treatment of diseases involves the use of
adoptive immunotherapy (See, e.a. Belldegrun et al.
(1989) Chapter 12, in Urolo~ic Oncology, Lepor et al.
(eds.), Rluwer Academic Publishers, Boston). Adoptive
immunotherapy is the passive transfer to an afflicted
host of in vitro expanded lymphoid cells that are
therapeutically effective in treating the disease via
destruction of the affected cells of the host by virtue
of specific interaction with the host's affected cells or
by furnishing therapeutically effective substances to the
host.
Adoptive immunotherapy involves the isolation of
lymphoid cells from an afflicted individual, the
selective expansion of subpopulations of lymphoid cells,
which have a high degree of reactivity or that have been
modified to have a high degree of reactivity against
cancerous or other disease affected cells, in vitro in
the presence of growth promoting substances, such as the
interleukins, that mediate the expansion of reactive
subpopulations of the cells populations and the
reintroduction of large numbers (1 x lOl to 5 x lOll) of
the expanded cells into the afflicted host, whereby the
in vitro expanded lymphoid cells destroy the cancerous
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or otherwise affected cells or furnish therapeutically
effective substances. For some methods of adoptive
immunotherapy the lymphoid cells are expanded in the
presence of target antigens or cells exhibiting such
target antigens as well as growth promoting substances.
The cells exhibiting such target antigens are generally
derived from the afflicted individual. Target antigens
can be isolated from the cells of an afflicted individual
or may be prepared by synthetic means, such as by
recombinant DNA technology or peptide synthesis. The use
of adoptive immunotherapy for the treatment of cancer has
been studied extensively. (See, e.a., Belldegrun et al.,
supra.~. Adoptive immunotherapy has been used to treat
cancers of the kidney, colon, lungs, renal and breast,
melanomas, lymphomas, and sarcomas (see, e.a.,
Belldegrun et al., supra., Topalian et al. ~1988) J.
Clin. Oncol. 5: 839-853, Rosenberg (1987a) U.S. Patent
No. 4,690,915, which disclosure is herein incorporated in
its entirety by reference thereto; see, also, Rosenberg,
20 et al.(1987b) New Eng. J. Med. 316: 889-897, Rosenberg
(1986a) at pp. 55-91 in Important Advances in Oncology,
DeVita et al. (eds.), JB Lippincott, New York), Yron et
al. (1980) J. Immunol. 125:238, and Rosenberg (1985)
Cancer 55: 1327).
Adoptive immunotherapy can also be used for the
treatment of other diseases, including viral diseases and
genetic defects. For example, lymphoid cells can be
isolated from a patient, modified by the incorporation of
cloned DNA into the DNA of the lymphoid cells, to encode
an enzyme that corrects a genetic defect or to encode a
therapeutically effective agent, cultured and then
reintroduced into the patient in whom the cloned DNA is
WO91/04317 PCT/US90/05051 ~
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expressed. Immunoreactlve lymphoid cells that have been
cultured in the presence of specific viral target
antigens and specific factors, such as IL-2, may be
useful for the treatment or immunization of individuals
against such virus.
As discussed above, some methods of adoptive
immunotherapy require the use of expanded subpopulations
of lymphoid cells that are produced from lymphoid cells
when the lymphoid cells have been cultured in the
presence of specific antigens, such viral antigens or the
antigenic portions of the immunologically active viral
proteins or antigens, or in the presence of cells bearing
such antigens, such as tumor cells or virally infected
cells. The antigen that is used may be that which is
pr~sented on a cell surface, such as an irradiated tumor
cell, it may be a purified antigen, or may be it may be
synthetic antigen, produced by methods such as by the
expression of cloned DNA or peptide synthesis. If the
particular antigenic source is tumor cells, then the
cells that are produced when the tumorous cells are
cultured in the presence of IL-2 are tumor infiltrating
lymphocytes (herein after TILs).
The mediators that are necessary for expansion
of such tumor specific lymphoid cells are growth
promoting substances and include mitogens, cytokines and
lymphokines. Mitogens are responsible for antigen
independent development of lymphoid cells, cytokines are
factors, such as lymphokines or monokines, that are
produced by cells that affect other cells, and
lymphokines are substances that are produced and secreted
by activated T lymphocytes and that affect other cell
types. In particular, it is known that certain
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lymphokines, such as IL-2, mediate specific expansion of
subpopulations of lymphoid cells that bear specific
phenotypic surface markers and that specifically
recognize certain antigens on the surfaces of affected
cells. The lymphokine, interleukin-2, hereinafter IL-2,
has been used to expand certain populations of
lymphocytes that have a high degree of antitumor
reactivity (see, e.q~, Rosenberg (1987a), supra., see,
also, Rosenberg (1987b), Rosenberg t1986a), supra., Yron
et al., supra., Rosenberg (1985), supra.,).
IL-2, which was originally identified as a T
cell growth factor, has been used to generate certain
lymphoid cells that possess antitumor reactivity against
the syngeneic or allogeneic tumor bearing host. For
example, incubation of resting lymphocytes, which are
obtained from tumor bearing hosts, including human and
murine hosts, in IL-2 for three to four days results in
the expansion of subpopulations o lymphocytes ~hat are
capable of lysing natural killer cell (hereinafter NK)-
resistant tumor cells, but not normal cells (see, e.g.,8elldegrun et al., supra.). This phenomenon is called
lymphokine activated killing (hereinafter LAK3 and the
lymphocytes that are responsible for this phenomenon
consist of two types of cells. The first type of cells
is called LAK cells and the second type of cells is
called TILs.
LAK cells can be obtained from both normal
individuals and from individuals afflicted with cancer or
other diseases. LAK cells appear to constitute a lytic
system of cells that is distinct from NK and cytolytic T
lymphocytes (hereinafter CTL) cells. LAK cell precursor
and effector cells possess phenotypes that are typical of
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NK cells (see, e.a., Phillips et al., (1986) J Exp. Med.
164: 814), but have the ability to lyse fresh,
noncultured NK-resistant allogeneic primary or metastatic
cancer cells, and can be generated from peripheral blood
lymphocytes (hereinafter PBL), thymus, spleen, lymph
node, bone marrow and thoracic duct cells (see, e.a,,
Belldegrun et al., supra., at pp. 215-216). LAK cells
also have the ability to lyse fresh autologous and
allogeneic tumor cells and many cultured cell lines.
Whereas LAK precursors have neither T nor B cell surface
markers, LAK cells appear to be Thy 1.2-positive and Ia-
negative and the majority of the cytotoxic activity
resides in the FcR-positive subpopulation (see, e.g.,
Lefor et al. (1989) at pp. 39-56 in Functions_of the
Natural Immune Svstem, Reynolds et al., eds., Plenum Pub.
Corp.). LAK cells have also been shown to mediate
antibody-dependent cellular cytotoxicity (ADCC) (see,
e.a., Lefor et al., supra.). It has ~een demonstrated
that IL-l and tumor necrosis factor (TNF) increase such
IL-2 induced ADCC activity and that such increase is
associated with a correlative increase in the lytic
potential of lymphoid cells that have been so induced
(see, e.a., Eisenthal et al. (1989) J. of Immunol. 142:
2307-2313).
TIL cells are lymphocytes that infiltrate into
tumors, against which a host's immune system is mounting
an immunological response, and can be isolated therefrom
(see, e.a., Yron et al., supra. and Anderson et al.,
supra.). TIL cells are found to have greater
specificity than LAK cells for autologous cells and
greater efficacy than LAK cells in adoptive immunotherapy
of cancer (see, e.a., Yron ek al., su~ra.). TIL cells
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have been obtained from resected human tumors, including
cancers of the kidney, colon, and breast, melanomas, and
sarcomas.
In vitro incubation of cells that have been
obtained from a tumor and grown in the presence of IL-2
results in the expansion of activated T cells within the
tumor and the destruction of tumor cells or tissue.
After 2-3 weeks of culture, the tumor cells have all been
destroyed and the culture consists of lymphoid cells that
have the phenotype of cytolytic T lymphocytes (CTL) (see,
e.a., Muul et al. (1987a) J. Immunol 138: 989, Topalian
et al., supra. and Itoh, et al. (1986) Cancer Res. 46:
301I). Some human TIL cells exhibit a high specificity
for their autologous tumors.
TIL cells also show promise for use in methods
of qenetic therapy (see, e.g. Culliton (1989), "News and
Comment" in Science 244: 1430-1433.) They provide a
source of autologous cells that can be modified by the
insertions of DNA encoding a desired protein, cultured,
and reintroduced into the patient. The desired protein
may be a therapeutically effective protein, such as tumor
necrosis factor, which is used in cancer therapy, CD4
receptor ~o which HIV binds, an enzyme, for which the
treated host is deficient, or a it may be a marker
protein, whereby the fate of the TIL cells in the treated
host may be studied.
In addition to LAK and TIL cells other types of
lymphoid cells have also been identified as possessing
antitumor reactivity. For example, ~LN (regional
draining lymph node) cells are a population of lymphoid
cells, which have antitumor reactivity, that are derived
from the regional draining lymph nodes of tumor bearing
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20666~
mice that have been immunized with weakly immunogenic
tumors (Stephenson et al. (1989) Surgery 105: 523-528).
RLN cells are therapeutic effector cells and represent a
different cell population than LAK cells.
Because many cancer patients do not respond to
adoptive immunotherapy, studies are underway to identify
other lymphokines, cytokines, and/or mitoqens that may be
useful alone or in combination with Il-2 in expanding
subpopulations of lymphoid cells for use as adoptive
immunotherapeutic agents. Although IL-2 has primarily
been used to generate such subpopulations of lymphoid
cells, other lymphokines, such as IL-4, IL-6 and other
interferons, and TNF have also been shown to be to be
useful in the production of in vitro expanded lymphoid
cells and may also prove to be useful in expanding
specific subpopulations of lymphoid cells. For example,
IL-4 (also called BSF-l) is a glycoprotein that is
derived from T cells and has been shown to induce LAK
activity if the lymphoid cells are first stimulated with
IL-2, but is inhibitory if the cells are not pre-
stimulated (Kawakami et al. (1989) J. of Immunol. 142:
3452-3461) IL-4 also has been shown to be capable of
stimulating the growth of TIL cells both alone and in
conjunction with IL-2. IL-4 appears to enhance the
growth of TIL cells and concomitantly inhibit the growth
of NKHI~ cells, which are responsible for non-specific
killer activity (Lotze et al. (1989) at pp. 167-179 in
Human Tumor Antiaens and S~ecific Tumor Therapy, Alan R.
Liss, Inc., see, also, Kawakami et al., (1988) J. of
Exptal. Med. 168: 2183-2191.).
The possibilities for uses of adoptive
immunotherapy are almost limitless. Not only can it be
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WO 91/04317 ~ PCI/USgO/0505~ ~
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used for the treatment of cancer by specific interactions
between the lymphoid cells and the tumor, but, as
discussed above, it can be used for the treatment of
genetic diseases and as a means of delivering antitumor
agents and other therapeutic agents. Lymphoid cells can
be removed from an individual who is suffering from a
genetic disease that results from an enzyme or hormone
deficiency. A wild type copy of the gene of interest
can be inserted into the lymphocytes and the lymphocytes
can be cultured in the presence of selective agents and
reintroduced into the afflicted individual.
Alternatively, the lymphoid cells can be genetically
modified to express a therapeutically effective
anticancer or antiviral agent, such as interferon or TNF,
and then reintroduced into the patient (See, e.a.,
Genetic Eng. News. Vol. 9, No. 3, March 1989 and p. 133
in Business Week/May 1, 1989).
In practicing adoptive immunotherapy it is,
however, necessary to develop methods not only for the
identification of therapeutically useful subpopulations
of lymphoid cells, but to develop methods for the
generation of large quantities of such cells. Adoptive
immunotherapy of human cancers and other disorders is a
highly promising treatment, but the inability to generate
clinically useful numbers of immunoreactive lymphoid
cells has been a major obstacle to the use of adoptive
immunotherapy (see, e.a. Rosenberg et al. (1986b) Science
233: 1318-1321 and Culliton ,su~ra.). There is, thus, a
need for methods for the large scale production of
lymphoid cells and for methods that can be readily
adapted as new cells and agents for their expansion are
identified.
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Currently, the generation of sufficient numbers
of expanded subpopulations of lymphoid cells, such as
TILs, for administration to patients having metastatic
disease is time consuming, inefficient, and prohibitively
expensive. It is accomplished by growing TILs, which are
derived fro~ a metastatic lesion, in plastic, gas
permeable culture bags, each of which holds about 1.5
liters of tissue culture medium that contains human serum
albumin and recombinant human IL-2 ( see, e.a., Muul et
al. (1987b) J. Immunol. Meth. 101: 171-181, see. also
Culliton, supra.) The cells grow and divide until they
reach a maximum density of, at most, 2-3 x 109 cells per
bag at which point they must be split into additional
culture bags. It, thus, may require 50 to 150 bags to
generate a sufficient number of cells a single clinical
treatment of one patient. Over a 4 to 6 week time
period the cells are grown and split into additional bags
until a sufficient number of cells for clinical treatment
are generated typically expanding into 100 to 150 bags,
containing approximately 1011 cells. The cells, which are
in a volume of 150 to 250 liters, must then be
centrifuged and maintained under sterile conditions. The
entire procedure, which generates enough cells for the
treatment of a single patient, uses enormous quantities
of mammalian cell culture medium, human serum albumin,
and IL-2, as well as a great deal of time and manpower.
Typically it requires about 5 hours to harvest the cells
for a single treatment. Because this cell concentration
method yields cells in dilute solution, is fraught with
opportunities for contamination of the cultured cells,
and is prohibitively expensive. Its use as a means to
generate the therapeutically necessary quantities of
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cells for clinical treatment is severely limited.
There is, thus, a need for the development of
methods that can be used to efficiently and cost
effectively grow the large numbers of biologically active
in_vitro expanded lymphoid cells that are suitable for
use in methods of adoptive immunotherapy. Further,
because of the wide range of disorders that can be
treated by this method, there is a need to develop
methods that can be readily adapted to changing protocols
and to the many protocols for which such expanded
populations of lymphoid cells will be used.
SUMMARY OF THE INVENTION
It is one object of this invention to provide an
improved method for the large-scale production of n
vitro expanded lymphoid cells that can be used in
adoptive immunotherapy, comprising (a) inoculating the
extra fiber space of a hollow fiber bioreactor with a
suspension of lymphoid cells in a growth-promoting factor
containing medium; (b~ perfusing said bioreactor with
tissue culture medium that contains an effective amount
of at least one growth promoting substance that
specifically expands a therapeutically useful
subpopulation of said lymphoid cells, wherein said
effective amount is an amount sufficient to effect said
specific expansion, said tissue culture medium sustains
the cell division and growth of said subpopulation, and
said therapeutic use is adoptive immunotherapy; and (c)
culturing said cells in said bioreactor in the presence
of said yrowth promoting substance for a time sufficient
to obtain a therapeutically effective number of said in
vitro expanded lymphoid cells.
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11
It is another o~ject of this invention to
provide an improved method for the production of in vitro
expanded lymphoid cells for use in methods of adoptive
immunotherapy, wherein said expanded lymphoid cells are
TIL cells, comprising (a) suspending cells that are
derived from a resected tumor tissue in cell tissue
culture medium; (b) culturing said suspension in the
presence of an effective amount at least one cytokine
that is capable of promoting the expansion of tumor
- 10 infiltrating lymphocytes, wherein said effective amount
is a amount-sufficient to effect the expansion of the
tumor infiltrating lymphocytes in said suspension; (c)
inoculating the extra fiber space of a hollow fiber
bioreactor that is a component of a hollow fiber culture
system with said cultured suspension of tumor
infiltrating lymphocytes: (d) perfusing said:bioreactor
with tissue culture medium that contains an effective
amount of at least one cytokine that is capable of
promoting the expansion of tumor infiltrating
lymphocytes, wherein said effective amount is an amount
sufficient to effect said specific expansion and said
tissue culture medium sustains the cell division and
growth of said tumor infiltrating lymphocytes; and (e)
culturing said tumor infiltrating lymphocytes in said
bioreactor in the presence of said tissue culture medium
for a time sufficient to obtain a therapeutically
effective number of said tumor infiltrating lymphocytes.
It is another object of this invention to
provide n vitro expanded lymphoid cells for use in
methods of adoptive immunotherapy.
It is another object of this invention to
provide in vitro expanded lymphoid cells for use in
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methods of adoptive immunotherapy, wherein said expanded
lymphoid cells are TIL cells.
It is another object of this invention to
provide in vitro expanded lymphoid cells for use in
methods of adoptive immunotherapy, wherein said expanded
lymphoid cells are lymphoid cells whose genomes have been
modified by the incorporation therein of cloned DNA.
It is another object of this invention to
provide a method for preparing a conditioned medium for
use in stimulating the growth of in vitro expanded
lymphoid cells and as a source of biologically active
growth promoting substances that specifically expand
therapeutically useful in vitro expanded lymphoid cells,
comprising removing the contents of the extra-fiber space
of a bioreactor in which in vitro expanded lymphocytes
have been cultured, pelleting and removing the ~ells from
said contents of the extra fiber space to produce an
extra fiber space cell supernatant; and dialyzing said
extra fiber space cell supernatant against tissue culture
medium to produce extra fiber space conditioned medium.
It is another object of this invention to
produce conditioned medium for use in stimulating the
growth of in vitro expanded lymphoid cells and as a
source of biologically active growth promoting substances
that specifically expand therapeutically useful i itro
expanded lymphoid cells.
It is another object of this invention to
provide an improved method for producing in vitro
expanded lymphoid cells, comprising culturing said cells
in the presence of an effective amount of extra fiber
space conditioned medium, wherein said amount is
effective to stimulate the rate of growth of said
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expanded cells at least about 50% more than the growth of
said cells in its absence
This invention significantly improves the
procedure for preparing therapeutically useful quantities
of in,,v,itro expanded lymphoid cells by providing an
improved method for culturing said cells that can be
adapted to the specific requirements of an adoptive
immunotherapeutic procedure, whereby lymphoid cells are
obtained from a patient, inoculated into a hollow fiber
bioreactor culture system and cultured in the presence
of an effective amount at least one growth promoting
substance that specifically expands a therapeutically
useful subpopulation of lymphoid cells.
In practicing this invention therapeutically
useful yields of biologically active therapeutically
effective in vitro expanded lymphoid cells are obtained ,
using a convenient method that not only significantly
reduces the costs associated with the production of such
cells but significantly increases the numbers of cells
that can be produced.
BRIEF DESCFcIPTION OF THE FIGURES
Figure 1 presents a typical growth curve for TIL
grown according to the methods of this invention. The TIL
increase in cell number versus time elapsed from the date
of excisional biopsy. Cells were obtained from the
tumor that had been excised from patient W. The culture
was initiated in gas permeable bags, when sufficient TIL
had been grown in standard culture flas~s from the
enzymatically dispersed tumor, which contained the TIL,
and then inoculated into the hollow fiber reactor on day
23 after excisional biopsy. -O-0-0- represents the
growth curve of cells grown in gas permeable bags as
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WOgl/04317 PCT/US90/0505 -
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14
measured by a~ increase in cell number, which is plotted
on the ordinate on the right side, versus duration of
culture, which is plotted on the abscissa as days. After
an initial lag period, the number of cells obtained from
patient W (W-TIL) increased exponentially over time. Time
= 0 indicates the date of excisional biopsy. TIL cells
were withdrawn from one bag on day 23 and were inoculated
into a CELLMAX~ hollow fiber bioreactor and harvested on
day 44 t~ -)- Glucose consumption, as measured by
a decrease in glucose concentration in the bioreactor
perfusate, which is plotted on the ordinate on the left
side, versus days in culture on the abscissa, increased
logarithmically with time(-o-o-o-). + represents the
replacement of the extra-fiber space (EFS) with fresh
complete media. The hollow fiber inoculum of 4.3 x 108
cells on day 23 yielded 5.4 x 101 cells at harvest.
Figure 2 depicts a growth curve showing G-TIL
(TIL obtained from patient G) increasing in number versus
time elapsed from the date of excisional biopsy. 0-o-
0- represents the growth cur~e of cells grown in gas
permeable bags as represented by an increase in cell
number. After an initial lag period, the number of cells
obtained from patient G increased exponentially over time
measured from the date of excisional biopsy. TILs were
2~ withdrawn from one bag on day 16, inoculated into a
CELLMAXsM hollow fiber bioreactor and harvested on day 30
~ ----). Glucose consumption, as measured by a
decrease in glucose concentration in the perfusate, which
is plotted on the ordinate on the left side versus
duration of culture, increased logarithmically with
time(-o-o-o-). ~ represents the replacement o* the EFS
with fresh complete medium. The inoculum of l.O x 108 TIL
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WO91/04317 PCT/US90/05051
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yielded a 1.5 x l01 TIL harvest. Perfusion of the hollow
fiber bioreactor was re-instituted on day 30, the day of
the first harvest, and the residual TIL expanded again to
l.5 x lo10 for a second harvest on day 51. Perfusion was
again re-instituted and the residual cells were once
again permitted to expand for a third harvest of 2.l x
l01 cells on day 73.
Figures 3 show scanning electron micrographs of
TIL cells grown by the methods of this invention from
patient W within the extra-fiber space of a CELLMAX~
hollow fiber bioreactor.
In Figure 3a the space between the large, ovoid
hollow fibers is filed with a nearly solid mass of TIL.
The ovoid shape of the normally cylindrical hollow fibers
and the space between the TIL mass and the fiber surfaces
are artifacts of histologic preparation.
Figure 3b shows a higher magnification so that
the individual cells near the outer surface of a single
hollow fiber can be seen.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
~nless defined otherwise, all technical and
scientific terms used herein have the same meaning as is
commonly understood by one of ordinary skill in the art
to which this invention belongs. All publications
mentioned herein are incorporated by reference thereto.
As used herein, lymphoid cells include lymphoid
cells that derived from any tissue in which lymphoid
cells are present. In general lymphoid cells are removed
from an individual who is to be treated. The lymphoid
cells may be derived from a tumor, peripheral blood, or
other tissues, such as the lymph nodes and spleen that
contain or produce lymphoid cells.
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As used herein, adoptive immunotherapy is a
process whereby in vitro expanded lymphoid cells are
transferred, administered or introduced into an
individual or host.
When the lymphoid cells are cultured in vitro
under appropriate conditions certain subpopulations
thereof are selectively expanded. The expanded
subpopulations of cells that are produced are herein
referred to as in vitro expanded lymphoid cells. The
subpopulation of cells is generally a heterogeneous
mixture of cells having different phenotypes, but it may
also consist of a homogeneous population of cells. The
particular mixture of cells that are produced is a
function of the starting material and the conditions
under which such cells are generated. The expanded
subpopulation can be used in adoptive immunotherapy
protocols. Upon introduction into an individual who is
being treated, the expanded subpopulation of cells
specifically recognizes and in some manner mediate
destruction of the host's afflicted cells-and/or produces
therapeutically effective agents. The conditions under
which such cells are produced include growth in the
presence of a cytokine or a mixture of cytokines, such as
IL-2, IL-1, IL-6 and IL-4. If the lymphoid cells are
cultured in the presence of a cytokine, then in vitro
expanded subpopulations of lymphoid cells that are
produced include activated lymphoid cells and, depending
upon the source thereof and the cytokine used, may
include LAK cells and TIL cells. If the lymphoid cells
that are expanded in the presence of the cytokine are
derived from a tumor, then the in vitro expanded lymphoid
subpopulation of cells that is produced are referred to
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as TIL cells. If the lymphoid cells are expanded in the
presence of a cytokine and target antigen, then the cells
that are produced are herein referred to as activated
lymphoid cells. If the target antigen is disposed on a
cancerous cell or is derived therefrom, then the in vitro
expanded lymphoid subpopulation of activated lymphoid
cells are TIL cells.
As used herein, therapeutically useful
subpopulations of in vitro expanded lymphoid cells are
those that can be used for adoptive immunotherapy.
As used herein, n vitro expanded lymphoid cells
are cells that are cultured for use in methods of
adoptive immunotherapy. Such cells constitute
subpopulations of lymphoid cells that are produced when
lymphoid cells are cultured in the presence of specific
mediators that induce expansion of at least one
particular subpopulation of lymphoid cells. Such
subpopulations include those that are immunologically
reactive with a diseased patient's affected cells. When
such cells are used as therapeutic agents they are
herein referred to as immunologically reactive lymphoid
cells. TILs and LAX cells, when used for adoptive
i~munotherapy of cancer are examples of immunologically
reactive cells. Other subpopulations of in vitro
expanded lymphoid cells, include lymphoid cells that have
been modified by genetic engineering to contain DNA that
encodes proteins that are not normally produced by said
lymphoid cells. Examples of such proteins include
traceable foreign marker proteins and therapeutically
effective substances, such as the CD4 protein to which
HIV binds and anti-cancer agents. In addition, such
cells may be genetically engineered to contain DNA
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encoding drug resistance or drug sensitivity so that such
cells may be selectively expanded or destroyed in vivo.
Other subpopulations include lymphoid cells that have
been cultured in vitro in the presence of specific target
antigens. The target antigen or antigens may be disposed
on the surface of a cell, such as a tumor cell, may be
from cells or prepared synthetically and introduced into
the tissue culture medium. Target antigens may be
synthesized by the methods of peptide synthesis or by
10 genetic engineering. In general in vitro expanded -
lymphoid cells a-re cells that have been cultured in the
presence of a target antigen specifically recognize and
in some manner mediate destruction of cells bearing the
target antigen or deliver a therapeutic agent to cells
that bear the target antigen.
As used herein, a target antigen is an antigen
that is specifically recognized by a subpopulation of in
vitro expanded lymphoid cells. An effective amount is at
least one target antigen is an amount that is sufficient
to select for the expansion of at least one subpopulation
of in vitro expanded lymphocytes that specifically
recognize said antigen.
As used herein, tumor-specific in vitro expanded
lymphoid cells are cells that specifically recognize
target antigens that are present on or in tumor cells.
TIL cells are tumor specific lymphoid cells. As used
herein a tumor-specific antigen is an antigen that is
disposed on the surface or inside of a tumor cell. Tumor
specific antigens may used in purified form, on
irradiated tumor cells, or they may be obtained by
purifying them from tumor cells or by synthesizing them
in vitro by methods, such as genetic engineering.
: . ,, '' .` . ~
WO91~317 PCT/US90/05051
2066~4~
19
As used herein, a growth promoting substance is
a substance that in some manner participates in or
induces cells to grow and/or divide. Growth promoting
substances include mitogens and cytokines. Examples of
growth promoting substances include the fibroblast growth
factors, epidermal growth factor, the products of
oncogenes, the interleukins, colony stimulating factors,
and any other of such factors that are known to those of
skill in the art.
As used herein, a mitogen is a substance that
induces cells to divide and in particular, as used
herein, are substances that stimulate a lymphocyte
population in an antigen-independent manner to
proliferate and differentiate into effector cells. ~
15 Examples of such substances include lectins and ~ -
lipopolysaccharides.
As used herein, a cytokine is a factor, such as
lymphokine or monokine~ that is produced by cells that
affect other cells.
As used herein, a lymphokine is a substance that
is produced and secreted by activated T lymphocytes and
that affects other cell types. The tumor necrosis
factor, the interleukins and the interferons are examples
of lymphokines. A monokine is a substance that is
secreted by monocytes or macrophages that affects other
cells.
As used herein, a therapeutically effective
number of in vitro expanded lymphoid cells is the number
of such cells that is at least sufficient to achieve the
desired therapeutic effect, when such cells are used in
a particular method of adoptive immunotherapy. For
example, a therapeutically effective amount of TILs for
'
WO 91 /0431 7 PCl tUS90/05051_
'~;0~
~0
a single treatment of a patient suffering from metastatic
cancer is at least about 101 to 1011 cells.
As used herein, a hollow cell fiber culture
system consists of a hollow fiber bioreactor a~ well as
pumping means for perfusing medium through said system,
reservoir means for providing and collecting medium, and
other components, including electronic controlling,
recording or sensing devices. . A hollow fiber bioreactor
is a cartridge that consists of a multitude of semi-
permeable tube-shaped fibers encased in a hollow shell.
The terms hollow fiber reactor and hollow fiber
bioreactor are used interchangeably.
As used herein, the extra fiber space (EFS) is
the space in which the cells grow within the shell of the
hollow fiber bioreactor that is external to the semi-
permeable fibers.
As used herein, the EFS cell supernatant is the
medium in which the cells in the EFS are growing. It
contains secreted cellular products, diffusible nutrients
and growth promoting substances, such as lymphokines and
cytokines, that mediate specific expansion of
subpopulations of in vitro expanded lymphoid cells.
Thus, as used herein, a hollow fiber bioreactor
consists of an outer shell casing, the semi-permeable
fibers, and the EFS, which contains the cells and the EFS
cell supernatant.
As used herein, EFS conditioned medium is the
EFS cell supernatant after it has been centrifuged to
remove any cells and particulate matter and dialyzed
against tissue culture medium.
As used herein, selective medium is a tissue
culture medium in which the in vitro expanded lymphoid
' '
', ' ' " ~ .
WO 91/0431, PCI`/US90/05051
2066~6~
21
cells are generated that contains the desired growth
promoting substance or substances and any other selective
agents, such as target antigens or agents designed to
select for growth of genetically engineered cells.
As used herein, complete AIM-V is a selective
medium that consists of the proprietary formula AIM-V
(GIBCO, Grand Island, N.Y.) and also contains 1000 units
of IL-2 /ml. (provided by cetus Corp., Emeryville, CA.),
lO ~g. gentamicin/ml. (GIBCO), 50 ~g. streptomycin/ml.
~GIBCO), 50 ~g penicillin/ml. (GIBCO), 1.25 ~g.
fungizone/ml. (Flow Laboratories, MacLean, VA.), 2.95 mg.
glucose/ml. and 2 mM. glutamine (Flow Laboratories).
Supplemented complete AIM-V consists of complete AIM-V
that is supplemented with 20% AIM-V supernatant that is
obtained from cultures of autologous LAX cells.
As used herein, AIM-V supernatant is prepared as
described in Muul et al. (1986) J. Immunol. Methods 88:
265). Briefly, LAK AIM-V supernatant is prepared by
growing peripheral blood lymphocytes in AIM-V or other
suitable tissue culture medium in the presence of IL-2
for 2 to 3 days and removing the cells by centrifugation
to obtain the supernatant.
Other suitable tissue culture media are well-
known and readily available to those of skill in the art
and may be readily substituted for AIM-V. For example,
a medium that consists of a 50-50 mixture of complete
AIM-V and RPMI having 10% heat-inactivated human serum,
and further supplemented with LAK supernatant may be
used.
As a first step when practicing any of the
embodiments of the invention disclosed herein lymphoid
cells that can be used to generate the desired
:: .
.'~ . .
WO 91/04317 ~ PCr/US90/05051 ~
206~64~ :
22
subpopulations of 1 _ itro expanded lymphoid cells must
be removed from an individual who is generally the
patient who is to be treated using an adoptive
immunotherapeutic method. Lymphoid cells are present in
many tissues in the body, include PBL, lymph nodes,
spleen cells, and any tissue in which an immune response
is being mounted. The lymphoid cells that are
contemplated for use in this invention are any whose
development and growth may be directed by appropriate
growth promoting substances to produce in vitro expanded
lymphoid cells that are suitable for any method of
adoptive i~munotherapy.
The cells obtained from the patient are
suspended in any cell culture medium that is suitable for
sustaining the growth of such mammalian cells. Cell
suspensions may be prepared from tumors or otherwise
affected tissue or from lymphoid cells. Such media are
readily available and the choice of an appropriate medium
is well within the level of skill in the art. The cells
are then plated into culture plates, or other suitable
means known to those of skill in the art, for further
expansion in an selective medium that also contains the
growth promoting substance(s) that is(are) necessary for
selective expansion of the desired subpopulation of
immunoreactive lymphocytes. Tha medium may also contain
target antigens. The cells are cultured and their
numbers expanded until a sufficient number are obtained.
Sometime at this point, the cells may be inoculated into
a gas permeable bag and grown in selective medium
containing the desired growth promoting substance and any
other desired selective agents until at least about lOa
cells are generated. Because the cells are cultured in
.... . .
.:~ '
.
WO9~/04317 PCT/US90/05051
23 20~4~ :
selective medium, the cells in suspension include
primarily the subpopulation(s) of interest. The cells
that have been generated are injected into a hollow
fiber bioreactor, and cultured under conditions that are
designed to expand the subpopulations thereof that can be
used in methods of adoptive immunotherapy.
~ ollow fiber bioreactors (abbreviated herein as
HF) are known to those of skill in the art (see, e.g.,
Knazek et al. U.S. Patent Nos. 4,220,725, 4,206,015,
4,200,689, 3,883,393, and 3,821,087, which disclosures
are herein incorporated by reference thereto). Hollow
fiber bioreactors have been used for the growth of
mammalian cells and for the production of biologically
active products that are secreted thereby (see, e.a.,
Knazek et al., supra., see, also, Yoshida et al., U.S.
Patent No. 4,391,912; Meyers et al., U.S. Patent No.
4,546,083; and Markus et al., TJ.S. Patent No. 4,301,249).
The hollow fiber bioreactor that is contemplated
for use in the practicing of this invention contains a
multitude of tube shaped semi-permeable membranes
(hereinafter called fibers) that are encased in a hollow
shell. Cultured cells grow and fill the spaces between
the fibers and are fed diffuse of or the flow of
nutrients from medium that is perfused through the lumina
of said membranes. An example of a hollow fiber
bioreactor that may be used in practicing this invention
is the hollow fiber bioreactor, B3, Cellco Advanced
Bioreactors, Inc., Kensington, MD, (see U.S. Application
Serial No. 07/23~,445, supra. for a complete description
thereof). The bioreactor, B3, contains about 8000 tube-
shaped, semi-permeable membranes, which provide a 1.1 m2
surface area. The fibers, which are each approximately
wo 91/01317 PCI/~JS90/05051--
20666~1~
24
250 ~m in diameter, are pulled through a polycarbonate
tube that is about 12 inches in length, and sealed at
each end in such a manner that liquid only flows through
the lumina of the fibers to exit at the opposite end of
the shell. The fiber walls nominally restrict passage to
substances having molecular weights less than a desired
cut-off range. The fibers divide the cartridge into the
extra-fiber space (EFS), typically about 50 ml. in
volume, and the volume within the fiber lumina. The
lo fibers and shell form a hollow fiber cartridge. Minimal
bulk flow of liquid occurs within the extra-fiber space,
which is also referred to as the extra-capillary or
shell-side space.
If desired, prior to use, selected target
antigens and/or the growth promoting substances may be
bound to the fibers. The fibers must be selected so
that the target antigen and/or growth promoting substance
can bind thereto. Binding may be irreversible and may be
accomplished by the use of cross-linking agents or other
methods known to those of skill in the ~rt or binding may
be reversible, such as by absorption of the antigen or
substance to the fiber.
The hollow fiber bioreactor is a component of a
hollow fiber cell culture system. A typical hollow
fiber cell culture system, such as the CELL~AX 100~
hollow fiber cell culture system (Cellco Advanced
Bioreactors, Inc., Kensington, MD.), which is described
in Knazek et al. U.S. Patent Application No. 07/238,445,
supra., which disclosure is herein incorporated in its
entirety by reference thereto, consists of a standard
glass media bottle, which serves as the reservoir,
stainless steel/Ryton gear pump, an autoclavable hollow
~, .
.
W09l/043l7 PCT/~S90/05051
20666~
fiber bioreactor, which consists of the fibers and shell
casing in which cells are cultured, and medical grade
silicone rubber tubing, or other connecting means, which
serves as a qas exchanger to maintain the appropriate pH
and PO2 Of the culture medium. All components are secured
to a stainless steel tray of sufficiently small
dimensions to enable four such systems to fit within a
standard tissue culture incubator. The pump speed and
automatic reverse of flow direction are determined by an
electronic control unit which is placed outside of the
incubator and is connected to the pump motor via a flat
ribbon cable which passes through the gasket of the
incubator door. The pump motor is magnetically coupled
to the pump and is lifted from the system prior to steam
autoclaving. Tissue culture medium, which may also
contain target antigens and/or growth promoting
substances, such as IL-2, is drawn from the reservoir,
pumped through the lumina of the hollow fibers, and then
passed through the gas exchange tubing in which it is re-
oxygenated and its pH readjusted prior to returning to
the reservoir for subsequent recirculation. The flow
rate can be increased as the number of cells increases
with time. Typically the initial flow rate of the medium
is adjusted to about 40 ml./min. and is increased over
time to about 300 ml./min. The direction of perfusion of
the medium through the hollow fiber lumina is
periodically and automatically reversed, typically every
ten minutes, in order to provide a more uniform
distribution of nutrient supply, waste dilution, and
cells within the space surrounding the hollow fibers.
The entire system is sterilized prior to cell
inoculation and is designed for operation in a standard
~ . :
W091/0~3l~ ~ PCT/US90/05051 ^
20~4~ -
26
air -Co2 tissue culture incubator. Upon inoculation, the
cells settle onto the surface of the hollow fibers,
through which nutrients pass to feed the cells and into
which metabolic waste products pass and are diluted into
the large volume of the recirculating perfusate. The
selected fiber should be semi-permeable to permit the
passage of nutrients into the EFS and should be of a
material on which or in the vicinity of which the cells
are able to grow. The fibers are made-of material, such
as DEAE-cellulose or polypropylene, that is porous and
suitable for the growth of mammalian cells. For example,
cellulosic hollow fibers 12 inches in length, whose walls
nominally restrict diffusion to substances having a
molecular weight less than 3000 Daltons are suitable for
use in practicing this invention. In some embodiments of
this invention the tumor cells or target antigens are
bound to the fibers, either reversibly or irreversibly,
so that the lymphoid cells are constantly grown in the
presence of the such antigens that are recognized by the
immunoreactive cells. In other embodiments, the growth
promoting substance is bound to the fibers. Binding may
be reversible, such as by adsorption, or irreversible if
a cross-linking agent is used to permanently affix the
antigen or growth promoting substance to the fiber.
Alternatively, the growth promoting substance and/or
antigen may also be included in the perfusate and/or in
the EFS.
A suspension of cells is inoculated into the
extra-fiber space (EFS) of a hollow fiber bioreactor
typically through one of two side ports. The lumina are
perfused with cell culture medium, which contains
diffusible nutrients and may also contain the growth
WO~l/04317 PCT/US90/OS051
2~6G4~
j
27
promoting substance(s), which specifically expand the
subpopulation(s) of lymphocytes that can be used in
adoptive immunotherapy and may also contain any target
antigens.
Selection of the growth promoting substance or
substances is a function of the subpopulation of lymphoid
cells that is desired. Such selection is within the
level of skill in the art and is dictated by the specific
subpopulation of lymphoid cells that is desired. For
example, if TIL cells are being grown, then IL-2, which
functions in some manner in directing the growth, and
possibility the development, of TIL cells from tumor
tissues, must be included in the culture medium. Growth
promoting substances, such as lymphokines, including IL-
2, are available to those of skill in the art. Many,
such as IL-2, have been cloned and expressed in
biologically active form. Recombinantly produced growth
promoting substances, such as recombinantly produced
interleukins, are suitable for use in this invention.
Means to clone DNA encoding such proteins and means to
produce biologically active proteins from such cloned DNA
are within the skill in the art. For example,
interleukins 1 through 6 have been cloned. Various
growth promoting substances and combinations thereof may
be used to expand desired subpopulations of lymphoid
cells.
In a typical embodiment of this invention the
cells are cultured in the presence of at least one growth
promoting substance that specifically expands at least
one immunoreactive subpopulation of lymphoid cells and in
any medium that is known to those of skill in the art to
be suitable for the growth of mammalian cells in vitro.
, . . . . .. - . .
'`~ ' ' ' ' ' ' . ' . ,` :. . ! . - ,
. . ' . ' .
',~ ' ,
. '~ , '
:
WO 91/~4317 ~ PCr/US90/05051 -.
~S6~
28
It is well-within the level of skill in the art to select
an appropriate culture medium. The growth promoting
substance that is contemplated for use in this invention
is selected for its ability to expand in vitro
subpopulations of lymphoid cells that specifically
recogni2e and mediate destruction of a patient's
afflicted cells, such as cancerous or virally infected
cells. In other embodiments of this invention the cells
are grown in the presence of at least one target antigen
lo in addition to at least one growth promoting substance.
In another embodiment of this invention the lymphoid
cells are isolated, modified by genetic engineering
methods, injected into the extra-fiber space of a hollow
fiber bioreactor and cultured under conditions that are
designed to expand the genetically modified subpopulation
thereof.
After inoculation, the culture medium is
continuously perfused through the hollow fiber bioreactor
by means of externally applied pressure, such as a pump.
A glass reservoir, the hollow fiber bioreactor, and
pumping means are connected by tubing, typically silicone
rubber, which simultaneously serves as a membrane gas
exchanger to replenish oxygen and, if the medium is
buffered with bicarbonate, to maintain the pH via CO2
transport into the perfusion medium. Medium that is
buffered with systems other than bicarbonate do not
necessarily require C02 in the incubator.
The in vitro expanded lymphoid cells grow and
divide and pile up upon each other until they fill up a
substantial portion of the extra-fiber space to form
nearly solid masses of cells. As the cells grow and
divide, the perfusate can be replaced and the EFS can be
:
- . . ~
'' ' . ,
.
WQ91/n43l7 PCT/US90/05051
2~6~4~
29
periodically drained.
The perfusing medium can be replaced by
replacing the reservoir ~ottle. After growth of the
cells has been established, it has been discovered that
it is not necessary to include human serum albumin in the
perfusion medium. The EFS can be drained periodically to
harvest the supernatant and/or to sample the Gells. When
the EFS is drained, any cells that have been drained can
be recovered and re-inoculated into the hollow fiber
bioreactor suspended in complete AIM-V or other serum
protein-containing medium.
The in vitro expanded lymphoid cells are
cultured in the hollow fiber bioreactor until the EFS
contains at least about lO10 to 1011 cell. The cells can
be harvested by shaking the bioreactor and pouring the
cells along with the EFS medium into a side port bottle.
In addition, the EFS cell supernatant, which is rich in
non-or poorly-diffusible cellular products, including
useful biologically active agents, such as IL-2 r~ceptors
and other growth promoting substances that are useful for
expanding desired subpopulations of lymphoid cells in
vitro, can be recovered for further processing in order
to purify-or partially purify said biologically active
agents. The cells can be spun down using a centrifuge or
by any other means known to those of skill in the art to
yield a cell pellet and the EFS cell supernatant, which
is enriched in biologically active molecules, such as IL-
2 receptors and growth promoting substances.
Aftex harvesting the cells, the growth of cells
remaining in the bioreactor can be re-instituted by
resuming perfusion of the culture medium, which contains
the growth promoting substance(s). The cells will then
WO 91~04317 PCI~VS90~05051--
2~6s64~
continue to divide and can be harvested. This step can
be repeated a plurality of times.
After harvest and pelleting of the cells, the
EFS cell supernatant can be dialyzed against fresh tissue
5 culture medium in order to produce EFS conditioned
medium, which can then be further processed or used
directly or diluted to stimulate the growth of cells,
such as TIL cells.
In one typical procedure using the methods of
10 this invention a tumor is excised from a patient
suffering from malignant melanoma, minced into small
piece~ and suspended in RPMI 1640 tissue culture medium
(Biofluids, Rockville, MD.) that contains 10 mg.
collagenase/ml., 1 mg. deoxyribonuclease/ml., and 2.5
15 units of hyaluronidase/ml. (Sigma). All operations in
which the cells are manipulated are performed using
sterile techniques in a laminar flow hood. The
suspension is stirred overnight, filtered through Nitex
Mesh and resuspended in LAK supernatant supplemented
20 complete AIM-V medium, which contains IL-2, cultured, and
is then plated onto culture plates at a density of about
5 x 105 cells/ml~ After about one week or when the cell
densities of TIL cells reached about 1-2 x 106 cells/ml.,
the cells are replated in fresh medium at densities of
25 about 5 x 105 cells/ml and after further growth the cells,
which are adjusted to a density of about 2 x 106
cells/ml., approximately 50 ml. are inoculated into a
hollow fiber bioreactor. Prior to use the hollow fiber
culture System is steam autoclaved, cOntlnuOu.lyperfu5ed with s
30 1.3 liters of recirculating deionized water, drained,
flushed, and perfused with complete AIM-V medium in both
the EFS and perfusate pathways.
~: ' ' - '-, ., ' ~: '
,.
' . . , :', , . ' ` " ` :'
.
WO9l/W317 PCTtVS90/05051
2~6~4~
31
The inoculated bioreactor is transferred to a
standard incubator where it is perfused with complete
AIM-V. Incubation continues for at least about 10 days
up to about 30 days until the number of cells in the
bioreactor reaches a therapeutically ef~ective amount of
cells, about lOl~ to lO~l cells. During the incubation
period the reservoir containing the perfusing medium is
changed in order to maintain a sufficiently high
concentration of glucose and other diffusible nutrients
in the EFS. If desired, the EFS can be periodically
drained during the incubation period in order to sample
the cells or to collect the EFS cell supernatant.
When the d~sired cell density is reached the
cells are harvested by vigorously shaking the hollow
fiber bioreactor and draining the EFS. The cells are
pelleted and the EFS cell supernatant collected for
further processing. The harvested cells possess the
morphological and biological characteristics of TIL
cells.
The EFS cell supernatant can be dialyzed against
fresh tissue culture medium to produce EFS conditioned
medium and added to newly seeded cells or order to
stimulate the growth of the newly seeded cells. The EFS
can also be added to the hollow fiber bioreactor, after
harvesting the cells, when growth is re-instituted in
order to stimulate the growth of the cells remaining
therein.
The following examples are included for
illustrative purposes only and are not intended to limit
the scope of the invention.
WO91~04317 ~ PCT/US90/0505~-
2 ~
32
EXAMP E_1
Prior to use eight hollow fiber culture systems
were steam autoclaved at 121 C for 20 minutes and then
perfused with 1.3 liters of deionized water overnight at
37 C. The perfusion pathway and extra-fiber space of
each system were drained and flushed with complete AIM-V
medium before replacing the reservoir bottle with a fresh
warmed 1 liter bottle of complete medium. All operations
were performed in a sterile laminar flow hood.
Tumors were excised from 8 patients (listed in column
1 of Table 1) who had metastatic melanoma. The tumors
were sterilely transported from the~ surgical suite to a
laminar flow hood, where the cells from each patient were
processed separately. The tumorous tissue from each
patient was minced into 1 mm. to 2 mm. pieces and
suspended in approximately 200 to 500 ml. of RPMI 1640
tissue culture medium (Biofluids, Rockville, MD.) that
contained 10 mg. collagenase/ml., 1 mg.
deoxyribonuclease/ml., and 2.5 units of hyaluronidase/ml.
(Sigma). Each suspension was gently stirred overnight at
room temperature and then ~iltered through sterile Nitex
mesh, washed twice, and resuspended in either LAX
supernatant supplemented complete AIM-V medium or in a
medium that consisted of a 50-50 mixture of complete AIM-
V and RPMI having 10% heat-inactivated human serum, and
20~ LAK supernatant and IL-2 (1000 units/ml.).
Each suspension of cells was plated into 6 well
culture plates (Costar Corp., Cambridge, MA.) at
densities of about 5 x 105 cells/ml. After about one week
or when the cell densities of TIL cells reached about 1-
2 x 106 cells/ml., the cells were replated in fresh medium
at densities of 5 x 105 ~IL/ml. After further growth the
,
.
. , . ~ .
:, -
. .
WoglJ043l7 PCT/US90/05051
2066~4~
33
cells were diluted into 0.5 to 1.5 liters of complete
AIM-V medium to a density of 5 x 105 cells/ml. The entire
volume was then injected into a 1.5 liter polyolefin
culture bag ~PL-732 plastic, Fenwal Laboratories,
Deerfield, IL.) and incubated in a flat position on a
perforated shelf without agitation at 37 C for 3-4 days
in a humidified 5% CO~air incubator. As the cells
multipliad, they were periodically diluted 1:3-4 in
complete AIM-V medium in new bags. Upon inoculation into
the bags the cells, after an initial lag phase, entered
a period of exponential growth.
During the exponential growth phase of the cells
in the bags, a small volume of the cell suspension was
withdrawn 17 to 33 days after excision of the tumor,
centrifuged at 400 x g. for 10 minutes at room
temperature. The pelleted cells were resuspended in
complete AIM-V medium in the 100 ml. glass side port
bottles The cells from each of the 8 patients with
metastatic melanoma were then inoculated into the hollow
fiber culture system. The cells that continued to be
cultured in the bags served as controls to which the
cells cultured according to the methods of this invention
were compared.
Cells that had been derived from each patient
(B, M, S, K, J, H, W, and G) were each inoculated into a
hollow fiber cartridge and into a bag. Inocula ranged
from 0.35 to 4.3 x 108 cells. The cells were injected
into the extra-fiber space of the hollow fiber bioreactor
through the side-ports that are each connected to the 100
ml glass bottle in which the TIL cells were suspended.
The bottles were gently pressurized with a 20 ml. plastic
syringe to force the cell suspension into the extra-fiber
W O 91/0~317 PC~r/US90/OSQ51--
2066~4~
34
space. The cells settled on or near the fibers from
which they received nutrient support by diffusion flow
from the perfusate Simultaneously, low molecular weight
metabolites diffused away ~rom the cells and throuqh the
fiber where they were diluted by the perfusate.
Two of the 8 cultures, S and J (see Table I) had
stopped growing in both the hollow fiber cultures and in
the control bags. Because the hollow fiber cultures and
bag cultures were maintained in separate incubators, it
would appear that the failure to grow was rela~ed to the
cells and not to the method by which the cells were being
cultured. The remaining 6 TIL cultures grew well in
both the hollow fiber culture system and the bags. Fig.
1 depicts a growth curve for the cells that were derived
from patient W.
The system was operated in a 37 C, humidified,
5%, COJair incubator. During the course of the process,
the reservoir bottle, which contained the perfusion
medium, complete AIM-V, was periodically replaced when
its glucose concentration decreased to the range of 100-
150 mg./dl. Other nutrients, as well as glucose, are
replenished by replacing the medium containing bottle
with a pre-warmed bottle containing fresh medium. The
number of times that the bottles were replaced is
indicated in column 8 of Table I. Thus, the amount of
medium used for each culture depended upon-the amount of
glucose consumed thereby and ranged from 8 to 27 liters
per hollow fiber culture system, which is equivalent to
about 2.4 to 6.6 liters of complete AIM-V per 101~ TIL
harvested (See Table I, infra.). The time required to
replace a reservoir bottle was less than 5 minutes.
It was discovered that once the cell culture was
.
.
WO91/04317 PCT/US90/05051
20~6~
initiated in the hollow fiber bioreactox, it was no
longer necessary to add human serum albumin to the
perfusion medium. The cells grew substantially as well
in the absence of human serum albumin in the perfusate as
in the presence thereof.
The extra fiber space was drained periodically
either to harvest the culture medium, which is enriched
in non-diffusible products that are secreted by TIL
cells, or to sample the cells in order to evaluate them
for number and functional characteristics. The number of
times the EFS was drained for each culture is indicated
in column 9 of Table I. Medium in the EFS was replaced
on the average, except for culture G, every 2.2 days.
The EFS of culture G was replaced 2 and 0 tim~s during
the respective 14 and 21 day culture periods.
The extra fiber space was drained in a laminar
flow hood by gently draining the extra-fiber fluid into
one of the two loading side port bottles. The medium
that was drained from the EFS was centrifuged at room
temperature for lO minutes at 200 x g, the cell pellet
was resuspended in fresh complete AIM-V medium and re-
inoculated into the EFS of the hollow fiber bioreactor so
that any TIL that had been flushed out would not be
discarded. The EFS cell supernatant was saved for
further processing. Draining, pelleting, resuspending,
and re-inoculation of the bioreactor took 20-30 minutes.
As the number cells in the bioreactor increased
over time the flow rate of the perfusate was increased
from 40 ml to 300 ml./minute. The direction of perfusion
was reversed every ten minutes. Incubation was continued
for 14 to 32 days (see Table I) at which time the cells
were ready to be harvested.
wos1/04317 PCT/US90/05051
2~666~ 36
EXAMPLE 2
After 14 to 32 days the entire hollow fiber
culture system was removed from the incubator an placed
in a laminar flow hood. The electronic control unit was
then reconnected to the pump motor and perfusion
continued at a rate of about 40 ml./minute in order to
prevent the cultured cells from becoming anoxic during
the harvest procedure. Approximately l/3 of the extra-
fiber medium was drained by gravity into a loading side
port bottle. The hollow fiber reactor was then shaken
vigorously in order to detach any cells from the fibers.
The remaining medium and cells in suspension were drained
into the side-port bottle. The procedure was then
repeated three times by adding 35 ml~ of fresh complete
medium into the side-port and injecting it into the
extra-fiber space prior to shaking the bioreactor. The
last two washes were accomplished by shaking the
cartridge with a hand held vibrator (Oster Corp.,
Milwaukee, WI.) for l minute to remove the more firmly
attached cells. The cells from the hollow fiber
cartridge were contained in a final volume of about 155
to 250 ml.; whereas, the equivalent number of cells grown
the bags are contained in at least about 10-20 gas
permeable bags or about 15-30 liters of medium.
After harvesting, the cells from each
bioreactor were centrifuged to form a final pellet, which
is ready for subsequent use. The entire harvesting
procedure took about 30 minutes and about 95% of the
cells in the bioreactor were recovered. Each hollow
fiber bioreactor yielded from l.5-5.4 x l010 cells.
The data for the cells from each of the 8
patients is summarized in Table I. The number of cells
.
. . .
:, ' . .
.
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wosl/o43l7 PCT/US90/05051
`. 20~4~
37
harvested from each hollow fiber culture system is
indicated in column 5.
EXAMPLE 3
1.5 x 101 TIL from patient G were harvested from
the bioreactor on day 30, 15 days after the bioreactor
was inoculated. Perfusion of the bioreactor with
complete AIM-V was re-instituted and the residual cells
within the EFS continued to divide and grow and yielded
an additional 1.5 x 101 cells on day 21 (see Table I and
Fig. 2). Perfusion was again re-instituted and the
residual cells were once again permitted to expand for a
third harvest of 2.1 x 101 cells on day 73.
After the final harvest, the entire CELLMAX~
hollow fiber culture system was steam-autoclaved at 121
C for 20 minutes. The system was then flushed with
deionized water and the silicone rubber tubing and hollow
fiber bioreactor were discarded. Cleaning, reassembly,
and re-autoclaving with fresh tubing and bioreactor in
place was performed before the system was reused for a
different patient.
EXAMPLE 4
The in vitro functional characteristics of cell
suspensions harvested or sampled from the bioreactors
were compared to the corresponding suspensions harvested
or sampled from the bags.
Cells were assayed periodically and after
harvesting for viability by diluting a sample in trypan
blue/normal saline solution (SIGMA) to a final
concentration of 0.04% gm./dl. Viability was estimated
by dye exclusion as observed under microscopic
examination. The viability of the cells, which is
presented in Table I varied from patient to patient. The
..
:
W091/0~317 PCT/~S90/05051 -
2 ~ 6 ~ 38
average viability of the cells grown in the hollow fiber
culture system was greater than 90%.
During the course of culturing, aliquots of
perfusate were assayed to determine the glucose and
lactate concentrations (Yellow Springs Instrument, CO.,
Yellow Springs, OH.), which permitted their rates of
consumption and production, respectively, to be measured.
See Figs. 1 and 2. The rates of glucose consumption by
TIL from different patients varied between 0.45 to 2.2
gms. per 101 TIL/24 hours (Table I, col. 7). The rates
of lactic acid production approximated the rates of
glucose consumption~ Such rates exhibited logarithmic
increases over time, doubling every 1.5-3.6 days. These
double times were commensurate with the doubling times,
1.5 to 3.2 days, of the cells grown in the bags.
Cytotoxicity of the cells was assessed by a
chromium release assay tested against targets that
consisted of autologous tumor cells, allogeneic tumor
cells, the NK-sensitive, K562 erythroleukemia cell line,
and the N~-resistant Daudi B-cell l~phoma line (see,
e.a., Topalian et al. (1987) J. Immunol. Meth. 102:
127). The results of the cytoxicity studies set forth in
Table II, which presents the results of measurements of
the cytolytic capacities of three dilutions of TIL taken
from simultaneous aliquots of bag and hollow fiber
cultures.
The percentage of specific lysis of R562 cells,
Daudi cells, autologous tumor cells, and allogeneic tumor
cells by various samples of the TIL cells from patients
M,K, K, W, and G taken at different times during the
growth period was measured by a chromium release assay.
The percentage specific lysis by LAK cells, which possess
.~ : ' '.'', ~
. . . .
,
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WO91/04317 PCT/US90/05051
- 2~66~
39
lytic capacity for all cell lines as well as autologous
and allogeneic tumor cells was also measured.
Because the K562 cell line is NK-sensitive it
serves to assess the activity of NK cells in the n vitro
expanded lymphoid cells that were derived from each
patient. NKHI~ cells, are responsible for non-specific
killer activity. The Daudi cell line, which is NK-
resistant but LAK sensitive, indicates the relative
amount of LAK activity present in a given sample.
Approximately 1O8 target cells were labelled with
400 ~Ci of sodium 5lCrO~ (New England Nuclear, Boston, MA.) ~-
in a 0.7 ml. volume for 1 hour, washed three times,
incubated for an additional 30 minutes at 37 C and washed
twice before use. Serial dilutions of the sampled
effector cells were plated with 5 x 108 target cells in
~riplicate at ratios of effector:target cell of 80:1,
20:1, and 5:1 in a total of 150 ~1 of culture medium in
96 well round bottom microtiter plates (Costar Corp.) and
incubated for 4 hours. Supernatants were then harvested
with the Skatron-Titertek system (Skatron, Lier, No-rway)
and counted in a gamma counter (LXB Instruments,
Gaithersburg, MD.). The amount of radioactivity that is
released by spontaneous target lysis was determined by
incubation of tumor target cells in the absence of
effector cells. The maximal amount of radioactivity
released, which represents maximal cell lysis, was
measured by incubating the target with 2% SDS.
Percentage of specific lysis, thus, equals:
(ex~erimental release - s~ontaneous release) x lOO
maximal release -spontaneous release
Although the experiment was somewhat limited in
scope, it appears from the data presented in Table II
.. . ~ .
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,
WO91/~317 5 PCT/ US90/05051--
2~664~
that the relative activities of the different cell types
in a TIL cell preparation that were expanded in the
hollow fiber bioreactor varied from patient to patient
and also over time in a single patient. See e.a., the
data obtained for the W-TILs set forth in Table II in
which the first set of measurements were obtained at day
7 after inoculation into the hollow fiber bioreactor and
the second set were obtained at day 28. If, however, the
data for each sample taken from either the bags or HF for
a single patient's cells taken on the same day are
compared, certain consistent differences between the
cells cultured in the HF and bags become apparent. NK
activity and LAK cell activity appear to be lower for the
cell populations grown in the ~F than for the cells grown
in the bags. Further, these differences do not seem to
correlate with a concomitant and equivalent decrease in
TIL activit~. Thus, the cells grown by the methods of
this invention appear to contain a lower percentage non-
TIL-associated activity.
The phenotypes of the sampled and harvested
cells from the bags and hollow fiber reactors were also
compared. The results of these experiments for patients
K and G are summarized in Table III.
Sampled or harvested cells were washed with cold
staining necium (Hank's buffered saline solution without
phenol red that contains 5% heat-inactivated fetal calf
serum and 0.02% sodium azide) and resuspended in medium
at concentrations of about 1 x 1O6 to 1 x 107 cells~ml.
Undiluted fluorescein-conjugated monoclonal antibodies
against human mononuclear cell antigens (Becton-
Dickinson, Mountain View, CA.) were added to 100 ~1
volumes of cell suspension at a concentration of 5%
.:
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. ' '~. , .
WO 91/04317 PCl`tUS90tO50~1
'.. , ,. ~S '
2b66SA~
41
(v/v). Monoclonal antibodies were used to ascertain what
surface antigens are present on the cell surface and to,
thus, functionally characterize the cells. The
antibodies tested were anti-Leu-4, which reacts against
T cells, anti-Leu-3a, which reacts against T
helper/inducer cells, anti-Leu-2b, which reacts against
T cytotoxic/suppressor cells, anti-Leu-5b, which reacts
against E rosette receptor-bearing cells, anti-Leu-7,
which reacts against NK and some T cells, anti-Leu-lla,
which reacts against NK cells and neutrophils, anti-Leu-
14 and anti-Leu-16, which react against B cells, anti-
HLA-DR, which reacts against activated T cells, B cells,
and monocytes/macrophages, anti-IL-2 receptor, which
reacts against activated T cells, and anti-Leu-M3, which
reacts against monocytes/macrophages. As negative
controls the antibodies used included anti-Thy-1.2, which
reacts against murine T cells, and phycoerthrosin. After
staining for 30-60 minutes at 4 C, the cells were washed
with staining medium, fixed with 1% paraformaldehyde ,
washed again, and resuspended in staining medium. Cells
that were so-labeled were stored at 4 C for 1-7 days.
Fluorescence analysis was performed using a FACS 440
microfluorometer that had been interfaced with a Consort
40 computer (Becton-Dickinson).
The results of these experiments indicate that
the surface antigen profile of the cells grown by the
method of this invention and those grown in the bags are
substantially identical. Surface antigens present on
the surface of cells grown in bags were also present on
those grown by the methods of this invention and the
surface antigens that were substantially absent on the
cells grown in the bags were absent from the cells grown
, ~ ' ,` " ,:
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W091/043l7 ~ PCT/US90/OS051--
%~3666~
42
in the bags were also substantially absent from the
surface of cells that were grown by the methods of this
invention.
TABLE ~
TIL HF* days Inoc- Harv- Viab- Gluc- #res- #EFS Tot.l Med./
inoc in ulum est x ility ose vr. chnge med. lolO
days cult. xlO-a 10 10 % gm/d. chnge harv.
B 33 210.5 4.5 85 ~.6 12 10 11.3 2.5
M 31 320.35 4.1 97 3.5 25 14 27 6.6 ~ -
S 1.3 ---
K 58 2910.0 26ml. 1.9 12 10 13
0.4 ---
H 24 26 1.8 2.8 91 1.7 9 12 10 3.6
W 24 22 4.3 S.4 93 2.4 12 7 13 2.4
G 17 14 1.0 1.5 87 2.9 7 2 8 5.0
G 31 21 Resi- 1.5 89 3.3 8 O 8.8 5.8
dual
* HF = hollow fiber culture system.
TIL = patient from which tumor was resected for
production of TILs; the cells from patients S
and J failed to grow in either the bags or HF.
HF inoc. days = number of days after the tumor was
excised from patient that cells inoculated into
HF device.
Days in cult. = days in culture in HF before harvest.
Inoculum x 10 8 = number of cells inoculated.
Viability = percentage viable at harvest.
Glucose gm/d. = glucose consumption rate by cells at
harvest.
# resvr. chnge = number of times medium in reservoir
was changed during course of HF culture.
# EFS chnge= number of time the extra fiber space
changed during course of HF culture.
Tot. 1 med. = total liters of culture medium used by
HF cultur~.
Med./101 harv. = liters of culture medium used per
101 cells harvested.
.......... ..
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,
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~9l/W3l7 PCT/~IS90/0505~
20S6~4~
-43-
TABLE II
T~et o~l Ta~ o~l T~r~ cell Tumor cell
Effector: ~562 Daudi lysis: Au- lysis~
T~ c~l tolocous Allogeneic
}?atio80:120:1 5:1 80:120:1 5:1 80:120:1 5:1 80:120:1 5:1
~ffector
cell:
LAK 44.4 lZ.9 6.928.4 9.1 5.7
M-TIL (HF) 6.6 5.3 .8 2.5 1.1 6.1
K'lIL (}:~y) 58.7 33,n 17.5 19.1 ll.S 3.7
K-TIL (HF) 21.6 11.2 7.4 4.8 5.0 4.5
LAK 65.2 23.0 7.343.6 14-1 8-4 17.2 8.9 4.3 4 6 1.13 6
H ~ L (~g) 7.4 5.8 .8 2.6 5.6 6.6 50.2 57.1 33.7 6 6 .1 ~7 3
H-TIL (HF) 2-3 9 7 -.8 2.8 2.8 49.9 42.9 39.6 .8 5 0 ~ .6
K-TIL (HF) 4.2 1.3 .8 1.8-2.1 -3.1 3.6 4 9 .1
LAK 81.0 44.0 ~8.038.8 18.6 6.0 5 1
W--IIL (~ag) 30.5 24.5 22.7 64.0 53.2 29-2 30 6 14 9 3 2 9 1 6.6 9
W-TIL (H~) 22.5 11.2 12.5 55.1 45.0 19.8 19.4 11 9 4.~ 2.0 4 1 5.8
LAK ~6.8 75.6 27.469.8 45.8 17.2 34.3 13 6 4.1 26 0 4 5
L (Bag) 15.0 6.9 .972.2 53.1 21.~ 15.4 7.9 2.8 . 11.7
W-TIL (HF) 8.6 2.5 .3 55.9 41.3 ~7.0 11.5 0 2.0 3 9 5 4 5 0
LAK 74.3 56.1 49.959.7 45.9 40.064.9 41.3 18.1 37.1 43.1 17.7
t;'llL (~ 3.5 J.55.221.3 4.9 2.651.1 32.0 22.33 9 2 92 5
G-TIL (HF) 11.1 10.1 6.1 3.9 9.6 ?.650-4 40.~ 38.2 3 43 2 3 6
TABLE III
Phenotypic Pr~
_X-HF X-8aq _ G-HF G-8aq
Leu 2 95.9 90.4 73.8 83.1
3 1.1 2.0 13.1 14.3
4 94.1 94.7 85.3 91.4
7 4.2 11.7 2.2 4.6
11 .6 .8 2.2 1.4
.3 g.s 3.7 0.4
16 3.7 4.0 0.1 0.8
1~ 8.3 20.4 6.5 7.1
m3 - .4 .3 .6 1.1
HLA-DR 80.7 85.6 89.3 91.1
TAC 2.4 4.7 27.3 30.4
SllBSTlT15Tr SHE~
, . , . , .
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WO9l/o43l7 PCT/US90/05051~
~ ` 2`~6S4~
!
EXAMPLE 5
As in Example 1, a hollow fiber culture system
is steam autoclaved at 121 c for 20 minutes and then
perfused with 1.3 liters of deionized water overnight at
37 C. The perfusion pathway and extra-fiber space of
each system are drained and flushed with complete AIM-V
medium before replacing the reservoir bottle with a fresh
warmed 1 liter bottle of complete medium. All operations
are performed in a sterile laminar flow hood.
: 10 A tumor is excised from a cancer patient and is
sterilely transported from the surgical suite to a
laminar flow hood, where the cells from the tumor are
minced into 1 mm. to 2 mm. pieces and suspended in
approximately 200 to 500 ml. of RPMI 1640 tissue culture
medium (Biofluids, Rockville, MD.) that contains 10 mg.
`~ collagenase/ml., 1 mg. deoxyribonuclease/ml.j and 2.5
units of hyaluronidase/ml. (Sigma). The suspension is
gently stirred overnight at room temperature and then
filtered through sterile Nitex mesh, washed twice, and
resuspended in LAK supernatant supplemented complete AIM-
. ~ medium as described in Example 1.
A portion of the suspension, at least about 50
ml., is irradiated with X-rays or other suitable means
- for a time sufficient to inactivate the tumor cells. The
irradiated suspension in a cryoprotective media, well
known to those knowledgable in the field, is stored in
liquid nitrogen. Shortly before use, the suspension is
warmed to about 37 C.
The remaining portion of suspended cells is
plated into a 6 well culture plates (Costar Corp.,
Cambridge, MA.) at densities of about 5 x 105 cells/ml and
~ after about one week or when the cell densities of TIL
.'':
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.: . . ,
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:,: . .... . .
WO 91/0431~ PCr/US90/05051
20~6~
cells reach about 1-2 x 106 cells/ml., the cells are
replated in fresh medium at densities of about 5 x 105
cell/ml and cultured until at least about 107 to 10~
cells are produced. The cells are pelleted and
resuspended in the suspension that contains the
irradiated tumor cells and the volume is adjusted to
about 100 ml. with complete AIM V medium. The mixture is
inoculated as described in Example 1 into the EFS of the
hollow fiber culture system. The cells settle on or near
the fibers.
The culture system is operated as described in
Example 1. Initially the perfusion medium is complete
AIM-V, which is periodically replaced when its glucose
concentration decreased to about 1 to 1.5 grams/liter.
Once the cell culture is established in the hollow fiber
bioreactor, it is no longer necessary to add human serum
albumin to the perfusion medium.
As the number cells in the bioreactor increased
over time the flow rate of the perfusate is increased
from 40 ml to 300 ml./minute. The direction of perfusion
is reversed every ten minutes. Incubation is continued
for 14 to 32 days at which time the cells are harvested
as described in Example 1.
EXAMPLE 6
As in Example 1, a hollow fiber culture system
is steam autoclaved at 121 C for 20 minutes and then
perfused with 1.3 liters of deionized water overnight at
37 C. The perfusion pathway and extra-fiber space of
each system are drained and flushed with complete AIM-V
medium before replacing the reservoir bottle with a fresh
warmed 1 liter bottle of complete medium. All operations
are performed in a sterile laminar flow hood.
; . , ~ ..... : .,
.'~' ~ - .
~.
WO91/~317 PCT/VS90/05051 _
2066~
46
A tumor is from a cancer patient and is
sterilely transported from the surgical suite to a
laminar flow hood, where the cells from the tumor are
minced into 1 mm. to 2 mm. pieces and suspended in
S approximately 200 to 500 ml. of RPMI 1640 tissue culture
medium (Biofluids, Rockville, MD.) that contains 10 mg.
collagenase/ml., 1 mg. deoxyribonuclease/ml., and 2.5
units of hyaluronidase/ml. (Sigma). The suspension is
gently stirred overnight at room temperature and then
filtered through sterile Nitex mesh, washed twice, and
resuspended in LAK supernatant supplemented complete AIM-
V medium as described in Example 1.
A portion of the suspension, at least aboul 50
ml., is removed. The suspension mixed with microcarrier
beads to which said cells bind to form a slurry. The
slurry is irradiated with X-rays for a time sufficient to
inactivate the tumor cells. The slurry is then
introduced into the bioreactor and the microcarrier beads
are permitted to settle onto or near the fibers. -
Alternatively, the portion of the suspension of
tumor cells is irradiated with X-rays for a time
sufficient to inactivate the tumor cells' growth. The
suspension of tumor cells are then introduced into the
EFS of the HF bioreactor and are permitted to settle onto
or near the fibers. A non-toxic cross-linking agent
introduced into the EFS and the tumor cells are
irreversibly bound to the fibers. After cross-linking the
EFS is drained and perfused with complete AIM-V medium.
The remaining portion of suspended cells is
plated into a 6 well culture plates (Costar Corp.,
Cambridge, MA.) at densities of about 5 x 105 cells/ml and
after about one week or when the cell densities of TIL
', .
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W~91/0~317 PCT/US90/05051
2~6664~
47
cells reach about 1-2 x 1O6 cells/ml., the cells are
replated in fresh medium at densities of about 5 x 105
cell/ml and cultured until at least about 107 to 10~
cells are produced. The cells are pelleted and
resuspended in the suspension that contains the
irradiated tumor cells and the volume is adjusted to
about lOO ml. with complete AIM V medium. The mixture is
inoculated as described in Example 1 into the EFS of the
hollow fiber culture system. The cells settle on or near
the fibers.
The culture system is operated as described in
Example 1. Initially the perfusion medium is complete
AIM-V, which is periodically replaced when its glucose
concentration decreased to about 1 to 1.5 grams/liter.
Once the cell culture is established in the hollow fiber
bioreactor, it is no longer necessary to add human serum
albumin to the perfusion medium.
As the number cells in the bioreactor increased
over time the flow rate of the perfusate is increased
20 from 40 ml to 300 ml./minute. The direction of perfusion
is reversed every ten minutes. Incubation is continued
for 14 to 32 days at which time the cells are harvested
as described in Example 1.
EXAMPLE 7
As in Example 1, a hollow fiber culture system
is steam autoclaved at 121 C for 20 minutes and then
perfused with 1.3 liters of deionized water overnight at
37 C. The perfusion pathway and extra-fiber space of
each system are drained an~ flushed with complete AIM-Y
medium before replacing the reservoir bottle with a fresh
warmed 1 liter bottle of complete medium. All operations
are performed in a sterile laminar flow hood.
.. . .
. . .
.
... : , - ' ;
. ?
W091/04317 PCT/US90tO5051
206664~ 48
A tumor is from a cancer patient and is
sterilely transported from the surgical suite to a
laminar flow hood, where the cells from the tumor are
minced into l mm. to 2 mm. pieces and suspended in
approximately 200 to 5no ml. of RPMI 1640 tissue culture
medium (Biofluids, Rockville, MD.) that contains lO mg.
collagenase/ml., l mg. deoxyribonuclease/ml., and 2.5
units of hyaluronidase/ml. ~Sigma). The suspension is
gently stirred overnight at room temperature and then
filtered through sterile Nitex mesh, washed twice, and
resuspended in LAK supernatant supplemented complete AIM-
V medium to as described in Example l.
The suspension of cells is plated into a 6 well
culture plates ~Costar Corp., Cambridge, MA~ at
densities of about 5 x 105 cells/ml and after about one
week or when the cell densities of cells reach about 1-2
x 106 cells/ml., the cells are replated in fresh medium at
densities of about 5 x 105 cell/ml and cultured until at
least about 107 to 108 cells are produced. The cells are
pelleted and resuspended in complete AIM V medium that
additionally contains about lOOO units/ml. of IL-4. The
is inoculated as described in Example l into the EFS of
the hollow fiber culture system. The cells settle on or
near the fibers.
The culture system is operated as described in
Example l. Initially the perfusion medium is either
complete AIM-V or complete AIM-V that also contains lOOO
units/ml. of IL-4. The perfusion medium is periodically
replaced when its glucose concentration decreased to
about l to l.5 grams/liter. Once the cell culture is
established in the hollow fiber bioreactor, it is no
longer necessary to add human serum albumin to the
.~ ,..
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~09lt~317 PCT/US90/05051
49 206664;~ -
perfusion medium.
As the number cells in the bioreactor increased
over time the flow rate of the perfusate is increased
from 40 ml to 300 ml./minute. The direction of perfusion
is reversed every ten minutes. Incubation is continued
for 14 to 32 days at which time the cells are harvested
as described in Example 1.
EXAMPLE 8
After removing the cells, which have been
cultured as in Example 1, and the EFS medium from the
hollow fiber bioreactor, the EFS and cells were
centrifuged at 200 x g to pellet the cells. The EFS cell
supernatant was dialyzed against fresh complete AIM V
medium for 24 hours at 4 C to produce what is herein
called EFS conditioned medium. 104 of the harvested cells
w~re seeded in flasks and grown in duplicate in either 5
ml. of EFS conditioned medium at dilutions of EFS
conditioned medium with complete AIM V of 1:10 and 1:100
EFS or in 5 ml. of dialyzed complete AIM V. It was
found that the EFS conditioned medium stimulated growth
of the cells. The data is presented in Table IV.
TABLE IV
STIMULATION OF GROWTH OF TIL BY EFS CELL SUPERNATANT
DILUTIONS OF EFS
1:100 1:10
Growth in
EFS conditioned medium 20 12.7
NO. CELLS X 104 16.8 12.7
_
Growth in dialyzed
Medium 9.2 10.0
NO. CELLS x 10 4 9.0 13.2
~ .
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W091/04317 PCT/US90/OSOSl-~
6~
Since modifications will be apparent to those of
skill in the art, it is intended that this invention be
limited only by the scope of the appended clai~s.
,- .
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