Ex vivo activation of immune cells

Disclosed is a process of activating patient-derived mononuclear cells by exposing the cells in vitro to substances wo generate immunoreactive cells. The ex vivo activated cells are then reinfused into the patient to enhance the immune system to treat various forms of cancer, infectious diseases, autoimmune diseases or immune deficiency diseases.

BACKGROUND OF THE INVENTION 
This invention relates to immunotherapy. 
Adoptive immunotherapy as an approach to treating cancer has been evolving 
rapidly over the past two decades. Beginning in the 1980's, IL-2 and 
lymphokine-activated killer (LAK) cell-based immunotherapies were shown to 
induce tumor responses in patients with melanoma and renal cell carcinoma 
(Rosenberg et al., 1985, N. Eng. J. Med. 313:1485-1492; Rosenberg et al., 
1986, Important Advances in Oncology, DeVita et al., eds, Philadelphia, 
Lipincott, p. 55; Schoof et al., 1988, Cancer Res. 48:5007-5010; West et 
al., 1987, N. Engl. J. Med. 316:898-905). However, these therapies require 
systemic administration of high dose IL-2 which usually is accompanied by 
severe toxicity (Siegel et al., 1991, J. Clin. Oncol. 9:694-704; Margolin 
et al., 1989, J. Clin. Oncol. 7:486-498). More recently, tumor 
infiltrating lymphocytes (TIL) isolated from surgically removed tumors 
were utilized for adoptive immunotherapy following ex vivo activation and 
expansion (Rosenberg et al., 1986, Science 223:1318-1321; Kradin et al., 
1989, Lancet 1:577-580; Speiss et al., 1987, J. Natl. Cancer Instit. 
79:1067-1075; Rosenberg et al., 1988, N. Engl. J. Med. 319:1676-1688). 
Although treatment of patients with TIL has been shown to achieve somewhat 
higher tumor response rates relative to LAK therapy for melanoma 
(Rosenberg et al., 1986, Science 223:1318-1321; Kradin et al., 1989, 
Lancet 1:577-580; Spiess et al., 1987, J. Natl. Cancer Instit. 
79:1067-1075; Rosenberg et al., 1988, N. Engl. J. Med. 319:1676-1688), 
this type of therapy has not shown consistent outcomes in clinical trials 
of other tumors. Furthermore, the limited availability of sufficient 
numbers of lymphocytes from autologous tumor tissue and other problems 
associated with long term, high volume cell culture have also restricted 
the application of TIL therapy. 
SUMMARY OF THE INVENTION 
The invention provides a novel, safe, and cost-effective method of 
nonspecifically enhancing a cell-mediated immune response to specific 
antigens or foreign substances in the body, including cancer cells. The 
process involves removing a patient's mononuclear cells and exposing the 
cells in vitro to substances which enhance the immune function of the 
cells. The ex vivo activated (EVA) cells are then reinfused into the 
patient to enhance the patient's immune responses and to treat various 
forms of cancer, infectious diseases, autoimmune diseases, or immune 
deficiency diseases. 
The invention features a process of producing a population of 
immunoreactive cells by (a) contacting a sample of mononuclear cells 
derived from a patient, e.g., peripheral blood mononuclear cells (PBMC), 
with OKT3 at or below 37.degree. C. to produce an OKT3-derived culture 
supernatant (T3CS); (b) removing the T3CS from the sample of 
patient-derived mononuclear cells; (c) determining the concentration of 
OKT3 in the T3CS, and if required, supplementing the T3CS with additional 
OKT3 to achieve a concentration of at least 0.1 ng/ml; (d) providing a 
second sample of mononuclear cells derived from the patient; and, (e) 
contacting the second sample of cells with the previously-generated T3CS 
for a period of time sufficient to yield a population of immunoreactive 
cells. 
A sample of patient-derived mononuclear cells may contain T cells, B cells, 
monocytes and macrophages as well as other immune cells such as 
polymorphonuclear leukocytes, neutrophils, eosinophils, natural killer 
cells, and stem cells. Mononuclear cells may be derived from the 
peripheral blood of the patient, or other sites, e.g., a tumor or 
tumor-draining lymph node. 
T3CS is a conditioned medium containing a mixture of autologous cytokines 
together with OKT3. The autologous cytokines mixture preferably promotes 
the growth and differentiation of Thl-type T cells, rather than Th2-type T 
cells. The OKT3 which is preferably in solution phase catalyzes the 
polyclonal activation of T cells, while the cytokines act synergistically 
as co-stimulants to optimize the overall degree of activation. The 
presence of both OKT3 and cytokines prevents the generation of T cells 
that are anergic or apoptotic and overcomes signal transduction defects in 
mononuclear cells derived from patients with cancer or chronic infectious 
diseases. 
By the term "immunoreactive cells" is meant polyclonal T cells that exist 
in a primed state of activation. Primed cells are multifunctional, i.e., 
they possess an enhanced capacity to proliferate and produce cytokines 
upon further stimulation. The primed state of activation of the 
immunoreactive cells induced by culture in the OKT3-autologous cytokine 
mixture can be identified by measuring the stable biochemical changes, 
e.g., expression of growth, differentiation, and activation markers, which 
occur both on the cell surface and intracellularly. Immunoreactive cells 
of the invention have enhanced immunologic effector function, e.g., helper 
activity (CD4.sup.+ T cells) or cytotoxicity (CD8.sup.+ T cells), compared 
to unprocessed patient-derived mononuclear cells. 
Immunoreactive cells have a low spontaneous level of immune function 
following processing, but are highly sensitized to respond to low doses of 
second signals upon further culture, or in vivo. The immunoreactive cells 
of the invention therefore require further exposure to an immune 
stimulant, such as an antigen; target cell, e.g., a tumor cell or 
virus-infected cell; an inflammatory molecule; an adhesion molecule; an 
immune cell, e.g., an accessory cell; a cytokine; or any combination 
thereof, to achieve full immunologic effector function. The immunoreactive 
cells of the invention are multifunctional, polyclonally-activated T cells 
which have been generated independent of disease-specific antigens 
utilizing a mixture of nonspecific lymphocyte activators, i.e., autologous 
cytokines, and a mouse monoclonal antibody, i.e, OKT3, as synergistic 
stimulants. 
Suppressor cells in a population of patient-derived mononuclear cells may 
be inactivated by contacting the second sample with a suppressor cell 
inhibitory compound, e.g., cimetidine, indomethacin, cyclophosphamide, 
ranitidine, pepsid, or any combination thereof. Other histamine type-2 
receptor blockers may also be used alone or in combination with the 
compounds listed above. Cimetidine and indomethacin are preferably used 
together at concentrations of 5.times.10.sup.-5 M (.+-.2-fold) and 
0.8.times.10.sup.-8 M (.+-.2-fold), respectively. 
The concentration of OKT3 used in the process of the invention is an amount 
that promotes activation of the patient's cells, but leaves minimal 
surface-bound OKT3 on the activated cell product. Minimizing the amount of 
surface-bound OKT3 on the immunoreactive cells in turn minimizes human 
anti-mouse antigen (HAMA) immune responses and rapid clearance of the 
immunoreactive cells from the circulation of a patient undergoing therapy 
with the immunoreactive cells of the invention. The concentration of OKT3 
is preferably greater than 0.1 ng/ml but less than 25 ng/ml, more 
preferably 1-25 ng/ml, and most preferably 10-15 ng/ml. In addition to 
OKT3, any compound that binds to the T cell receptor or the T cell 
receptor-associated CD3 molecule on the cell surface may be used to 
stimulate the first sample of patient-derived mononuclear cells to produce 
T3CS. 
Culture of the first sample with OKT3 is carried out for a period of time 
sufficient to produce a mixture of nonspecific lymphocyte activators 
capable of promoting the OKT3-catalyzed activation and differentiation of 
the second sample of patient-derived mononuclear cells into a population 
of immunoreactive cells. The culture period may range from 1 to 7 days and 
is preferably 3 days. The length of culture of the first sample may be 
adjusted, e.g., prolonged, to achieve the desired concentration of a 
nonspecific lymphocyte activator, e.g., a cytokine, e.g., tumor necrosis 
factor-alpha (TNFA) or interleukin-2 (IL-2), in the T3CS. 
Culture of the second sample of patient-derived cells with T3CS is carried 
out for a period of time sufficient to produce a population of 
immunoreactive cells. As discussed above, the immunoreactive cells are in 
a primed state of activation, i.e., the cells are no longer in a resting 
state but require an additional stimulus, e.g., exposure to an antigen or 
other immune stimuli, to achieve a fully activated state characterized by 
enhanced immune function compared to unprocessed cells. Full activation of 
immunoreactive cells may be measured by expression of new cell surface 
markers, e.g., CD25, secretion of lymphokines, e.g., IFN.gamma., GM-CSF, 
or TNF.alpha., cellular proliferation, or cellular differentiation into 
effector cells, e.g., cytolytic T cells or helper T cells. 
Culture of patient-derived mononuclear cells with T3CS may be carried out 
for a period of 1 to 30 days, preferably 5 days. In the absence of 
antigen, the length of the culture period may be as short as 1-3 days; in 
the presence of antigen, the cells may be co-cultured for a period of 1 to 
30 days. The length of culture may be adjusted, e.g., prolonged, to 
achieve the desired level of activation of the immunoreactive cells. 
The invention also features a process of producing a population of 
immunoreactive cells by (a) providing a first sample of mononuclear cells 
derived from a patient; (b) determining the concentration of Fc-receptor 
positive accessory cells in the first sample, and if required, 
supplementing the first sample with a second sample of mononuclear cells 
derived from the same patient to achieve a concentration of 0.1-50% 
Fc-receptor positive accessory cells in the first sample; (c) contacting 
the first sample with OKT3 at or below 37.degree. C. to produce a T3cs; 
(d) removing the T3CS from the first sample; (e) determining the 
concentration of OKT3 in the T3CS, and if required, supplementing the T3CS 
with OKT3 to achieve a concentration of at least 0.1 ng/ml; (f) providing 
a third sample of mononuclear cells derived from the same patient; (g) 
contacting the third sample with T3CS for a period of time sufficient to 
activate the third sample in vitro to yield a population of immunoreactive 
cells. The Fc-receptor positive cells are preferably monocytes, but may be 
granulocytes or dendritic cells. The concentration of patient-derived 
monocytes is preferably 0.1-50%, more preferably 1-30%, more preferably 
5-15%, and most preferably 10% of the cells in the sample. The second 
sample of patient-derived mononuclear cells may be enriched for monocytes 
using cell fractionation techniques known in the art, e.g, panning or 
FACS, prior to augmenting the concentration of monocytes in the first 
sample. 
In another aspect, the process is carried out by: (a) contacting a first 
sample of mononuclear cells derived from a patient with OKT3 at or below 
37.degree. C. to produce a T3CS; (b) removing the T3CS from the first 
sample; (c) determining the concentration of tumor necrosis factor-alpha 
(TNF.alpha.) in the T3CS, and if required, supplementing the T3CS with 
TNF.alpha. to achieve a concentration of at least 5 pg/ml; (d) providing a 
second sample of mononuclear cells derived from the same patient; (e) 
inactivating suppressor cells in the second sample; and, (f) contacting 
the second sample with T3CS and for a period of time sufficient to 
activate the second sample in vitro to yield a population of 
immunoreactive cells. The concentration of TNF.alpha. is preferably in the 
range of 100 pg/ml to 100 ng/ml, more preferably in the range of 100 pg/ml 
to 3000 pg/ml, and most preferably in the range of 500 pg/ml to 1000 
pg/ml. The concentration of a cytokine, e.g., TNF.alpha., may be augmented 
by (1) altering the length of the T3CS-generation step, (2) supplementing 
T3CS with purified non-recombinant cytokine, (3) supplementing T3CS with a 
recombinant cytokine, or (4) increasing the concentration of a 
cytokine-producing patient-derived mononuclear cell. The T3CS generation 
step (step (a)) of the process may be carried out for 1-7 days and is 
preferably carried out for 3 days. 
In yet another aspect, the process requires after step (b), determining the 
concentration of both TNF.alpha. and OKT3, and if required, supplementing 
the T3CS with TNF.alpha. to achieve a concentration of at least 5 pg/ml 
TNF.alpha. and with OKT3 to achieve a concentration of at least 0.1 ng/m 
OKT3. 
The levels o 
The levels of interferon-gamma (IFN-.gamma.), interleukin-4 (IL-4), and 
interleukin-10 (IL-10) in T3CS may be adjusted to achieve optimal 
generation of a particular type of immunoreactive cell, e.g., a Thi-type T 
cell. Accordingly, the process may include the steps of: (a) contacting a 
first sample of mononuclear cells derived from a patient with OKT3 at or 
below 37.degree. C. to produce a T3CS; (b) removing the T3CS from the 
first sample; (c) determining the concentration of IFN-.gamma., IL-4, and 
IL-10 in the T3CS, and if required, adjusting the concentration of 
IFN-.gamma. to at least 500 pg/ml, the concentration of IL-4 to less than 
or equal to 20 pg/ml, and the concentration of IL-10 to less than or equal 
to 20 pg/ml; (d) providing a second sample of mononuclear cells derived 
from the same patient; (e) inactivating suppressor cells in the second 
sample; and, (f) contacting the second sample with T3CS and for a period 
of time sufficient to activate the second sample in vitro to yield a 
population of immunoreactive cells. 
The process of the invention may also include a step in which the state of 
activation of the immunoreactive cells is evaluated prior to 
administration of the cells to the patient. The cells may be evaluated 
phenotypically or functionally, i.e., by measuring the expression of a 
cell surface marker indicative of cell activation and/or differentiation 
or by measuring cell proliferation in response to an additional immune 
stimulus, e.g, antigen or phorbol myristate acetate (PMA). For example, 
following cell processing according to the invention, the number of 
CD25-positive cells in the population of immunoreactive T cells may be 
measured and the cells discarded if the number is less than 10% of the 
total number of T cells in the population. Preferably the number of 
CD25.sup.+ cells is at least 20% of the total number of T cells in the 
population. 
The level of activation of the immunoreactive cells may also be evaluated 
by measuring proliferation in response to further stimulation by immune 
stimulants. The cells are discarded if the level of proliferation, e.g., 
the amount of .sup.3 H-thymidine incorporated into cellular DNA, is less 
than twice the level of proliferation of a sample of unprocessed 
mononuclear cells. The level of proliferation of the immunoreactive cells 
is at least twice that of unprocessed cells and preferably is 5-fold 
greater than that of unprocessed patient-derived cells. If necessary, the 
level of activation of the immunoreactive cells may be adjusted, e.g., to 
a higher level of activation, by altering the length of the patient-derive 
mononuclear cell-T3CS co-culture period to generate the immunoreactive 
cells, or alternatively, by adding fresh T3CS. 
The mixture of nonspecific autologous lymphocyte activators and OKT3, i.e., 
T3CS, preferably contains: interleukin-1-alpha (IL-1.alpha.), 
interleukin-1-beta (IL-1.beta.), interleukin-6 (IL-6), interleukin-8 
(IL-8), TNF.alpha., tumor necrosis factor-beta (TNF.beta.), 
interferon-gamma (IFN.gamma.), granulocyte macrophage-colony stimulating 
factor (GM-CSF), monocyte/macrophage colony stimulating factor (M-CSF) and 
OKT3. In preferred embodiments, T3CS contains 12.7 ng/ml (.+-.10-40%) OKT3 
in addition to autologous cytokines in the following amounts 
(.+-.10%-40%): IL-1.alpha. (105 pg/ml), IL-162 (1433 pg/ml), IL-6 (808 
pg/ml), IL-8 (213 ng/ml), TNF.alpha. (570 pg/ml), TNF.beta. (171 pg/ml), 
IFN.gamma. (14350 pg/ml), M-CSF (1193 pg/ml), and GM-CSF (840 pg/ml). 
IL-2, interleukin-3, IL-4, interleukin-7, IL-10, IL-12, T cell growth 
factor-beta (TGF.beta.), and granulocyte-colony stimulating factor may 
each be present in T3CS at a concentration of less than 20 pg/ml. 
Preferably, these cytokines are present at a concentration of less than 5 
pg/ml. At least a 20% increase in the number of CD25.sup.+ T cells in the 
first sample of patient-derived mononuclear cells following a 
T3CS-generation culture compared to a sample of uncultured mononuclear 
cells is predictive of sufficient production of autologous cytokines. 
To avoid differentiation of the patient-derived mononuclear cells into 
lymphokine-activated killer (LAK) cells, the concentration of IL-2 must be 
less than 100 units/ml. The concentration of IL-2 in T3CS is preferably 
less than 50 units/ml, and more preferably in the range of 10-20 units/ml, 
and most preferably in the range of 1-5 ng/ml (1 unit of IL-2 is 
approximately equal to 250 pg/ml). 
To generate optimal levels of antigen-independent nonspecific lymphocyte 
activators, production of T3CS is preferably carried out at a temperature 
greater than 29.degree. C. but less than 37.degree. C., e.g., 35.degree. 
C., for a period of 2 days. Co-culture of patient-derived mononuclear 
cells with T3CS to generate the immunoreactive cells may also be carried 
out at sub-physiologic temperature, e.g., a temperature greater than 
29.degree. C. but less than 37.degree. C. 
Following the incubation of cells with T3CS, the cells may be removed from 
the T3CS and contacted with IL-2, preferably in an amount which is 
sufficient to bind to at least 25% of the IL-2 receptors on the surface of 
the immunoreactive cells; more preferably, the amount of IL-2 is 
sufficient to saturate the IL-2 receptors on the surface of the 
immunoreactive cells. Contacting the immunoreactive cells with IL-2 is 
preferably done at 4.degree. C., e.g., during storage or delivery of the 
cells prior to administration to the patient. 
The invention also features a process of producing a population of 
antigen-specific polyclonal T cells by (a) contacting a first sample of 
mononuclear cells derived from a patient with OKT3 at or below 37.degree. 
C. to produce a T3CS; (b) removing the T3CS from the first sample; (c) 
determining the concentration of OKT3 in the T3CS, and if required, 
supplementing the T3CS with additional OKT3 to achieve a concentration of 
at least 0.1 ng/ml; (d) providing a second sample of mononuclear cells 
derived from the same patient; (e) contacting the second sample with T3CS 
and an antigen for a period of time, e.g., 1-30 days, sufficient to 
activate the second sample in vitro to yield a population of 
antigen-specific polyclonal T cells. The concentration of OKT3 in the T3CS 
is preferably 0.1-1 ng/ml. To achieve the desired concentration, T3CS may 
be supplemented with OKT3, or OKT3 may be removed from T3CS using methods 
known in the art, such as chromatography, antibody-mediated depletion, or 
filtration. The antigen may be in the form of a natural or synthetic 
peptide, cell extract, a purified antigen, or a recombinantly expressed 
antigen and may be a tumor antigen, bacterial antigen, viral antigen, or 
autoantigen. 
In another aspect, the invention provides an immunoreactive mononuclear 
cell produced by the inventive process. The cell is preferably a T cell, 
more preferably a Th1-type T cell. The T cell preferably expresses at 
least 10%, more preferably at least 75%, and most preferably at least 100% 
more cell-surface CD25 than an unprocessed mononuclear cell, e.g., a 
mononuclear cell in a resting state. The cell of the invention is 
preferably in a primed state, i.e., the cell proliferates at a rate that 
is at least twice that of an unprocessed T cell when contacted with an 
immune stimulant. The invention also includes a mixture of immunoreactive 
cells produced by the inventive process at least 75% of which are T 
lymphocytes, e.g., Th1-type T cells. The cells of the invention can be 
used to treat any condition characterized by sub-optimal immune 
responsiveness. 
Another aspect of the invention features a process for producing a mixture 
of autologous nonspecific lymphocyte activators by collecting mononuclear 
cells from the blood of a patient afflicted with cancer or an infectious 
disease, inactivating suppressor T cells in the sample of mononuclear 
cells, and contacting the mononuclear cells with a compound that binds to 
the T cell receptor or the T cell receptor-associated CD3 molecule at or 
below 37.degree. C., e.g., 29.degree.-36.degree. C., e.g., 35.degree. C. 
for 2 days. The T cell receptor-binding compound may bind to the alpha 
chain or beta chain of a T cell receptor (or alternatively to the gamma or 
delta chain). The CD3-binding compound is preferably soluble OKT3, but may 
be any ligand that binds to the CD3 molecule on the surface of the cell. 
The cells may be contacted with the CD3-binding compound in the absence or 
in the presence of an antigen; the CD3-binding compound may be removed 
from the cell culture supernatant following the production of the mixture 
of autologous cytokines. In preferred embodiments, the mixture contains 
autologous cytokines in the following amounts (.+-.10%-40%): IL-1.alpha. 
(105 pg/ml), IL-1.beta. (1433 pg/ml), IL-6 (808 pg/ml), IL-8 (213 ng/ml), 
TNF.alpha. (570 pg/ml), TNF.beta. (171 pg/ml), IFN.gamma. (14350 pg/ml), 
M-CSF (1193 pg/ml), and GM-CSF (840 pg/ml), either in the presence or 
absence of 12.7 ng/ml (.+-.10-40%) OKT3. 
The invention also includes the immunoreactive cells of the invention 
together with a pharmaceutically acceptable carrier or diluent for patient 
administration. 
In yet another aspect, the invention features a method of treating a tumor 
or a viral pathogen in a patient by administering to the patient the 
immunoreactive cells of the invention. A suppressor cell inhibiting 
compound, e.g., cimetidine, indomethacin, or both, may be concurrently 
administered to the patient. The method may be used to treat any type of 
cancer including both solid tumors and hematologic tumors, e.g., renal 
cell carcinoma, breast carcinoma, prostate carcinoma, colo-rectal 
carcinoma, pancreatic carcinoma, ovarian carcinoma, melanoma and non-small 
cell carcinoma of the lung as well as leukemias and lymphomas. The methods 
and compositions of the invention represent a promising approach to tumors 
not treatable by conventional forms of therapy such as chemotherapy, 
radiation therapy, or surgery. 
Mononuclear cells taken from a patient afflicted with a complex chronic 
viral disease may also be processed according to the invention to yield 
immunoreactive cells which can then be returned to the patient to augment 
the patient's immune response to the pathogen. Patients infected with 
pathogenic viruses, e.g., hepatitis B virus, hepatitis C virus, recurrent 
herpesvirus (herpes simplex virus, varicella zoster virus, 
cytomegalovirus), papilloma virus, Epstein Barr viurs and HIV (HIV-1 and 
HIV-2), may be treated in this manner. 
Other features and advantages of the invention will be apparent from the 
following detailed description, and from the claims.

PROCESSING OF PERIPHERAL BLOOD MONONUCLEAR CELLS 
Mononuclear cells to be processed according to the invention can be 
obtained from patients, e.g., those afflicted with a malignant tumor or an 
infectious disease such as hepatitis B. Peripheral blood or a mononuclear 
cell-enriched population of cells (obtained using known methods, e.g., 
apheresis) is taken from a patient, and a portion of the sample is mixed 
with an anticoagulant, e.g., heparin, sodium citrate, 
ethylenediaminetetraacetic acid, sodium oxalate. The blood-anticoagulant 
mixture then is diluted in a physiologically acceptable solution such as 
sodium chloride or phosphate buffered solution. Mononuclear cells are 
recovered by layering the blood-anticoagulant composition onto a 
centrifugation separation medium such as Ficoll-Hypaque (Pharmacia 
Corporation) or Lymphocyte Separation Medium (Litton Bionetics 
Corporation). The layered mixture then is centrifuged, and the interface 
containing the mononuclear cells is collected and washed. 
The suppressor cells in the mononuclear cell population may be functionally 
inactivated by contacting the mononuclear cells with an agent that has a 
specific affinity for or effect upon suppressor cells. A particularly 
suitable composition for inactivating suppressor cells is an H2 receptor 
antagonist, such as cimetidine; a suitable composition for inactivating 
the suppressor activity of monocytes is indomethacin. Following suppressor 
cell inactivation, the mononuclear cells are suspended in a culture medium 
containing a mitogenic compound which binds to the T cell receptor or the 
T cell receptor-associated CD3 molecule, e.g., a CD3-binding compound, 
e.g., OKT3, to produce T3CS. Cimetidine may be used in the inventive 
process to inactivate suppressor cells. The addition of cimetidine to the 
medium when PBMC are cultured in the T3CS resulted in increased activation 
of T cells as measured by enhanced proliferative responses of 
immunoreactive cells upon further stimulation with PMA (data not shown). 
In each sample, the concentration of mononuclear cells can be in the range 
of about 0.5-5.0.times.10.sup.6 cells/ml, preferably 1-2.times.10.sup.6 
cells/ml. Although any standard tissue culture medium can be utilized in 
the process of this invention, the cells are preferably cultured under 
serum-free conditions at 37.degree. C. using a standard tissue culture 
medium, e.g., AIM V medium available from Gibco-BRL, Grand Island, N.Y. 
Mononuclear cells are generally cultured with OKT3 for a period of 3 days 
to generate T3CS. The T3CS may be used immediately or stored frozen and 
then thawed for use. 
The concentration of cytokines, e.g., TNF.alpha., or OKT3 concentration in 
the T3CS may be measured by any conventional means such as 
radioimmunoassay or enzyme-linked immunosorbent assay (ELISA) using 
antibodies specific for those components. 
EXAMPLE 1 
Characterization of T3CS 
Generation of T3CS 
Peripheral blood mononuclear cells (PBMC) were obtained from patients with 
mRCC by leukopheresis and fractionated using ficoll density separation. 
The cells from the mononuclear fraction were cultured at 1.times.10.sup.6 
/ml in AIM V medium (Gibco-BRL, Grand Island, N.Y.) with 25 ng/ml OKT3 
(Orthoclone OKT3; Ortho Pharmaceutical Corporation, Raritan, N.J.) for 3 
days in Lifecell.RTM. bags. To inhibit suppressor cell activity, 50 .mu.M 
cimetidine (Tagamet.RTM.; Smith Kline Beecham Pharmaceutical, Cidra, Pa.) 
and 10 nM indomethacin (Indocine; Merck Sharp & Dohme, West Point, Pa.) 
were also added to the culture medium. At the end of the culture, the 
culture bags were centrifuged at 1100.times.g for 20 min at room 
temperature, and the supernatants were collected, aliquoted, and stored at 
-70.degree. C. 
Composition of T3CS 
The composition of the T3CS was determined by ELISA analysis using 
Quantikine kits from R&D Systems (Minneapolis, Minn.) for IL-.alpha.a, 
IL-2, 3, 4, 6, 7, 8, TNF.alpha., TNF.beta., GM-CSF, TGF.beta. and G-CSF, 
IFN.gamma. kits from Endogen (Boston, Mass.) and Gibco (Grand Island, 
N.Y.), IL-10 kits from Biosource International (Camarillo, Calif.), and 
IL-1.beta. kits from Cistron Biotechnology (Pine Brook, N.J.). IL-12 was 
determined by a bioassay (phytohemagglutinin (PHA) blast proliferation). 
The amount of OKT3 present in T3CS was determined by ELISA using the 
following reagents purchased from Vector Laboratories, Inc. (Burlingame, 
Calif.): Horse anti-mouse IgG to capture the OKT3 mouse mAb, biotinylated 
horse anti-mouse IgG to detect the captured mAb and ABC reagent consisting 
of avidin-conjugated horseradish peroxidase to amplify the signal. 
Ortho-phenylenediamine dihydrochloride (Sigma Chemical Co., St. Louis, 
Mo.) was used as a substrate. 
Polyclonal Activation of PBMC 
To evaluate EVA cells, cultures were carried out under the same conditions 
as used in processing patients-derived cells except on a smaller scale (5 
ml). Excess PBMC obtained from mRCC patients were isolated by ficoll 
density gradient and were cultured at 2.times.10.sup.6 /ml for 5 days in 
complete AIM V medium (containing 50M cimetidine and 10 nM indomethacin) 
with either 25% (vol/vol) T3CS, or various concentrations of OKT3. Cells 
cultured with medium alone served as control. After incubation, the cells 
were washed and resuspended at a concentration of 10.times.10.sup.6 /ml in 
"infusion medium" (1% human serum albumin and 0.5% dextrose in Lactated 
Ringers solution). The cells were then stored overnight at 4.degree. C. 
prior to analysis (to simulate overnight storage for final QA/QC and 
shipping to clinical sites). 
Phenotypic Analysis of PBMC and EVA Cells 
Cell phenotypes were determined by flow cytometry. Immunofluorescent 
staining was carried out using mAb specific for cell surface antigens, 
e.g., Coulterclones T3-RD1/CD3-RD1, T4-RD1, T8-RD1 and -FITC, IL-2R-FITC, 
I3-FITC and 2H4-RD1 (Coulter Corporation, Hialeah, FL), and Dako 
UCHL1-FITC (Dako corporation, Carpintera, Calif.). Appropriate 
isotype-matched labels were used as negative controls. Cells were 
resuspended at 2.times.10.sup.6 /ml in AIM V medium and aliquoted to 100 
.mu.l per tube. Two to four .mu.ls of labeled mabs were added to the cells 
according to the manufacturers' recommendation. The cells were then 
incubated with the Abs at 4.degree. C. for 30 min, washed once with 500 
.mu.l of cold PBS, and resuspended at a concentration of 4.times.10.sup.5 
cells/ml in PBS for immediate analysis using a Coulter Epics Profile II 
flow cytometer. 
PMA Assay 
Proliferative responses to PMA were used to measure the degree of cell 
activation. The PBMC or EVA cells were resuspended to 1.times.10.sup.6 
cells/ml in AIM V medium with or without 1 ng/ml PMA, and cultured in 
triplicate in 96-well flat-bottom plates (Costar, Cambridge, Mass.) at 
37.degree. C. with 5% CO.sub.2 for 48 hours. The cells were pulsed with 
.sup.3 H-thymidine (1 .mu.Ci /well) for the last 6 hours of culture, and 
harvested onto filtermats. The amount of radioactivity incorporated into 
the cells was determined by liquid scintillation counting. 
Depletion of OKT3 or Cytokines From T3CS 
T3CS was divided into 6 ml aliquots, and incubated with the appropriate 
neutralizing antibody (Goat anti-human, from R&D Systems) at 37.degree. C. 
for one hour. The amount of antibody used for depletion was determined by 
the level of the particular cytokine known to be present and the antibody 
activity required for neutralization as specified by the manufacturer. 
Specifically, 125 .mu.l of antibody was added for the depletion of 
IL-1.beta. or TNF.alpha., 20 .mu.l for IL-6, 60 .mu.l for IFN, 100 .mu.l 
for GM-CSF, and 250 .mu.l for IL-8. After incubation, 1 ml of pre-washed 
magnetic beads (Advanced Magnetics, Cambridge, Mass.) conjugated with 
either rabbit-anti-goat IgG (for cytokine depletion) or goat-anti-Mouse 
IgG (for OKT3 depletion) was added to the samples (2 ml of beads were used 
for multiple-cytokine depletion) and incubated with the cells at room 
temperature for 20 min. The cytokine-bound beads were then removed by 
placing the tube in a magnetic holder for 5 min. The cytokine-depleted 
T3CS was carefully transferred to a fresh labeled tube. The depletions 
were monitored by ELISA analysis. 
Cytokine Composition of T3CS 
As described above, T3CS was generated by culturing PBMC isolated from mRCC 
patients with 25 ng/ml OKT3 for 3 days. Aliquots of randomly selected mRCC 
patient T3CS (N=33-42) were analyzed for the presence of 17 different 
cytokines and OKT3 by ELISA. As shown in Table 1, T3CS contained a mixture 
of monokines and lymphokines including IL-1.alpha., IL-1.beta., IL-6, 
IL-8, TNF.alpha. and .beta., IFN.gamma., M-CSF, and GM-CSF. Several other 
cytokines, including IL-2, 3, 4, 7, 10, 12, TGF.beta., and G-CSF, were 
either undetectable or detected at very low levels in all samples tested. 
The cytokine profile indicated that the cells involved in the PBMC 
activation process were predominantly monocytes and Th1-type T cells 
(Seder et al., 1994, Ann. Rev. Immunol. 12:635-673) or T cells that had 
differentiated into Th1-type cytokine producers during the cultures. 
Although kinetic studies indicated that significant amounts of IL-2 
(15-433 pg/ml) were present in all samples of T3CS analyzed during the 
first 24 hours of the culture, the level of IL-2 dropped sharply by day 2 
(0-132 pg/ml). IL-2 levels became undetectable by day 3 when the culture 
supernatant was harvested. The drop in IL-2 is likely to be due to active 
consumption of the cytokine during cell culture. The mean OKT3 
concentration in T3CS was 12.7 ng/ml. T3CS contains significantly lower 
levels of IL-2 than conventional conditioned media, e.g., PHA-generated 
cell supernatants, Concanavalin-A-generated cell supernatants, or mixed 
lymphocyte culture supernatants. Cytokine levels in T3CS from the majority 
patients fell into acceptable ranges, i.e., sufficient amounts of the key 
cytokines were present to produce immunoreactive cells in subsequent PBMC 
cultures. T3CS from a small number of patients contained low levels of 
certain cytokines. As discussed above, the concentration of cytokines may 
be augmented, if necessary, by carrying out the T3CS generating step for a 
longer period of time or alternatively, by adding recombinant or 
nonrecombinant cytokines. 
TABLE 1 
______________________________________ 
COMPOSITION OF CONDITIONED MEDIUM (T3CS) 
Cytokines: Mean N 
______________________________________ 
IL-1.alpha. 
(pg/ml) 105 33 
IL-1.beta. (pg/ml) 1433 36 
IL-6 (pg/ml) 808 39 
IL-8 (ng/ml) 213 39 
TNF.alpha. (pg/ml) 570 39 
TNF.beta. (pg/ml) 171 39 
INF.gamma. (pg/ml) 14350 40 
GM-CSF (pg/ml) 840 39 
M-CSF (pg/ml) 1193 39 
OKT3 (ng/ml) 12.7 42 
______________________________________ 
IL-2, 3, 4, 7, 10, 12, TGFB and GCSF are below detectable levels (&lt;3-8 
pg/ml) in &gt;90% samples tested. 
Accessory Cell Requirement for Production of Immunoreactive Cells 
According to the invention, the concentration of patient-derived monocytes 
in a sample of patient-derived mononuclear cells, is preferably 0.1-50%, 
more preferably 1-30%, more preferably 5-15%, and most preferably 10%. 
To evaluate the role of monocytes in cultures of patient-derived 
mononuclear cells, monocytes were depleted from patient-derived 
mononuclear cells using the well-known methods of adherence, e.g., to 
plastic plates, or incubation with L-leucine methyl ester. Incubation of a 
monocyte-depleted sample of patient-derived mononuclear cells with T3CS 
failed to yield the immunoreactive cells of the invention. In addition, 
blocking of the Fc receptor on the surface of Fc-receptor positive 
accessory cells, e.g., by adding an excess of soluble human polyclonal IgG 
to the patient-derived mononuclear cell-T3CS co-culture, inhibited the 
T3CS-catalyzed generation of immunoreactive cells by 93%. These data 
indicate that Fc-receptor positive accessory cells, i.e., monocytes, 
granulocytes or dendritic cells, are required to generate immunoreactive 
cells using the methods of the invention when OKT3 is used in solution 
phase. 
If a sample of patient-derived mononuclear cells has a sub-optimal 
concentration of accessory cells, the sample of patient-derived 
mononuclear cells may be enriched for monocytes, granulocytes, or 
dendritic cells. 
Role of Soluble OKT3 
The OKT3 used in the process of the invention is preferably in solution 
phase rather than solid phase. One advantage of using soluble OKT3 in the 
inventive process is that soluble OKT3 mediates a more physiological 
interaction between Fc-receptor-bearing accessory cells, e.g., monocytes, 
and T cells. By forming a bridge between these cells, a full complement of 
costimulatory signals is initiated, thus minimizing the potential for the 
generation of incompletely (anergic) or aberrantly (apoptotic) activated T 
cells. 
Enhancement of OKT3-Induced T Cell Activation by Autologous Cytokines 
The role of cytokines in the induction of T cell activation was compared to 
that of OKT3 alone. PBMC from seven mRCC patients were cultured for 5 days 
in AIM-V medium containing either 2.5 ng/ml OKT3 alone or 25% (vol/vol) 
T3CS that was first depleted of OKT3 and then reconstituted with 2.5 ng/ml 
of fresh antibody. This depletion/reconstitution approach was undertaken 
to assure that the amount of biologically active OKT3 in the T3CS would be 
identical to the OKT3 control. To avoid the bias from a single aliquot of 
T3CS, a pool made from three different mRCC patients was used to stimulate 
the PBMC from all seven patients. 
The level of T cell activation was determined by the expression of cell 
surface IL-2 receptor (CD25) as measured by flow cytometry. FIGS. 1A-1E 
are representative immunofluorescence histograms that demonstrate that 
CD25 expression on T cells stimulated with the autologous cytokine-OKT3 
mixture was significantly higher than that on T cells stimulated with OKT3 
alone. 
Primed Activation State of Immunoreactive Cells 
The degree of T cell activation was also assessed functionally by 
measurement of the proliferative response of EVA cells upon further 
stimulation by PMA, a protein kinase C activator. 
FIG. 4 shows the results of an experiment in which immunoreactive cells 
were contacted with an immune stimulant, e.g., various concentrations of 
PMA. In the absence of further stimulation by PMA, immunoreactive cells 
displayed very little spontaneous proliferation or cytokine secretion when 
cultured at 37.degree. C. in AIM V medium alone. However, when contacted 
with 1-10 ng/ml of PMA, immunoreactive cells (but not unprocessed PBMC) 
displayed an enhanced capacity to proliferate and produce IFN.gamma. (as 
well as other Th1-type cytokines such as TNF.alpha., TNF.beta., and GM-CSF 
(data not shown)). These results are in contrast to those obtained with 
mononuclear cells activated by a conventional stimulant, such as PHA, 
which possess high levels of spontaneous proliferation and cytokine 
production following an initial 5-day activation culture (data not shown). 
This enhanced proliferation and cytokine secretion upon contact with an 
immune stimulant and concomitant absence of or low levels of spontaneous 
proliferation and cytokine secretion indicates that the immunoreactive 
cells of the invention are in a primed state of activation. 
Similarly, an increase in proliferation was observed when EVA cells were 
contacted with another immune stimulant, i.e., IL-2 (FIG. 5). These data 
confirm the primed nature of the immunoreactive cells and suggest that the 
immunoreactive cells of the invention could be effectively co-administered 
with a low dose of IL-2. 
Enhanced Effector Function of EVA Cells: Cytolytic T Cells 
The ability of the T3CS to support the generation of cytotoxic EVA cells 
was evaluated. As shown in FIG. 7, cytotoxicity was determined using a 
conventional chromium-51 release assay with the K562 leukemia cell line 
and allogeneic human renal carcinoma cell line 769P as targets. As shown 
in FIG. 7, EVA cells, i.e., T cells which have been polyclonally-activated 
independent of tumor-associated antigens according to the invention, 
possessed a greatly enhanced cytotoxicity toward both tumor lines compared 
to unprocessed PBMC. These in vitro results suggest that EVA cells are 
capable of directly killing tumor cells in vivo. 
Enhanced Effector Function of EVA Cells: Helper T Cells 
In addition to enhanced cytolytic function, the immunoreactive cells of the 
invention were found to have enhanced helper function. The ability of T3CS 
to support the generation of EVA cells with helper cell function was 
determined by measurement of the ability of irradiated EVA cells to 
enhance the proliferative response of unprocessed mononuclear cells upon 
stimulation by a recall antigen, e.g., an influenza antigen. As shown in 
FIG. 6, EVA cells added to cultures at low levels (5-10%) provided helper 
signals to unprocessed patient-derived mononuclear cells (presumably 
through the secretion of Th1-type cytokines) resulting in a significant 
increase in proliferation. These in vitro results suggest that EVA cells 
are capable of amplifying and broadening their effects in vivo through the 
production of cytokines. 
The ability of EVA cells to proliferate and to produce a variety of 
cytokines (IL-2, GM-CSF, IFN.gamma., TNF.alpha.) in vitro in response to 
further stimulation by such agents as PMA and IL-2, as well as to lyse 
tumor cell targets, is greatly enhanced compared to the PBMC from which 
they were derived. The lowered activation threshold of the EVA cells 
exhibited in vitro suggests that once they are reinfused into patients, 
they are likely to demonstrate enhanced responsiveness to immunological 
signals, such as weakly immunogenic tumor antigens which normally are 
non-stimulatory to unprocessed cells. 
Phenotypic Characterization of Immunoreactive Cells 
Following short term (5-day) culture of patient-derived mononuclear cells 
in the autologous cytokine mixture/OKT3-containing conditioned medium, the 
resulting EVA cells were analyzed for the expression of cell surface 
antigens. EVA cells expressed enhanced levels of a variety of activation 
and/or differentiation markers on their cell surface including cytokine 
receptors, e.g., CD25, major histocompatibility complex (MHC) antigens, 
e.g., MHC class II, adhesion molecules/homing receptors, e.g., CD44/Leu8, 
costimulatory molecules, e.g., CD28, and markers of primed or memory T 
cells, e.g., CD45RO, as shown in Table 5. 
Generation of Immunoreactive Cells: Synergistic Effects of Autoloa 
Cytokines Plus OKT3 
OKT3 was depleted from the T3CS in order to determine the relative effects 
of the anti-CD3 monoclonal antibody and the autologous cytokines on the 
induction of CD25 expression on the surface of T lymphocytes (FIG. 2A) and 
the proliferation of EVA cells upon further stimulation by PMA (FIG. 2B). 
Following depletion of OKT3 from the T3CS, there was little or no 
measurable activation of T cells, i.e., the autologous cytokines were not 
capable of stimulating resting PBMC in the absence of OKT3. However, when 
mRCC patient PBMC were cultured with complete T3CS, the cytokines 
functioned in synergy with OKT3 resulting in large increases in T cell 
activation relative to the levels achieved with OKT3 alone. Taken 
together, these results indicate that OKT3 catalyzes the generation of 
immunoreactive cells, while the autologous cytokines function as 
costimulants to optimize the overall T cell activation process. 
TABLE 5 
______________________________________ 
EVA CELL PRODUCT IDENTITY 
% (+) 
LYMPHOCYTES 
EVA 
PBMC CELLS 
______________________________________ 
CD3(+) - T CELLS 75% 84% 
CD4(+) - HELPER T CELLS 
48% 60% 
CD8(+) - CYTOTOXIC T CELLS 
20% 25% 
CD3(+)/CD25.sup.(+) - ACTIVATED T CELLS 
3% 44% 
(IL2 RECEPTOR - EARLY STAGE MARKER) 
CD3(+)/