U.S. patent application number 11/078897 was filed with the patent office on 2005-07-28 for modified rapid expansion methods ("modified-rem") for in vitro propagation of t lymphocytes.
This patent application is currently assigned to Targeted Genetics Corporation. Invention is credited to Clary, Kim W., Flyer, David C..
Application Number | 20050164387 11/078897 |
Document ID | / |
Family ID | 21893787 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050164387 |
Kind Code |
A1 |
Flyer, David C. ; et
al. |
July 28, 2005 |
Modified rapid expansion methods ("modified-REM") for in vitro
propagation of T lymphocytes
Abstract
The present invention provides a modified rapid expansion method
(termed "low-PBMC-REM" or "modified-REM"), for quickly generating
large numbers of T lymphocytes, including cytolytic and helper T
lymphocytes, without using the large excesses of peripheral blood
mononuclear cells (PBMC) or EBV-transformed lymphoblastoid cells
(LCL) characteristic of high-PBMC-REM. Clonal expansions of greater
than 500-fold can be achieved within a single stimulation cycle of
about 8-14 days.
Inventors: |
Flyer, David C.; (Redmond,
WA) ; Clary, Kim W.; (Seattle, WA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Assignee: |
Targeted Genetics
Corporation
Seattle
WA
|
Family ID: |
21893787 |
Appl. No.: |
11/078897 |
Filed: |
March 10, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11078897 |
Mar 10, 2005 |
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09956581 |
Sep 17, 2001 |
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6890753 |
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09956581 |
Sep 17, 2001 |
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08793707 |
Mar 3, 1997 |
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6316257 |
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08793707 |
Mar 3, 1997 |
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PCT/US97/03293 |
Mar 3, 1997 |
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60037333 |
Mar 4, 1996 |
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Current U.S.
Class: |
435/372 |
Current CPC
Class: |
C07K 2317/74 20130101;
C12N 5/0636 20130101; C12N 2501/599 20130101; C07K 16/2896
20130101; A61K 2039/505 20130101; C12N 2502/11 20130101; C12N
2501/515 20130101; C12N 2501/23 20130101; C12N 2501/58
20130101 |
Class at
Publication: |
435/372 |
International
Class: |
C12N 005/08 |
Claims
1. A method for rapidly expanding an initial T lymphocyte
population in culture medium in vitro, comprising the steps of:
adding an initial T lymphocyte population to a culture medium in
vitro; adding to the culture medium a non-dividing mammalian cell
line expressing at least one T-cell-stimulatory component, wherein
said cell line is not an EBV-transformed lymphoblastoid cell line
(LCL); and incubating the culture.
2. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is selected from the group consisting
of an Fc-.gamma. receptor, a cell adhesion-accessory molecule and a
cytokine.
3. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is selected from the group consisting
of an Fc-.gamma. receptor, a cell adhesion-accessory molecule and a
cytokine, and wherein said initial T lymphocyte population is
expanded at least 200-fold after an incubation period of less than
about two weeks.
4. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is selected from the group consisting
of an Fc-.gamma. receptor, a cell adhesion-accessory molecule and a
cytokine, and wherein said initial T lymphocyte population is
expanded at least 500-fold after an incubation period of less than
about two weeks.
5. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is selected from the group consisting
of an Fc-.gamma. receptor, a cell adhesion-accessory molecule and a
cytokine, and wherein said initial T lymphocyte population is
expanded at least 1000-fold after an incubation period of less than
about two weeks.
6. A rapid expansion method of claim 1, further comprising the step
of adding anti-CD3 monoclonal antibody to the culture medium
wherein the concentration of anti-CD3 monoclonal antibody is at
least about 1.0 ng/ml.
7. A rapid expansion method of claim 1, further comprising the step
of adding IL-2 to the culture medium, wherein the concentration of
IL-2 is at least about 10 units/ml.
8. A rapid expansion method of claim 1, wherein said mammalian cell
line comprises at least one cell type that is present at a
frequency at least three times that found in human peripheral blood
mononuclear cells (human PBMCs).
9. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is selected from the group consisting
of an Fc-.gamma. receptor and a cell adhesion-accessory
molecule.
10. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is selected from the group consisting
of a cell adhesion-accessory molecule and a cytokine.
11. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is selected from the group consisting
of an Fc-.gamma. receptor and a cytokine.
12. A rapid expansion method of claim 1, wherein said mammalian
cell line expresses a cell adhesion-accessory molecule.
13. A rapid expansion method of claim 12, wherein said cell
adhesion-accessory molecule is selected from the group consisting
of Class II MHC, Class I MHC, ICAM 1, ICAM 2, ICAM 3, CD58, CD72,
fibronectin, ligand to CD27, CD80, CD86 and hyaluronate.
14. A rapid expansion method of claim 1, wherein said mammalian
cell line expresses a cytokine.
15. A rapid expansion method of claim 1, wherein said
T-cell-stimulatory component is a molecule that binds to CD21.
16. A rapid expansion method of claim 14, wherein said cytokine is
selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-7,
IL-12 and IL-15.
17. A rapid expansion method of claim 1, further comprising the
step of adding a soluble T-cell-stimulatory factor to the culture
medium.
18. A rapid expansion method of claim 17, wherein said soluble
T-cell-stimulatory factor is selected from the group consisting of
a cytokine, an antibody specific for a T cell surface component,
and an antibody specific for a component capable of binding to a T
cell surface component.
19. A rapid expansion method of claim 17, wherein said soluble
T-cell-stimulatory factor is a cytokine selected from the group
consisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15.
20. A rapid expansion method of claim 17, wherein said soluble
T-cell-stimulatory factor is an antibody specific for a T cell
surface component, and wherein said T cell surface component is
selected from the group consisting of CD4, CD8, CD11a, CD2, CD5,
CD49d, CD27, CD28 and CD44.
21. A rapid expansion method of claim 17, wherein said soluble
T-cell-stimulatory factor is an antibody specific for a component
capable of binding to a T cell surface component, and wherein said
T cell surface component is selected from the group consisting of
CD4, CD8, CD11a, CD2, CD5, CD49d, CD27, CD28 and CD44.
22. A rapid expansion method of claim 17, wherein said soluble
T-cell-stimulatory factor is a molecule that binds to CD21.
23. A rapid expansion method of claim 22, wherein said molecule
that binds to CD21 is an anti-CD21 antibody.
24. A rapid expansion method of claim 1, further comprising the
step of adding to the culture a multiplicity of peripheral blood
mononuclear cells (PBMCs).
25. A rapid expansion method of claim 24, wherein the ratio of
PBMCs to initial T cells to be expanded is less than about
40:1.
26. A rapid expansion method of claim 24, wherein the ratio of
PBMCs to initial T cells to be expanded is less than about
10:1.
27. A rapid expansion method of claim 24, wherein the ratio of
PBMCs to initial T cells to be expanded is less than about 3:1.
28. A rapid expansion method of claim 1, further comprising the
step of adding to the culture a multiplicity of EBV-transformed
lymphoblastoid cells (LCLs).
29. A rapid expansion method of claim 28, wherein the ratio of LCLs
to initial T cells to be expanded is less than about 10:1.
30. A rapid expansion method of claim 1, wherein the initial T
lymphocyte population comprises at least one human CD8+
antigen-specific cytotoxic T lymphocyte (CTL).
31. A rapid expansion method of claim 1, wherein the initial T
lymphocyte population comprises at least one human CD4+
antigen-specific helper T lymphocyte.
32. A method of genetically transducing a human T cell, comprising
the steps of: adding an initial T lymphocyte population to a
culture medium in vitro; adding to the culture medium a
non-EBV-transformed mammalian cell line expressing a
T-cell-stimulatory component; and incubating the culture; and
adding a vector to the culture medium.
33. A genetic transduction method of claim 32, wherein the vector
is a retroviral vector containing a selectable marker providing
resistance to an inhibitory compound that inhibits T lymphocytes,
and wherein the method further comprises the steps of: continuing
incubation of the culture for at least one day after addition of
the retroviral vector; and adding said inhibitory compound to the
culture medium after said continued incubation step.
34. A genetic transduction method of claim 32, further comprising
adding a multiplicity of human PBMCs.
35. A genetic transduction method of claim 34, wherein the ratio of
PBMCs to initial T cells is less than about 40:1.
36. A genetic transduction method of claim 32, further comprising
adding non-dividing EBV-transformed lymphoblastoid cells (LCL).
37. A genetic transduction method of claim 36, wherein the ratio of
LCL to initial T cells is less than about 10:1.
38. A method of generating a REM cell line capable of promoting
rapid expansion of an initial T lymphocyte population in vitro,
comprising the steps of: depleting one or more cell types from a
human PBMC population to produce a cell-type-depleted PBMC
population, using said cell-type-depleted PBMC population in place
of non-depleted PBMCs in an hp-REM protocol to determine the
contribution of the depleted cell type to the activity provided by
the non-depleted PBMCs, identifying a T cell stimulatory activity
provided by said depleted cell type, and transforming a mammalian
cell line with a gene allowing expression of said T cell
stimulatory activity.
39. A method of generating a REM cell line according to claim 38,
wherein said T-cell-stimulatory component is selected from the
group consisting of an Fc-.gamma. receptor, a cell
adhesion-accessory molecule and a cytokine.
40. A REM cell line capable of stimulating rapid expansion of an
initial T lymphocyte population in vitro, comprising a mammalian
cell line generated according to the method of claim 38.
41. A REM cell line according to claim 40, wherein said cell line
expresses a cell adhesion-accessory molecule.
42. A REM cell line according to claim 41, wherein said cell
adhesion-accessory molecule is selected from the group consisting
of Class II MHC, Class I MHC, ICAM 1, ICAM 2, ICAM 3, CD58, CD72,
fibronectin, ligand to CD27, CD80, CD86 and hyaluronate.
43. A REM cell line according to claim 40, wherein said cell line
expresses an Fc-.gamma. receptor.
44. A REM cell line according to claim 40, wherein said cell line
expresses at least one T cell stimulatory cytokine.
45. A REM cell line according to claim 44, wherein said T cell
stimulatory cytokine is selected from the group consisting of IL-1,
IL-2, IL-6, IL-7, IL-12 and IL-15.
46. A REM cell line according to claim 40, wherein said cell line
expresses a molecule that binds to CD21.
47. A culture medium capable of rapidly expanding an initial T
lymphocyte population in vitro comprising a REM cell line according
to claim 40.
48. A culture medium according to claim 47, further comprising an
exogenous cytokine.
49. A culture medium according to claim 47, further comprising a
multiplicity of exogenous cytokines, wherein said multiplicity
comprises at least one interleukin.
50. A culture medium according to claim 49, wherein said
interleukin is selected from the group consisting of IL-1, IL-2,
IL-6, IL-7, IL-12 and IL-15.
51. A culture medium according to claim 47, further comprising a
molecule that binds to CD21.
52. A culture medium according to claim 51, wherein said molecule
that binds to CD21 is an anti-CD21 antibody.
53. A culture medium according to claim 49, further comprising an
anti-CD3 monoclonal antibody.
Description
FIELD OF THE INVENTION
[0001] This invention relates to improved methods for culturing T
lymphocytes, including human antigen-specific cytolytic and helper
T lymphocytes. The methods of the present invention result in the
very rapid and efficient expansion of T cells which are useful, for
example, in cellular immunotherapy.
BACKGROUND
[0002] T lymphocytes are formed in the bone marrow, migrate to and
mature in the thymus and then enter the peripheral blood and
lymphatic circulation. T lymphocytes can be phenotypically
subdivided into several distinct types of cells including: helper T
cells, suppressor T cells, and cytotoxic T cells. T lymphocytes,
unlike B lymphocytes, do not produce antibody molecules, but
express a heterodimeric cell surface receptor that can recognize
peptide fragments of antigenic proteins that are attached to
proteins of the major histocompatibility complex (MHC) expressed on
the surfaces of target cells; see, e.g., Abbas, A. K., Lichtman, A.
H., and Pober, J. S., Cellular and Molecular Immunology, 1991, esp.
pages 15-16.
[0003] T lymphocytes that can be expanded according to the present
invention are of particular interest in the context of cellular
"immunotherapy". As used herein, cellular immunotherapy refers to
any of a variety of techniques involving the introduction of cells
of the immune system, especially T lymphocytes, into a patient to
achieve a therapeutic benefit. Such techniques can include, by way
of illustration, "immuno-restorative" techniques (involving, e.g.,
the administration of T cells to a patient having a compromised
immune system); "immuno-enhancing" techniques (involving, e.g., the
administration of T cells to a patient in order to enhance the
ability of that patient's immune system to avoid or combat a cancer
or a pathogen such as a virus or bacterial pathogen); and
"immuno-modulating" techniques (involving, e.g., the administration
of T cells to a patient in order to modulate the activity of other
cells of the patient's immune system, such as in a patient affected
by an autoimmune condition).
[0004] Cytotoxic T lymphocytes (CTLs) are typically of the CD3+,
CD4-, CD8+ phenotype and lyse cells that display fragments of
foreign antigens associated with class I MHC molecules on their
cell surfaces. CTLs that are CD3+, CD4+, CD8- have also been
identified. Target cells for CTL recognition include normal cells
expressing antigens after infection by viruses or other pathogens;
and tumor cells that have undergone transformation and are
expressing mutated proteins or are over-expressing normal
proteins.
[0005] Most "helper" T cells are CD3+, CD4+, CD8-. Helper T cells
recognize fragments of antigens presented in association with class
II MHC molecules, and primarily function to produce cytokines that
amplify antigen-specific T and B cell responses and activate
accessory immune cells such as monocytes or macrophages. See, e.g.,
Abbas, A. K., et al., supra. Helper T cells can also participate in
and/or augment cytolytic activites.
[0006] In addition to conventional helper T cells and cytolytic or
"killer" T cells, it will also be useful to be able to rapidly
expand other T cell populations. For example, T cells expressing
the gamma/delta T cell receptor represent a relatively small
portion of the human T cell population, but are suspected to play a
role in reactivity to viral and bacterial pathogens as well as to
tumor cells (see, e.g., W. Haas et al. 1993. Annu. Rev. Immunol.
11: 637). Another T cell population of potential clinical
importance is the population of CD1-restricted T cells. CD1 is an
MHC-like molecule that shows limited polymorphism and, unlike
classical MHC molecules which "present" antigenic peptides, CD
molecules bind lipoglycans and appear to be important in the
recognition of microbial antigens (see, e.g., P. A. Sieling et al.
1995. Science 269: 227; and E. M. Beckman et al. 1994. Nature 372:
691).
[0007] T lymphocytes are thus key components of the host immune
response to viruses, bacterial pathogens and to tumors. The
significance of properly functioning T cells is made quite clear by
individuals with congenital, acquired or iatrogenic T cell
immunodeficiency conditions (e.g., SCID, BMT, AIDS, etc.) which can
result in the development of a wide variety of life-threatening
infections or malignancies. Persons with diseases that are related
to a deficiency of immunologically-competent T lymphocytes, or
persons with conditions that can be improved by administering
additional T lymphocytes, can thus be benefited by cellular
immunotherapies, as referred to above. T cells for use in such
therapies can be derived from the immunodeficient host, or from
another source (preferably a compatible donor). The latter source
is of course especially important in situations in which an
immunodeficient host has an insufficient number of T cells, or has
T cells that are insufficiently effective. In either case, it is
difficult to obtain sufficient numbers of T cells for effective
administration; and thus target T cells must first be grown to
large numbers in vitro before administration to a host.
[0008] After undergoing such cellular immunotherapy, hosts that
previously exhibited, e.g., inadequate or absent responses to
antigens expressed by pathogens or tumors, can express sufficient
immune responses to become resistant or immune to the pathogen or
tumor.
[0009] Adoptive transfer of antigen-specific T cells to establish
immunity has been demonstrated to be an effective therapy for viral
infections and tumors in animal models (reviewed in Greenberg, P.
D., Advances in Immunology (1992)). For adoptive immunotherapy to
be effective, antigen-specific T cells usually need to be isolated
and expanded in numbers by in vitro culture, and following adoptive
transfer such cultured T cells must persist and function in vivo.
For treatment of some human diseases, the use in immunotherapy of
cloned antigen-specific T cells which represent the progeny of
single cells, offers significant advantages because the specificity
and function of these cells can be rigorously defined and precise
dose:response effects readily evaluated. Riddell et al., were the
first to adoptively transfer human antigen-specific T cell clones
to restore deficient immunity in humans. Riddell, S. R. et al.,
"Restoration of Viral Immunity in Immunodeficient Humans by the
Adoptive Transfer of T Cell Clones", Science 257: 238-240 (1992).
In that study, Riddell et al. used adoptive immunotherapy to
restore deficient immunity to cytomegalovirus in allogeneic bone
marrow transplant recipients. Cytomegalovirus-specific CD8+
cytotoxic T cell clones were isolated from three CMV seropositive
bone marrow donors, propagated in vitro for 5 to 12 weeks to
achieve numerical expansion of effector T cells, and then
administered intravenously to the respective bone marrow transplant
(BMT) recipients. The BMT recipients were deficient in CMV-specific
immunity due to ablation of host T cell responses by the
pre-transplant chemoradiotherapy and the delay in recovery of donor
immunity commonly observed after allogeneic bone marrow transplant
(Reusser et al. Blood, 78: 1373-1380, 1991). Riddell et al. found
that no toxicity was encountered and that the transferred T cell
clones provided these immunodeficient hosts with rapid and
persistent reconstitution of CD8+ cytomegalovirus-specific CTL
responses.
[0010] Riddell et al. (J. Immunology, 146: 2795-2804, 1991) used
the following procedure for isolating and culturing the CD8+
CMV-specific T cell clones: peripheral blood mononuclear cells
(PBMCs) derived from the bone marrow donor were first cultured with
autologous cytomegalovirus-infected fibroblasts to activate
CMV-specific CTL precursors. Cultured T cells were then
restimulated with CMV-infected fibroblasts and the cultures
supplemented with .gamma.-irradiated PBMCs. 2-5 U/ml of
interleukin-2 (IL-2) in suitable culture media was added on days 2
and 4 after restimulation to promote expansion of CD8+ CTL (Riddell
et al., J. Immunol., 146: 2795-2804, 1991). To isolate T cell
clones, the polyclonal CD8+ CMV-specific T cells were plated at
limiting dilution (0.3-0.6 cells/well) in 96-well round bottom
wells with either CMV-infected fibroblasts as antigen-presenting
cells (Riddell, J. Immunol., 146: 2795-2804, 1991); or anti-CD3
monoclonal antibody to mimic the stimulus provided by
antigen-presenting cells. (Riddell, J. Imm. Methods, 128: 189-201,
1990). Then, .gamma.-irradiated peripheral blood mononuclear cells
(PBMC) and EBV-transformed lymphoblastoid cells (LCL) were added to
the microwells as feeder cells. Wells positive for clonal T cell
growth were evident in 10-14 days. The clonally derived cells were
then propagated to large numbers initially in 48- or 24-well plates
and subsequently in 12-well plates or 75-cm.sup.2 tissue culture
flasks. T cell growth was promoted by restimulation every 7-10 days
with autologous CMV-infected fibroblasts and .gamma.-irradiated
feeder cells consisting of PBMC and LCL, and the addition of 25-50
U/ml of IL-2 at 2 and 4 days after restimulation.
[0011] A major problem that exists in the studies described above,
and in general in the prior art of culturing T cells, is the
inability to grow large quantities of human antigen-specific T cell
clones in a timely fashion. It is not known if the slow growth of T
cells in culture represents an inherent property of the cell cycle
time for human lymphocytes or the culture conditions used. For
example, with the culture method used in the CMV adoptive
immunotherapy study described above, three months were required to
grow T cells to achieve the highest cell dose under study which was
1.times.10.sup.9 T cells/cm.sup.2. This greatly limits the
application of adoptive immunotherapy for human viral diseases and
cancer since the disease process may progress during the long
interval required to isolate and grow the specific T cells to be
used in therapy. Based on extrapolation from animal model studies
(reviewed in Greenberg, P. D., Advances in Immunology, 1992), it is
predicted that in humans doses of antigen-specific T cells in the
range of 10.sup.9-10.sup.10 cells may be required to augment immune
responses for therapeutic benefit.
[0012] However, rapidly expanding antigen-specific human T cells in
culture to achieve such high cell numbers has proven to be a
significant obstacle. Thus, with the exception of the study by
Riddell et al., supra, (in which several months were taken to grow
a sufficient number of cells) studies of adoptive immunotherapy
using antigen-specific T cell clones have not been performed. The
problem of producing large numbers of cells for adoptive
immunotherapy was identified in U.S. Pat. No. 5,057,423. In this
patent, a method for isolating pure large granular lymphocytes and
a method for the expansion and conversion of these large granular
lymphocytes into lymphokine activated killer (LAK) cells is
described. The methods are described as providing high levels of
expansion, i.e. up to 100-fold in 3-4 days of culture. Although LAK
cells will lyse some types of tumor cells, they do not share with
MHC-restricted T cells the properties of recognizing defined
antigens and they do not provide immunologic memory. Moreover, the
methods used to expand LAK cells, which predominantly rely on high
concentrations of IL-2 do not efficiently expand antigen-specific
human T cells (Riddell et al., unpublished); and those methods can
render T cells subject to programmed cell death (i.e. apoptosis)
upon withdrawal of IL-2 or subsequent stimulation via the T cell
receptor (see the discussion of the papers by Lenardo et al, and
Boehme et al., infra). Earlier methods that relied on the use of
lectins, such as concanavalin A or phytohemagglutinin (see, e.g.,
Van de Griend et al., Transplantation 38: 401-406 (1984), and Van
de Griend et al., J. Immunol. Methods 66: 285-298 (1984)), are even
less satisfactory because the use of such non-specific stimultory
lectins tends to induce a number of phenotypic changes in the
stimulated cells that make them quite different from T cells
stimulated via the CD3 receptor.
[0013] The inability to culture antigen-specific T cell clones to
large numbers has in part been responsible for limiting adoptive
immunotherapy studies for human diseases such as cancer (Rosenberg,
New Engl. J. Med., 316: 1310-1321, 1986; Rosenberg, New Engl. J.
Med., 319: 1676-1680, 1988) and HIV infection (Ho M. et al., Blood
81: 2093-2101, 1993) to the evaluation of activated polyclonal
lymphocyte populations with poorly defined antigen specificities.
In such studies, polyclonal populations of lymphocytes are either
isolated from the blood or the tumor filtrate and cultured in high
concentrations of the T cell growth factor IL-2. In general, these
cells have exhibited little if any MHC-restricted specificity for
the pathogen or tumor and in the minority of patients that have
experienced therapeutic benefit, it has been difficult to discern
the effector mechanism involved. Typically, adoptive immunotherapy
studies with non-specific effector lymphocytes have administered
approximately 2.times.10.sup.10 to 2.times.10.sup.11 cells to the
patient. (See, e.g., U.S. Pat. No. 5,057,423, at column 1, lines
40-43).
[0014] The development of efficient cell culture methods to rapidly
grow T lymphocytes will be useful in both diagnostic and
therapeutic applications. In diagnostic applications, the ability
to rapidly expand T cells from a patient can be used, for example,
to quickly generate sufficient numbers of cells for use in tests to
monitor the specificity, activity, or other attributes of a
patient's T lymphocytes. Moreover, the capability of rapidly
achieving cell doses of 10.sup.9-10.sup.10 cells will greatly
facilitate the applicability of specific adoptive immunotherapy for
the treatment of human diseases.
[0015] There are several established methods already described for
culturing cells for possible therapeutic use including methods to
isolate and expand T cell clones. Typical cell culture methods for
anchorage-dependent cells, (i.e., those cells that require
attachment to a substrate for cell proliferation) are limited by
the amount of surface area available in culture vessels used (i.e.,
multi-well plates, petri dishes, and culture flasks). For
anchorage-dependent cells, the only way to increase the number of
cells grown is generally to use larger vessels with increased
surface area and/or use more vessels. However, hematopoietic cells
such as T lymphocytes are anchorage-independent. They can survive
and proliferate in response to the appropriate growth factors in a
suspension culture without attachment to a substrate. Even with the
ability to grow antigen-specific lymphocytes in a suspension
culture, the methods reported to date have not consistently
produced rapid numerical expansion of T cell clones. For example,
in a study of T cells conducted by Gillis and Watson, it was found
that T cells cultured at low densities, i.e., 5.times.10.sup.3 to
1.times.10.sup.4 cell/ml in the presence of the T cell growth
factor IL-2, proliferated rapidly over a seven day period and
eventually reached a saturation density of 3-5.times.10.sup.5
cells/ml. Gillis, S. and Watson, J. "Interleukin-2 Dependent
Culture of Cytolytic T Cell Lines", Immunological Rev., 54: 81-109
(1981). Furthermore, Gillis and Watson also found that once cells
reached this saturation concentration, the cells would invariably
die. Gillis et al., id.
[0016] Another study reported three different methods for
establishing murine T lymphocytes in long-term culture. Paul et
al., reported that the method most widely used is to grow T
lymphocytes from immunized donors for several weeks or more in the
presence of antigen and antigen-presenting cells (APCs) to provide
the requisite T cell receptor signal and co-stimulatory signals,
and with the addition of exogenous growth factors before attempting
to clone them, Paul, W. E., et al., "Long-term growth and cloning
of non-transformed lymphocytes", Nature, 294: 697-699, (1981). T
cells specific for protein antigens are then cloned by limiting
dilution with antigen and irradiated spleen cells as a source of
APCs. A second method involved growing T cells as colonies in soft
agar as soon as possible after taking the cells from an immunized
donor. The T cells were stimulated in an initial suspension culture
with antigen and a source of APCs, usually irradiated spleen cells.
In this second approach, it was found that, after 3 days, the cells
were distributed in the upper layer of a two-layer soft agar
culture system. The colonies were picked from day 4 to 8 and then
expanded in long-term cultures. The third approach involved
selecting cells for their functional properties rather than their
antigenic specificity and then growing them with a series of
different irradiated feeder cells and growth factor containing
supernatants. Paul, W. E. et al., "Long-term growth and cloning of
non-transformed lymphocytes", Nature, 294: 697-699, (1981). It is
apparent that with each of these methods, it is not possible to
expand individual T cell clones from a single cell to
10.sup.9-10.sup.10 cells in a timely manner. Thus, despite the
ability to clone antigen-specific T cells, and convincing evidence
of the therapeutic efficacy of T cell clones in accepted animal
models, the technical difficulty in culturing human T cells to
large numbers has impeded the clinical evaluation and application
of cellular immunotherapeutic procedures.
[0017] Yet another concern with cultured T cells is that they must
remain capable of functioning in vivo in order to be useful in
immunotherapeutic procedures. In particular, it has been observed
that antigen-specific T cells which were grown long term in culture
in high concentrations of IL-2 may develop cell cycle abnormalities
and lose the ability to return to a quiescent phase when IL-2 is
withdrawn. In contrast, the normal cell cycle consists of four
successive phases: mitosis (or "M" phase) and three phases which
make up the "interphase" stage. During the M phase, the cell
undergoes nuclear division and cytokinesis. The interphase stage
consists of the G1 phase in which the biosynthetic activities
resume at a high rate after mitosis; the S phase in which DNA
synthesis occurs and the G2 phase which continues until mitosis
commences. While in the G1 phase, some cells appear to cease
progressing through the division cycle; and are said to be in a
"resting" or quiescent state (denoted as the "G0" state). Certain
environmental factors (such as a lack of growth factors in serum or
confluence of cell cultures) may cause cells to enter the quiescent
state. Once the factor is restored, the cell should resume its
normal progress through the cycle. However, cells grown in culture
may be unable to enter the quiescent phase when the growth factor
is removed, resulting in the death of these cells. This growth
factor dependence is particularly relevant to cultured T cells. T
lymphocytes that are exposed over a long term to high
concentrations of IL-2 to promote cell growth often will die by a
process called apoptosis if IL-2 is removed or if they are
subsequently stimulated through the T cell receptor, i.e., if they
encounter specific antigens. (see, e.g., Lenardo M. J., Nature,
353: 858-861, 1991; Boehme S. A. and Lenardo M. J., Eur. J.
Immunol., 23: 1552-1560, 1992). Therefore, the culture methods used
to propagate LAK cells or TIL-cells, and prior methods to culture T
cells which predominantly rely on high long-term concentrations of
IL-2 to promote expansion in vitro, may render many of the cells
susceptible to apoptosis, thus limiting or eliminating their
usefulness for cellular immunotherapy.
[0018] It may also be advantageous in cellular immunotherapy
studies to use gene transfer methods to insert foreign DNA into the
T cells to provide a genetic marker, to facilitate evaluation of in
vivo migration and survival of transferred cells, or to confer
functions that may improve the safety and efficacy of transferred T
cells. An established method for stable gene transfer into
mammalian cells is the use of amphotropic retroviral vectors (see,
e.g., Miller A D, Current Topics in Microbiology and Immunology
158: 1-24, 1992). The stable integration of genes into the target
cell using retrovirus vectors requires that the cell be actively
cycling, specifically that these cells transit M phase of the cell
cycle. Prior studies have introduced a marker gene into a small
proportion of polyclonal T cells driven to proliferate with high
doses of IL-2, and these cells were reinfused into humans as tumor
therapy and provided a means of following the in vivo survival of
transferred cells. (Rosenberg et al. New Engl. J. Med., 323:
570-578, 1990). However, for human T cells (which cycle slowly when
grown with standard techniques) the efficiency of stable gene
transfer is very low, in the range of 0.1-1% of T cells. (Springett
C M et al. J. Virology, 63: 3865 , 1989). Culture methods which
more efficiently recruit the target T cells into the S and G2-M
phases of the cell cycle may increase the efficiency of gene
modification using retrovirus-mediated gene transfer (Roe T. et
al., EMBO J, 2: 2099-2108, 1993), thus improving the prospects for
using genetically-modified T cells in cellular immunotherapy or
using T cells to deliver defective genes in genetic deficiency
diseases.
[0019] The rapid expansion method described by S. Riddell et al.
(in PCT Publication WO 96/06929, published 7 Mar. 1996),
hereinafter referred to as "high-PBMC REM" or "hp-REM" was
developed to provide functional, antigen-specific T cell clones for
use in clinical adoptive immunotherapy protocols. The hp-REM
protocol was designed to provide maximal T cell expansion in a
limited amount of time without loss of T cell function and
specificity. Generally, the hp-REM protocol involves the steps of
adding an initial T lymphocyte population to a culture medium in
vitro; adding to the culture medium a disproportionately large
number of non-dividing peripheral blood mononuclear cells ("PBMC")
as feeder cells such that the resulting population of cells
contains at least about 40 PBMC feeder cells (preferably at least
about 200, more preferably at least about 400) for each T
lymphocyte in the initial population to be expanded; and incubating
the culture. In preferred embodiments of the hp-REM protocol, the T
cells to be expanded are also exposed to a disproportionately large
number of EBV-transformed lymphoblastoid cells ("LCL"), to an
anti-CD3 monoclonal antibody (e.g., OKT3) (to activate the T cells
via the T cell antigen receptor), and to the T cell growth factor
interleukin-2 (IL-2).
[0020] In the hp-REM protocol, T cells are generally expanded using
a vast excess of feeder cells consisting of peripheral blood
mononuclear cells (PBMC) and possibly also EBV-transformed
lymphoblastoid cells (EBV-LCL). T cells to be expanded typically
represent less than about 0.2% of the cells in the hp-REM culture
method. As described, the T cells can be activated through the T
cell antigen receptor using an anti-CD3 monoclonal antibody (e.g.
OKT3) and T cell proliferation can be induced using IL-2. Such
hp-REM culture conditions were reported to result in a level of T
cell expansion 100 to 200-fold greater than that reported by
others.
[0021] However, for most uses, it would be preferable to avoid the
use of large excesses of feeder cells (i.e. PBMC and EBV-LCL) in
the preparation of T cells destined for clinical use. For example,
PBMCs are derived from human blood and could represent a potential
source of adventitious agents (e.g. human imunodeficiency virus,
type 1 and 2; human T cell leukemia virus I, type 1 and 2; and
hepatitis virus, such as hepatitis B, C and G), and EBV-LCL could
represent a potential source of Epstein-Barr virus. In addition,
the large-scale application of the hp-REM protocol would require a
large supply of human peripheral blood to provide adequate numbers
of feeder cells.
[0022] It would therefore be particularly advantageous to reduce
the numbers of such feeder cells required or to replace them
entirely. With these concerns in mind, the methods of the present
invention (hereinafter referred to as "low-PBMC-REM" or
"modified-REM") are designed to achieve rapid in vitro expansion of
T cells without using the vast excess of PBMC and/or EBV-LCL feeder
cells that are the key characteristic of the hp-REM protocol.
BRIEF SUMMARY OF THE INVENTION
[0023] This invention provides a method for rapidly producing large
numbers of T cells, including human antigen-specific cytolytic and
helper T cells, isolated from an initial population of T cells,
without using the vast excess of PBMC and/or EBV-LCL feeder cells
that are the key characteristic of the hp-REM protocol. While the
methods of the present invention are applicable to the rapid
expansion of T lymphocytes, generally, the rapid expansion method
will be especially advantageous in situations in which an
individual T cell clone must be expanded to provide a large
population of T lymphocytes. Thus, the present invention provides
an especially important tool in the context of human adoptive
immunotherapy, as has been exemplified in studies (using hp-REM,
described below) involving human bone marrow transplant recipients
at the Fred Hutchinson Cancer Research Center. The present
invention also provides a method to improve the efficiency of
stable gene transfer into T lymphocytes, as exemplified below.
[0024] Accordingly, one object of the invention is to rapidly
expand T lymphocytes to large numbers in vitro without using the
vast excess of PBMC and/or EBV-LCL feeder cells that are the key
characteristic of the hp-REM protocol. Such rapidly expanded T cell
populations can be used, inter alia, for infusion into individuals
for the purpose of conferring a specific immune response, as
exemplified herein. T cells that can be expanded using the present
invention include any of the various T lymphocyte populations
described herein (see, e.g., the discussion above regarding CTLs,
helper T cells and other T lymphocytes, and the potential uses of
such cells in immunotherapeutic techniques).
[0025] Another object of the invention is to use the method to grow
T cells in a manner which facilitates the stable introduction of
foreign genetic material which can be used to alter the function of
T cells to be used in cellular immunotherapies, as described above,
or to otherwise overcome for a defective or inadequate gene in the
host.
[0026] A number of preferred embodiments of the present invention
are described in the following enumeration:
[0027] 1. A method (referred to herein as "low-PBMC-REM" or
"modified-REM") for rapidly expanding an initial T lymphocyte
population in culture medium in vitro, comprising the steps of:
adding an initial T lymphocyte population to a culture medium in
vitro; adding to the culture medium a non-dividing mammalian cell
line expressing at least one T-cell-stimulatory component, wherein
said cell line is not an EBV-transformed lymphoblastoid cell line
(LCL); and incubating the culture. REM cultures will generally be
incubated under conditions of temperature and the like that are
suitable for the growth of T lymphocytes. For the growth of human T
lymphocytes, for example, the temperature will generally be at
least about 25 degrees Celsius, preferably at least about 30
degrees, more preferably about 37 degrees. Descriptions of suitable
media and other culture conditions are well-known in the art, and
are also exemplified herein.
[0028] 2. A rapid expansion method according to the preceding item,
wherein said T-cell-stimulatory component is selected from the
group consisting of an Fc-1 receptor, a cell adhesion-accessory
molecule and a cytokine.
[0029] 3. A rapid expansion method according to any of the
preceding items, wherein said T-cell-stimulatory component is
selected from the group consisting of an Fc-1 receptor, a cell
adhesion-accessory molecule and a cytokine, and wherein said
initial T lymphocyte population is expanded at least 200-fold after
an incubation period of less than about two weeks.
[0030] 4. A rapid expansion method according to any of the
preceding items, wherein said T-cell-stimulatory component is
selected from the group consisting of an Fc-.gamma. receptor, a
cell adhesion-accessory molecule and a cytokine, and wherein said
initial T lymphocyte population is expanded at least 500-fold after
an incubation period of less than about two weeks.
[0031] 5. A rapid expansion method according to any of the
preceding items, wherein said T-cell-stimulatory component is
selected from the group consisting of an Fc-1 receptor, a cell
adhesion-accessory molecule and a cytokine, and wherein said
initial T lymphocyte population is expanded at least 1000-fold
after an incubation period of less than about two weeks.
[0032] 6. A rapid expansion method according to any of the
preceding items, further comprising the step of adding anti-CD3
monoclonal antibody to the culture medium wherein the concentration
of anti-CD3 monoclonal antibody is at least about 1.0 ng/ml.
Typically, a concentration of about 10 ng/ml is employed although
much lower levels can be used, as illustrated below.
[0033] 7. A rapid expansion method according to any of the
preceding items, further comprising the step of adding IL-2 to the
culture medium, wherein the concentration of IL-2 is at least about
10 units/ml. Typically, a concentration of about 25 units/ml is
used. Preferably, the incubation is continued for at least about 9
days and wherein the step of adding IL-2 to the culture medium is
repeated after each 3-5 day interval. Typically, IL-2 is added on
day 0, again on day 5 or 6, and again on day 8 or 9.
[0034] 8. A rapid expansion method according to any of the
preceding items, wherein said mammalian cell line comprises at
least one cell type that is present at a frequency at least twice
that found in human peripheral blood mononuclear cells (human
PBMCs); preferably at least three times, at least ten times, or at
least fifty times the frequency generally found in human PBMCs.
[0035] 9. A rapid expansion method according to any of the
preceding items, wherein said T-cell-stimulatory component is
selected from the group consisting of an Fc-.gamma. receptor and a
cell adhesion-accessory molecule.
[0036] 10. A rapid expansion method according to any of the
preceding items, wherein said T-cell-stimulatory component is
selected from the group consisting of a cell adhesion-accessory
molecule and a cytokine.
[0037] 11. A rapid expansion method according to any of the
preceding items, wherein said T-cell-stimulatory component is
selected from the group consisting of an Fc-.gamma. receptor and a
cytokine.
[0038] 12. A rapid expansion method according to any of the
preceding items, wherein said mammalian cell line expresses a cell
adhesion-accessory molecule.
[0039] 13. A rapid expansion method according to any of the
preceding items, wherein said cell adhesion-accessory molecule is
selected from the group consisting of Class II MHC, Class I MHC,
ICAM 1, ICAM 2, ICAM 3, CD58, CD72, fibronectin, ligand to CD27,
CD80, CD86 and hyaluronate.
[0040] 14. A rapid expansion method according to any of the
preceding items, wherein said mammalian cell line expresses a
cytokine. Preferably the cytokine is an interleukin.
[0041] 15. A rapid expansion method according to any of the
preceding items, wherein said T-cell-stimulatory component is a
molecule that binds to CD21.
[0042] 16. A rapid expansion method according to any of the
preceding items, wherein said cytokine is selected from the group
consisting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-IS.
[0043] 17. A rapid expansion method according to any of the
preceding items, further comprising the step of adding a soluble
T-cell-stimulatory factor to the culture medium.
[0044] 18. A rapid expansion method according to any of the
preceding items, wherein said soluble T-cell-stimulatory factor is
selected from the group consisting of a cytokine, an antibody
specific for a T cell surface component, and an antibody specific
for a component capable of binding to a T cell surface
component.
[0045] 19. A rapid expansion method according to any of the
preceding items, wherein said soluble T-cell-stimulatory factor is
a cytokine selected from the group consisting of IL-1, IL-2, IL-4,
IL-6, IL-7, IL-12 and IL-15.
[0046] 20. A rapid expansion method according to any of the
preceding items, wherein said soluble T-cell-stimulatory factor is
an antibody specific for a T cell surface component, and wherein
said T cell surface component is selected from the group consisting
of CD4, CD8, CD11a, CD2, CD5, CD49d, CD27, CD28 and CD44.
[0047] 21. A rapid expansion method according to any of the
preceding items, wherein said soluble T-cell-stimulatory factor is
an antibody specific for a component capable of binding to a T cell
surface component, and wherein said T cell surface component is
selected from the group consisting of CD4, CD8, CD11a, CD2, CD5,
CD49d, CD27, CD28 and CD44.
[0048] 22. A rapid expansion method according to any of the
preceding items, wherein said soluble T-cell-stimulatory factor is
a molecule that binds to CD21.
[0049] 23. A rapid expansion method according to any of the
preceding items, wherein said molecule that binds to CD21 is an
anti-CD21 antibody.
[0050] 24. A rapid expansion method according to any of the
preceding items, further comprising the step of adding to the
culture a multiplicity of peripheral blood mononuclear cells
(PBMCs). Preferably, PBMC are irradiated with gamma rays in the
range of about 3000 to 3600 rads, more preferably at about 3300
rads.
[0051] 25. A rapid expansion method according to any of the
preceding items, wherein the ratio of PBMCs to initial T cells to
be expanded is less than about 40:1.
[0052] 26. A rapid expansion method according to any of the
preceding items, wherein the ratio of PBMCs to initial T cells to
be expanded is less than about 10:1.
[0053] 27. A rapid expansion method according to any of the
preceding items, wherein the ratio of PBMCs to initial T cells to
be expanded is less than about 3:1.
[0054] 28. A rapid expansion method according to any of the
preceding items, further comprising the step of adding to the
culture a multiplicity of EBV-transformed lymphoblastoid cells
(LCLs). Preferably, PBMC are irradiated with gamma rays in the
range of about 6000 to 10,000 rads, more preferably at about 8000
rads.
[0055] 29. A rapid expansion method according to any of the
preceding items, wherein the ratio of LCLs to initial T cells to be
expanded is less than about 10:1.
[0056] 30. A rapid expansion method according to any of the
preceding items, wherein the initial T lymphocyte population
comprises at least one human CD8+ antigen-specific cytotoxic T
lymphocyte (CTL). In preferred embodiments of the present
invention, the CTL is specific for an antigen present on a human
tumor or encoded by a pathogen such as a virus or bacterium.
[0057] 31. A rapid expansion method according to any of the
preceding items, wherein the initial T lymphocyte population
comprises at least one human CD4+ antigen-specific helper T
lymphocyte.
[0058] 32. A method of genetically transducing a human T cell,
comprising the steps of: adding an initial T lymphocyte population
to a culture medium in vitro; adding to the culture medium a
non-EBV-transformed mammalian cell line expressing a
T-cell-stimulatory component; and incubating the culture; and
adding a vector to the culture medium. A vector refers to a unit of
DNA or RNA in a form which is capable of being introduced into a
target cell. Transduction is used generally to refer to the
introduction of such exogenous DNA or RNA into a target cell and
includes the introduction of heterologous DNA or RNA sequences into
target cells by, e.g., viral infection and electroporation. A
currently preferred method of transducing T lymphocytes is to use
retroviral vectors, as exemplified herein.
[0059] 33. A genetic transduction method according to item 32,
wherein the vector is a retroviral vector containing a selectable
marker providing resistance to an inhibitory compound that inhibits
T lymphocytes, and wherein the method further comprises the steps
of: continuing incubation of the culture for at least one day after
addition of the retroviral vector; and adding said inhibitory
compound to the culture medium after said continued incubation
step. Preferably, the retroviral vector contains both a positive
and a negative selectable marker. Preferred positive selectable
markers are derived from genes selected from the group consisting
of hph, neo, and gpt, and preferred negative selectable markers are
derived from genes selected from the group consisting of cytosine
deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Especially
preferred markers are bifunctional selectable fusion genes wherein
the positive selectable marker is derived from hph or neo, and the
negative selectable marker is derived from cytosine deaminase or a
TK gene.
[0060] 34. A genetic transduction method according to any of items
32-33, further comprising adding a multiplicity of human PBMCs.
[0061] 35. A rapid expansion method according to any of items
32-34, wherein the ratio of PBMCs to initial T cells is less than
about 40:1.
[0062] 36. A genetic transduction method according to any of items
32-35, further comprising adding non-dividing EBV-transformed
lymphoblastoid cells (LCL).
[0063] 37. A rapid expansion method according to any of items
32-36, wherein the ratio of LCL to initial T cells is less than
about 10:1.
[0064] 38. A method of generating a REM cell line capable of
promoting rapid expansion of an initial T lymphocyte population in
vitro, comprising the steps of: depleting one or more cell types
from a human PBMC population to produce a cell-type-depleted PBMC
population, using said cell-type-depleted PBMC population in place
of non-depleted PBMCs in an hp-REM protocol to determine the
contribution of the depleted cell type to the activity provided by
the non-depleted PBMCs, identifying a T cell stimulatory activity
provided by said depleted cell type, and transforming a mammalian
cell line with a gene allowing expression of said T cell
stimulatory activity.
[0065] 39. A method of generating a REM cell line according to item
38, wherein said T-cell-stimulatory component is selected from the
group consisting of an Fc-.gamma. receptor, a cell
adhesion-accessory molecule and a cytokine.
[0066] 40. A REM cell line capable of stimulating rapid expansion
of an initial T lymphocyte population in vitro, comprising a
mammalian cell line generated according to a method according to
the preceding item 38 or item 39.
[0067] 41. A REM cell line according to item 40, wherein said cell
line expresses a cell adhesion-accessory molecule.
[0068] 42. A REM cell line according to any of items 40-41, wherein
said cell adhesion-accessory molecule is selected from the group
consisting of Class II MHC, Class I MHC, ICAM 1, ICAM 2, ICAM 3,
CD58, CD72, fibronectin, ligand to CD27, CD80, CD86 and
hyaluronate.
[0069] 43. A REM cell line according to any of items 40-42, wherein
said cell line expresses an Fc-.gamma. receptor.
[0070] 44. A REM cell line according to any of items 40-43, wherein
said cell line expresses at least one T cell stimulatory
cytokine.
[0071] 45. A REM cell line according to any of items 4044, wherein
said T cell stimulatory cytokine is selected from the group
consisting of IL-1, IL-2, IL-6, IL-7, IL-12 and IL-15.
[0072] 46. A REM cell line according to any of items 40-44, wherein
said cell line expresses a molecule that binds CD21. As used
herein, a molecule that binds CD21 can be a natural or synthetic
molecule known or determined to bind to the CD21 cell surface
determinant. Molecules known to bind to CD21 include anti-CD21
antibodies, as well as molecules such as C3d, C3dg, iC3b and EBV
gp350/220, and derivatives thereof.
[0073] 47. A culture medium capable of rapidly expanding an initial
T lymphocyte population in vitro comprising a REM cell line
according to any of items 4046.
[0074] 48. A culture medium according to item 47, further
comprising an exogenous cytokine.
[0075] 49. A culture medium according to any of items 4748, further
comprising a multiplicity of exogenous cytokines, wherein said
multiplicity comprises at least one interleukin.
[0076] 50. A culture medium according to any of items 4749, wherein
said interleukin is selected from the group consisting of IL-1,
IL-2, IL-6, IL-7, IL-12 and IL-15.
[0077] 51. A culture medium according to any of items 47-50,
further comprising a molecule that binds to CD21. As used herein, a
molecule that binds CD21 can be a natural or synthetic molecule
known or determined to bind to the CD21 cell surface determinant.
Molecules known to bind to CD21 include anti-CD21 antibodies, as
well as molecules such as C3d, C3dg, iC3b and EBV gp350/220, and
derivatives thereof that bind to CD21.
[0078] 52. A culture medium according to item 51, wherein said
molecule that binds to CD21 is an anti-CD21 antibody.
[0079] 53. A culture medium according to any of items 49-52,
further comprising an anti-CD3 monoclonal antibody.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS AND APPLICATIONS OF
THE INVENTION
[0080] The invention described herein provides methods for rapidly
expanding populations of T lymphocytes, including human cytotoxic T
lymphocytes and helper T lymphocytes, which can be particularly
useful in cellular immunotherapy of human diseases, without using
the vast excess of PBMC and/or EBV-LCL feeder cells that are the
key characteristic of the hp-REM protocol.
[0081] The T cells will be referred to as "target T cells". In
general, target T cells are added in small numbers to a culture
vessel containing standard growth medium that has been supplemented
with components that stimulate rapid expansion in vitro (REM) as
described herein. Preferably, human recombinant IL-2 or another
suitable IL-2 preparation is added in low concentrations at 3-5 day
intervals (typically on "day 0" (i.e. at culture initiation) or
"day 1" (the day following inititiation), again on day 5 or 6, and
again on day 8 or 9). REM protocols result in a rapid expansion of
T cells, typically in the range of a 500- to 3000-fold expansion
within 8 to 14 days. Such methods can thus achieve expansion rates
that are approximately 100- to 1000-fold more efficient for each
stimulation cycle than previously-described methods of culturing
human T cells.
[0082] Furthermore, REM protocols are applicable to the rapid
expansion of any T cell sub-population including helper T cells and
cytolytic T cells; and to T cell clones of many different antigenic
specificities (e.g., to cytolytic or helper T cells specific for
CMV, HIV, or other viral, bacterial, or tumor-derived antigens). In
addition, REM protocols can be used for both small scale growth
(e.g. to rapidly expand T cells from 10.sup.4 to 10.sup.7 cells);
or for large-scale expansions (e.g. to rapidly expand T cells from
10.sup.6 to greater than 10.sup.10 cells); depending on the size of
culture vessel chosen.
[0083] REM protocols thus make it possible to efficiently expand T
cell clones for use in adoptive immunotherapies by dramatically
shortening the time required to grow the numbers of cells required
to restore, enhance, or modulate human immunity. In the study by
Riddell et al. (Science 257: 238-240, 1992), once T cell clones
were isolated it was necessary to culture the clones for twelve
weeks and to pool multiple clones to achieve the highest
administered cell dose of 1.times.10.sup.9 CD8+ CMV-specific T
cells/m.sup.2 body surface area. Using REM protocols, the expansion
of individual T cell clones to greater than 10.sup.9 cells can be
accomplished in less than three weeks.
[0084] With respect to the rapid expansion methods (i.e. "REM"
technology), the following abbreviations are used to distinguish
the various REM protocols referred to herein. The basic Riddell
protocol (as described above and in the cited Riddell patent
application), which uses a disproportionately large number of PBMC
feeder cells (and preferably also EBV-LCL feeder cells) is referred
to as "high-PBMC REM" or simply "hp-REM". Conversely, the methods
of the present invention, which do not employ such large excesses
of PBMC feeder cells (and preferably no EBV-LCL feeder cells) are
referred to as "low-PBMC REM" or "modified-REM". Such methods are
described in detail below.
[0085] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, cell biology, recombinant DNA, and immunology, which
are within the skill of the art. Such techniques are explained
fully in the literature. See e.g., Sambrook, Fritsch, and Maniatis,
Molecular Cloning: A Laboratory Manual, Second Edition (1989);
Animal Cell Culture (R. I. Freshney, Ed., 1987); Gene Transfer
Vectors for Mammalian Cells (J. M. Miller and M. P. Calos eds.
1987); Handbook of Experimental Immunology, (D. M. Weir and C. C.
Blackwell, Eds.); Current Protocols in Molecular Biology (F. M.
Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Siedman, J.
A. Smith, and K. Struhl, eds., 1987); Current Protocols in
Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.
Shevach and W. Strober, eds., 1991); Oligonucleotide Synthesis (M.
J. Gait Ed., 1984); and the series Methods in Enzymology (Academic
Press, Inc.).
[0086] All patents, patent applications, and publications mentioned
herein, both supra and infra, are hereby incorporated herein by
reference.
[0087] As an aid in understanding this invention, the following is
a list of some abbreviations commonly used herein:
1 CTL cytotoxic T lymphocyte(s) APC antigen-presenting cell(s) CMV
cytomegalovirus HIV human immunodeficiency virus EBV Epstein Barr
virus hIL-2 human interleukin-2 MHC major histocompatibility
complex PBMC peripheral blood mononuclear cell(s) EBV-LCL
EBV-transformed lymphoblastoid cell line (sometimes abbreviated as
simply "LCL") PBS phosphate buffered solution REM rapid expansion
method hp-REM high-PBMC REM lp-REM low-PBMC or "modified" REM
[0088] A "cytokine," as used herein, refers to any of a variety of
intercellular signaling molecules (the best known of which are
involved in the regulation of mammalian somatic cells). A number of
families of cytokines, both growth promoting and growth inhibitory
in their effects, have been characterized including, for example:
interleukins (such as IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9 (P40), IL-10, IL-11, IL-12, IL-13,
IL-14, and IL-15); CSF-type cytokines such as GM-CSF, G-CSF, M-CSF,
LIF, EPO, TPO ("thrombopoietin"), TNF-.alpha., and TNF-.beta.);
interferons (such as IFN-.alpha., IFN-.beta., IFN-.gamma.);
cytokines of the TGF-.beta. family (such as TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, inhibin A, inhibin B, activin A, activin
B); growth factors (such as EGF, VEGF, SCF ("stem cell factor" or
"steel factor"), TGF-.alpha., aFGF, bFGF, KGF, PDGF-A, PDGF-B,
PD-ECGF, INS, IGF-1, IGF-II, NGF-.beta.); .alpha.-type intercrine
cytokines (such as IL-8, GRO/MGSA, PF-4, PBP/CTAP/.beta.TG, IP-10,
MIP-2, KC, 9E3); and .beta.-type intercrine cytokines (such as
MCAF, ACT-2/PAT 744/G26, LD-78/PAT 464, RANTES, G26, I309, JE,
TCA3, MIP-1.alpha.,B, CRG-2); and chemotactic factors (such as
NAP-1, MCP-1, MIP-1.alpha., MIP-1.beta., MIP-2, SIS.beta.,
SIS.delta., SIS.epsilon., PF-4, PBP, .gamma.IP-10, MGSA). A number
of other cytokines are also known to those of skill in the art. The
sources, characteristics, targets and effector activities of these
cytokines have been described and, for many of the cytokines, the
DNA sequences encoding the molecules are also known; see, e.g., R.
Callard & A. Gearing, The Cytokine Facts Book (Academic Press,
1994), and the particular publications reviewed and/or cited
therein, which are hereby incorporated by reference in their
entirety. As referenced in catalogs such as The Cytokine Facts
Book, many of the DNA and/or protein sequences encoding such
cytokines are also generally available from sequence databases such
as GENBANK (DNA); and/or SWISSPROT (protein). Typically, cloned DNA
encoding such cytokines will already be available as plasmids,
although it is also possible to synthesize polynucleotides encoding
the cytokines based upon the published sequence information.
Polynucleotides encoding the cytokines can also be obtained using
polymerase chain reaction (PCR) methodology, as described in the
art. See, e.g., Mullis & Faloona, Met. Enzymology, 155: 355
(1987). The detection, purification, and characterization of
cytokines, including assays for identifying new cytokines effective
upon a given cell type, have also been described in a number of
publications as well as the references referred to herein. See,
e.g., Lymphokines and Interferons, 1987; and DeMaeyer, E., et al.,
"Interferons and Other Regulatory Cytokines," (John Wiley &
Sons 1988).
[0089] A mammalian "cell line", as used herein, refers to a
population of mammalian cells (preferably human cells) that have
undergone repeated propagation in vitro; as distinguished from
"primary cells" taken from an individual such as a human.
Generally, a mammalian cell line will have been propagated in vitro
for at least about 10 generations, more typically at least about 40
generations, most typically at least about 100 generations. Most
preferably, the mammalian cell line can be propagated and
maintained long term (i.e., at least several months in vitro,
preferably at least a year). Such cell lines would include, but are
not limited to, "clonal" lines (in which all of cells of the
population are derived from a single ancestral cell). Conversely, a
mixed peripheral blood population such as PBMCs would not
constitute a mammalian cell line. A mammalian cell line for use in
the present invention may, however, contain a cell type found in
peripheral blood but in that case the cell type will generally be
present at a frequency much higher than is normally found in human
peripheral blood mononuclear cells (at least twice the frequency
generally found in human peripheral blood mononuclear cells;
preferably at least five times, at least ten times, at least twenty
times or at least fifty times the frequency generally found in
human peripheral blood mononuclear cells). A particular "cell type"
might be, for example, one of the cell types typically found in
peripheral blood (such as B lymphocytes, monocytes, cytotoxic T
lymphocytes, helper T lymphocytes, granulocytes, eosinophils or NK
cells); or of a cell type not normally found in peripheral blood
(such as fibroblasts, endothelial cells, etc.); or a more specific
subpopulation of such a cell type (e.g. a subpopulation that is
relatively homogeneous with respect to antigen-specificity or
expression of a particular receptor). Thus, a cell line might be
relatively homogeneous with respect to attributes such as
antigen-specificity or cell surface receptors/ligands, as discussed
in more detail below. By way of illustration, a receptor-specific
monocyte line refers to a population of cells in vitro in which the
majority of cells are monocytes possessing a particular cell
surface receptor (which cell line might have been obtained for
example by transforming a population of monocytes with genes
expressing the particular receptor). Again, by way of illustration,
an antigen-specific CTL cell line refers to a population of cells
in vitro in which the majority of cells are cytotoxic T lymphocytes
specific for a particular antigen such as a viral, bacterial or
tumor antigen (which cell line might have been obtained for example
by exposing a population of T cells to repeated stimulation with a
particular antigen and subsequently enriching for antigen-specific
CTLs).
[0090] Preferably, such a cell line for use with the present
invention will be rendered non-dividing prior to use in the
modified-REM culture (e.g., by irradiation). However, one can
alternatively (or in addition) employ a cell line that is dividing
(preferably at a rate similar to or slower than the expanding T
cells) but which can be subsequently eliminated by virtue of its
having a negative selectable marker (e.g., a suicide gene that can
be used to inhibit or kill cells carrying the gene, or a cell
surface marker that can be used to isolate and/or eliminate cells
carrying the marker). In the latter case, the cell line can be
allowed to expand to some degree in the REM culture before being
negatively selected.
[0091] Preferably, mammalian cell lines to be used with the present
invention are relatively homogeneous lines (i.e. at least 50% of
the cells are of a particular cell type, more preferably at least
70%, at least 90%, at least 95% or at least 99% of the cells are of
a particular cell type). It should be noted, however, that T cells
to be expanded by exposure to such a cell line might also be
exposed to additional cell lines (at the same time or in sequence).
Thus, by way of illustration, a modified-REM culture (containing a
T lymphocyte population to be expanded) might be exposed to one
mammalian cell line or to several such lines. For modified-REM, T
cells to be expanded will be exposed to at least one such mammalian
cell line and/or to a non-cellular mixture of factors (including,
e.g., cytokines, antibodies, soluble ligands, etc.), as discussed
herein.
[0092] The T cells to be propagated in culture (i.e., the "target"
T-cells) can be obtained from the subject to be treated.
Alternatively, T cells can be obtained from a source other than the
subject to be treated, in which case the recipient and transferred
cells are preferably immunologically compatible (or the receipient
is otherwise made immuno-tolerant of the transferred cells).
Typically, the target T cells are derived from tissue, bone marrow,
fetal tissue, or peripheral blood. Preferably, the cells are
derived from peripheral blood. If the T cells are derived from
tissues, single cell suspensions can be prepared using a suitable
medium or diluent.
[0093] Mononuclear cells containing the T lymphocytes can be
isolated from the heterogenous population according to any of the
methods well known in the art. As illustrative examples,
Ficoll-Hypaque gradient centrifugation, fluorescence-activated cell
sorting (FACs), panning on monoclonal antibody coated plates,
and/or magnetic separation techniques can be used (separately or in
combination) to obtain purified populations of cells for expansion
according to the present invention. Antigen-specific T cells can be
isolated by standard culture techniques known in the art involving
initial activation of antigen-specific T cell precursors by
stimulation with antigen-presenting cells and, for a clonal
population, by limiting dilution cultures using techniques known in
the art, such as those described in Riddell and Greenberg (J.
Immunol. Meth., 128: 189-201, 1990); and Riddell et al. (J.
Immunol., 146: 2795-2804, 1991). See also, the Examples below. T
cell clones isolated in microwells in limiting dilution cultures
typically have expanded from a single cell to 2.times.10.sup.4 to
5.times.10.sup.5 cells after 14 days.
[0094] For expansion, T cells can be placed in appropriate culture
media in plastic culture vessels with T cell stimulatory components
as described herein. The initial phase of rapid expansion is
generally carried out in a culture vessel, the size of which
depends upon the number of target cells, and which may typically be
a 25 cm.sup.2 flask. The size of the culture vessel used for
subsequent cycles of T cell expansion depends on the starting
number of T cells and the number of cells needed. Typical starting
cell numbers for different sized culture vessels are as follows:
5.times.10.sup.4 to 2.times.10.sup.5--approximate- ly 25 cm.sup.2
flask; 2.times.10.sup.5 to 5.times.10.sup.5--approximately 75.sup.2
cm flask; 5.times.10.sup.5 to 1.times.10.sup.6--approximately
225-cm.sup.2 flask; and 1.times.10.sup.6 to
2.times.10.sup.6--roller bottle. The approximate initial volume of
media used with each flask is: 25 cm.sup.2--20-30 ml; 75
cm.sup.2--60-90 ml; 225 cm.sup.2--100-200 ml; roller bottle--500
ml.
[0095] For even larger-scale expansions, a variety of culture means
can be used, including for example, spinner flasks, cell culture
bags, and bioreactors (such as hollow-fiber bioreactors).
[0096] As used herein, "feeder cells" are accessory cells that
provide co-stimulating functions in conjunction with T cell
receptor activation (which can be achieved by ligation of the T
cell receptor complex with anti-CD3 monoclonal antibody). PBMC
feeder cells for use in REM can be obtained by techniques known in
the art, for example by leukaphoresis, which is a standard medical
procedure with minimal risks (see, e.g., Weaver et al., Blood 82:
1981-1984, 1993); and these feeder cells can be stored by
cryopreservation in liquid nitrogen until use. LCL can be generated
from peripheral blood B cells by transformation with EBV, for
example the B95-8 strain of EBV, using standard methods (see, e.g.,
Crossland et al., J. Immunol. 146: 4414-20, 1991), or by
spontaneous outgrowth in the presence of cyclosporin A. Such LCL
cells will grow rapidly and indefinitely in culture.
[0097] Prior to adding any feeder cells to the culture vessel
(whether PBMCs or cells derived from a cell line as described
herein), such feeder cells are preferably prevented from undergoing
mitosis. Techniques for preventing mitosis are well known in the
art and include, for example irradiation. For example, any PBMCs
can be irradiated with gamma rays in the range of about 3000 to
4000 rads (preferably PBMCs are irradiated at about 3600 rads); any
LCL can be irradiated with gamma rays in the range of about
6000-12,000 rads (preferably LCL are irradiated at about 10,000
rads); and any cells derived from other cell lines can also be
irradiated with gamma rays in the range of about 6000-12,000 rads.
As discussed above, negatively selectable feeder cells can also be
used.
[0098] Since the antigen specificity of the T cell clone is
generally defined prior to expanding the cell in the culture
system, either autologous or allogeneic feeder cells can be used to
support T cell growth. The ability to use allogeneic feeder cells
is important in situations in which the patient is infected with a
virus that is present in PBMC, e.g., HIV, that could therefore
contaminate the T cell cultures. In such circumstances, the use of
allogeneic feeder cells derived from an individual that is screened
and deemed to be a suitable blood donor by American Red Cross
criteria can be used in the culture method.
[0099] The T cell receptor activation signal (normally provided by
antigen and antigen-presenting cells) can be achieved by the
addition anti-CD3 monoclonal antibodies to the culture system. The
anti-CD3 monoclonal antibody most commonly used is "OKT3", which is
commercially available from Ortho Pharmaceuticals in a formulation
suitable for clinical use. The use of anti-CD3 (".alpha.CD3") mAb
rather than antigen as a means of ligating the T cell receptor
bypasses the need to have a source of antigen-presenting cells,
which for virus-specific T cells would require maintaining large
numbers of suitable autologous cells and infecting these cells in
vitro with high titer virus. A concentration of anti-CD3 monoclonal
antibody of at least about 0.5 ng/ml, preferably at least about 1
ng/ml, more preferably at least about 2 ng/ml, promoted the rapid
expansion of the T cells such that a 500- to 3000-fold expansion
can be achieved within about 10 to 13 days of growth. Typically, a
concentration of about 10 ng/ml anti-CD3 monoclonal antibody was
used.
[0100] Of course, as an alternative to anti-CD3 monoclonal
antibody, the T cell receptors can be activated and the cells
stimulated by the addition of antigen-presenting cells, as
described in Riddell et al., J. Immunol. 146: 2795-2904, 1991.
Suitable antigen-presenting cells include, for example, viral
infected cells, tumor cells, and cells pulsed with the relevant
peptide antigen.
[0101] The culture media for use in the methods of the invention
can be any of the commercially available media, preferably one
containing: RPMI, 25 mM HEPES, 25 .mu.M 2-mercaptoethanol, 4 mM
L-glutamine, and 11% human AB serum. Fetal calf serum can be
substituted for human AB serum. Preferably, after addition of any
feeder cells, anti-CD3 monoclonal antibody, and culture media are
added to the target T cells, and the mixture is allowed to incubate
at 37.degree. C. in a 5% CO.sub.2 humidified atmosphere under
standard cell culture conditions which are well known in the art.
Typically, such conditions may include venting, and addition of
CO.sub.2 if necessary (e.g., 5% CO.sub.2, in a humidified
incubator).
[0102] Preferably, the medium is also supplemented with
interleukin-2 (IL-2). Typically recombinant human IL-2 is used,
although a functional equivalent thereof may also be used.
Preferably, IL-2 is added on day 1, and is re-added at 3-5 day
intervals. Thus, IL-2 was generally added on day 1, on day 5 or 6,
and again on day 8 or 9. Expansion can be improved by using an IL-2
concentration of at least about 5 U/ml, more preferably at least
about 10 U/ml. Generally, a concentration of about 25 U/ml can be
used.
[0103] As described in Riddell et al., supra, antigen-specific T
cells expanded using REM retained their antigen-specific
functionality. For example, four different HIV-specific CD8+
cytotoxic T cell clones retained the ability to kill virus-infected
cells expressing the relevant antigen (i.e. HIV), and did not
acquire non-specific cytolytic activities against irrelevant
virus-infected or transformed target cells. Similarly, four
different CMV-specific CD8+ cytotoxic T cell clones retained the
ability to kill CMV-infected cells, and did not acquire
non-specific cytolytic activities against irrelevant virus-infected
or transformed target cells. These characteristics were also
applicable to CD4+ helper T cells. Thus, antigen-specific CD4+ T
cells propagated using REM retained the ability to proliferate in
response to the appropriate viral antigens and appropriate
antigen-presenting cells (APC). Furthermore, antigen-specific T
cells cultured under REM were also capable of entering a quiescent,
non-dividing phase of the cell cycle; and were capable of remaining
viable for at least 4 weeks in vitro. Thus, aliquots of T cells can
be removed from the cultures at the end of a stimulation cycle
(generally day 12-14), and placed in a culture vessel with a
roughly equal number of irradiated PBMC (without anti-CD3 mAb,
antigen or IL-2).
[0104] The addition of irradiated PBMC as feeder cells during
storage of expanded populations improved the ability of the T cells
to enter a resting phase and to remain viable. Preferably, the
ratio of PBMC feeder cells to resting T cells during storage is at
least about 2:1. Without the addition of PBMC feeder cells,
viability of the T cells generally drops significantly (typically
to levels of about 10% or less).
[0105] As described in Riddell et al., supra, T cells expanded by
REM assumed a small round morphology and 60-95% remained viable by
trypan blue dye exclusion even after 28 days in culture. T cells
propagated by hp-REM also entered a resting phase upon IL-2
withdrawal; and they did not undergo programmed cell death (i.e.
apoptosis) upon restimulation via the antigen-specific T cell
receptor. Upon restimulation (e.g. with anti-CD3 mAb or antigen),
the T cells reacquired responsiveness to IL-2, and can enter the S
and G2 phases of the cell cycle and increased in cell number. Such
characteristics are believed to be important for in vivo survival
of the cells and for the efficacy of cellular immunotherapy. In
contrast, certain previously-described methods for the propagation
of T cells have been reported to cause apoptotic cell death in a
proportion of cells after cytokine withdrawal or T cell receptor
restimulation (see, e.g., Boehme S A and Lenardo M J, Eur. J.
Immunol., 23: 1552-1560, 1992).
[0106] There are a number of different circumstances in which the
introduction of functional genes into T cells to be used in
immunotherapy may be desirable. For example, the introduced gene or
genes may improve the efficacy of therapy by promoting the
viability and/or function of transferred T cells; or they may
provide a genetic marker to permit selection and/or evaluation of
in vivo survival or migration; or they may incorporate functions
that improve the safety of immunotherapy, for example, by making
the cell susceptible to negative selection in vivo as described by
Lupton S. D. et al., Mol. and Cell Biol, 11: 6 (1991); and Riddell
et al., Human Gene Therapy 3: 319-338 (1992); see also the
publications of WO/92 08796 and WO/94 28143 by Lupton et al.,
describing the use of bifunctional selectable fusion genes derived
from fusing a dominant positive selectable marker with a negative
selectable marker.
[0107] Various infection techniques have been developed which
utilize recombinant infectious virus particles for gene delivery.
This represents a currently preferred approach to the transduction
of T lymphocytes of the present invention. The viral vectors which
have been used in this way include virus vectors derived from
simian virus 40 (SV40) (see, e.g., Karlsson et al., Proc. Natl.
Acad. Sci. USA 84 82: 158, 1985); adenoviruses (see, e.g., Karlsson
et al., EMBO J. 5: 2377, 1986); adeno-associated virus (AAV) (see,
e.g., B. J. Carter, Current Opinion in Biotechnology 1992, 3:
533-539); and retroviruses (see, e.g., Coffin, 1985, pp. 17-71 in
Weiss et al. (eds.), RNA Tumor Viruses, 2nd ed., Vol. 2, Cold
Spring Harbor Laboratory, New York). Thus, gene transfer and
expression methods are numerous but essentially function to
introduce and express genetic material in mammalian cells. A number
of the above techniques have been used to transduce hematopoietic
or lymphoid cells, including calcium phosphate transfection (see,
e.g., Berman et al., supra, 1984); protoplast fusion (see, e.g.,
Deans et al., supra, 1984); electroporation (see, e.g., Cann et
al., Oncogene 3: 123, 1988); and infection with recombinant
adenovirus (see, e.g., Karlsson et al., supra; Reuther et al., Mol.
Cell. Biol. 6: 123, 1986); adeno-associated virus (see, e.g.,
LaFace et al., supra); and retrovirus vectors (see e.g., Overell et
al., Oncogene 4: 1425, 1989). Primary T lymphocytes have been
successfully transduced by electroporation (see, e.g., Cann et al.,
supra, 1988) and by retroviral infection (see e.g., Nishihara et
al., Cancer Res. 48: 4730, 1988; Kasid et al., supra, 1990; and
Riddell, S. et al., Human Gene Therapy 3: 319-338, 1992).
[0108] Retroviral vectors provide a highly efficient method for
gene transfer into eukaryotic cells. Moreover, retroviral
integration takes place in a controlled fashion and results in the
stable integration of one or a few copies of the new genetic
information per cell.
[0109] Retroviruses are a class of viruses which replicate using a
virus-encoded, RNA-directed DNA polymerase, or reverse
transcriptase, to replicate a viral RNA genome to provide a
double-stranded DNA intermediate which is incorporated into
chromosomal DNA of an avian or mammalian host cell. Most retroviral
vectors are derived from murine retroviruses. Retroviruses
adaptable for use in accordance with the present invention can,
however, be derived from any avian or mammalian cell source. These
retroviruses are preferably amphotropic, meaning that they are
capable of infecting host cells of several species, including
humans. A characteristic feature of retroviral genomes (and
retroviral vectors used as described herein) is the retroviral long
terminal repeat, or LTR, which is an untranslated region of about
600 base pairs found in slightly variant forms at the 5' and 3'
ends of the retroviral genome. When incorporated into DNA as a
provirus, the retroviral LTR includes a short direct repeat
sequence at each end and signals for initiation of transcription by
RNA polymerase II and 3' cleavage and polyadenylation of RNA
transcripts. The LTR contains all other cis-acting sequences
necessary for viral replication.
[0110] A "provirus" refers to the DNA reverse transcript of a
retrovirus which is stably integrated into chromosomal DNA in a
suitable host cell, or a cloned copy thereof, or a cloned copy of
unintegrated intermediate forms of retroviral DNA. Forward
transcription of the provirus and assembly into infectious virus
occurs in the presence of an appropriate helper virus or in a cell
line containing appropriate sequences enabling encapsidation
without coincident production of a contaminating helper virus. Mann
et al. (Cell 33: 153, 1983) describe the development of cell lines
(e.g., .PSI.2) which can be used to produce helper-free stocks of
recombinant retrovirus. These cells lines contain integrated
retroviral genomes which lack sequences required in cis for
encapsidation, but which provide all necessary gene product in
trans to produce intact virions. The RNA transcribed from the
integrated mutant provirus cannot itself be packaged, but these
cells can encapsidate RNA transcribed from a recombinant retrovirus
introduced into the same cell. The resulting virus particles are
infectious, but replication-defective, rendering them useful
vectors which are unable to produce infectious virus following
introduction into a cell lacking the complementary genetic
information enabling encapsidation. Encapsidation in a cell line
harboring trans-acting elements encoding an ecotropic viral
envelope (e.g., .PSI.2) provides ecotropic (limited host range)
progeny virus. Alternatively, assembly in a cell line containing
amphotropic packaging genes (e.g., PA317, ATCC CRL 9078; Miller and
Buttimore, Mol. Cell. Biol. 6: 2895, 1986) provides amphitropic
(broad host range) progeny virus. Such packing cell lines provide
the necessary retroviral gag, pol and env proteins in trans. This
strategy results in the production of retroviral particles which
are highly infectious for mammalian cells, while being incapable of
further replication after they have integrated into the genome of
the target cell. The product of the env gene is responsible for the
binding of the retrovirus to viral receptors on the surface of the
target cell and therefore determines the host range of the
retrovirus. The PA 317 cells produce retroviral particles with an
amphotropic envelope protein, which can transduce cells of human
and other species origin. Other packaging cell lines produce
particles with ecotropic envelope proteins, which are able to
transduce only mouse and rat cells.
[0111] Numerous retroviral vector constructs have been used
successfully to express many foreign genes (see, e.g., Coffin, in
Weiss et al. (eds.), RNA Tumor Viruses, 2nd ed., vol. 2 (Cold
Spring Harbor Laboratory, New York, 1985, pp. 17-71). Retroviral
vectors with inserted sequences are generally functional, and few
sequences that are consistently inhibitory for retroviral infection
have been identified. Functional polyadenylation motifs inhibit
retroviral replication by blocking retroviral RNA synthesis, and
there is an upper size limit of approximately 11 kb of sequence
which can be packaged into retroviral particles (Coffin, supra,
1985); however, the presence of multiple internal promoters,
initially thought to be problematic (Coffin, supra, 1985), was
found to be well tolerated in several retroviral constructs
(Overell et al., Mol. Cell. Biol. 8: 1803, 1983).
[0112] Retroviral vectors have been used as genetic tags by several
groups to follow the development of murine hematopoietic stem cells
which have been transduced in vitro with retrovirus vectors and
transplanted into recipient mice (Williams et al., Nature 310: 476,
1984; Dick et al., Cell 42: 71, 1985; Keller et al., Nature 318:
149, 1985). These studies have demonstrated that the infected
hematopoietic cells reconstitute the hematopoietic and lymphoid
tissue of the recipient animals and that the cells display a normal
developmental potential in vivo. The marked cells can be visualized
using any of a number of molecular biological techniques which can
demonstrate the presence of the retroviral vector sequences, most
notably Southern analysis and PCR (polymerase chain reaction). The
ability to mark cells genetically using retroviral vectors is also
useful in clinical settings in which the technique can be used to
track grafts of autologous cells. This approach has already been
used to track TILs (tumor-infiltrating lymphocytes) in patients
given TIL therapy for terminal cancer treatment by Rosenberg et al.
(N. Engl. J. Med. 323: 570, 1990). The transduction of these cells
with the marker gene was not associated with in vitro cellular
dysfunction (Kasid et al., Proc. Natl. Acad. Sci. USA 87: 473,
1990).
[0113] Many gene products have been expressed in retroviral
vectors. This can either be achieved by placing the sequences to be
expressed under the transcriptional control of the promoter
incorporated in the retroviral LTR, or by placing them under the
control of a heterologous promoter inserted between the LTRs. The
latter strategy provides a way of coexpressing a dominant
selectable marker gene in the vector, thus allowing selection of
cells which are expressing specific vector sequences.
[0114] It is contemplated that overexpression of a stimulatory
factor (for example, a lymphokine or a cytokine) may be toxic to
the treated individual. Therefore, it is within the scope of the
invention to include gene segments that cause the T cells of the
invention to be susceptible to negative selection in vivo. By
"negative selection" is meant that the infused cell can be
eliminated as a result of a change in the in vivo condition of the
individual. The negative selectable phenotype may result from the
insertion of a gene that confers sensitivity to an administered
agent, for example, a compound. Negative selectable genes are known
in the art, and include, inter alia the following: the Herpes
simplex virus type I thymidine kinase (HSV-1 TK) gene (Wigler et
al., Cell 11: 223, 1977) which confers ganciclovir sensitivity; the
cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the
cellular adenine phosphonbosyltransferase (APRT) gene, bacterial
cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:
33 (1992)).
[0115] In addition, it is useful to include in the T cells a
positive marker that enables the selection of cells of the negative
selectable phenotype in vitro. The positive selectable marker may
be a gene which, upon being introduced into the host cell expresses
a dominant phenotype permitting positive selection of cells
carrying the gene. Genes of this type are known in the art, and
include, inter alia, hygromycin-B phosphotransferase gene (hph)
which confers resistance to hygromycin B, the aminoglycoside
phosphotransferase gene (neo or aph) from Tn5 which codes for
resistance to the antibiotic G418, the dihydrofolate reductase
(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug
resistance (MDR) gene.
[0116] Preferably, the positive selectable marker and the negative
selectable element are linked such that loss of the negative
selectable element necessarily also is accompanied by loss of the
positive selectable marker. Even more preferably, the positive and
negative selectable markers are fused so that loss of one
obligatorily leads to loss of the other. An example of a fused
polynucleotide that yields as an expression product a polypeptide
that confers both the desired positive and negative selection
features described above is a hygromycin phosphotransferase
thymidine kinase fusion gene (HyTK). Expression of this gene yields
a polypeptide that confers hygromycin B resistance for positive
selection in vitro, and ganciclovir sensitivity for negative
selection in vivo. See Lupton S. D., et al., Mol. and Cell. Biology
11: 3374-3378, 1991. In addition, in preferred embodiments, the
polynucleotides of the invention encoding the chimeric receptors
are in retroviral vectors containing the fused gene, particularly
those that confer hygromycin B resistance for positive selection in
vitro, and ganciclovir sensitivity for negative selection in vivo,
for example the HyTK retroviral vector described in Lupton, S. D.
et al. (1991), supra. See also the publications of PCT/US91/08442
and PCT/US94/05601, by S. D. Lupton, describing the use of
bifunctional selectable fusion genes derived from fusing a dominant
positive selectable markers with negative selectable markers.
[0117] Preferred positive selectable markers are derived from genes
selected from the group consisting of hph, neo, and gpt, and
preferred negative selectable markers are derived from genes
selected from the group consisting of cytosine deaminase, HSV-1 TK,
VZV TK, HPRT, APRT and gpt. Especially preferred markers are
bifunctional selectable fusion genes wherein the positive
selectable marker is derived from hph or neo, and the negative
selectable marker is derived from cytosine deaminase or a TK
gene.
[0118] A variety of methods can be employed for transducing T
lymphocytes, as is well known in the art. Typically, one can carry
out retroviral transductions as follows: on day 1 after stimulation
using REM as described herein, one can provide the cells with 20-30
units/ml IL-2; on day 1, 2, or 3, one half of the medium can be
replaced with retroviral supernatant prepared according to standard
methods and the cultures supplemented with S .mu.g/ml polybrene and
20-30 units/ml IL-2; on day 4, the cells are washed and placed in
fresh culture medium supplemented with 20-30 units/ml IL-2; on day
5, the exposure to retrovirus can be repeated; on day 6, the cells
can be placed in selective medium (containing, e.g., an antibiotic
corresponding to an antibiotic resistance gene provided in the
retroviral vector) supplemented with 30 units/ml IL-2; on day 13,
viable cells can be separated from dead cells using Ficoll Hypaque
density gradient separation and then the viable cells can be
subcloned using REM.
[0119] Using an antigen-specific CTLs (55E1, an EBV-specific CD8+
clonal line) and a retroviral vector (LAPSN, Clowes et al., 1994,
J. Clin. Invest. 93: 644 (which allowed for monitoring of alkaline
phosphatase expression by flow cytometry)), high transduction
frequencies can be achieved when the cells are exposed to vector on
day 1, 2 or 3 after initiation of REM.
[0120] As described above, T cells prepared according to the
invention can be used to restore, enhance, and/or modulate immunity
in recipient individuals. By "immunity" is meant a lessening of one
or more physical symptoms associated with a response to infection
by a pathogen, or to a tumor, to which the lymphocyte response is
directed. The amount of cells administered is usually in the range
present in normal individuals with immunity to the pathogen. Thus,
CD8+ CD4- cells are usually administered by infusion, with each
infusion in a range of at least 10.sup.6 to 10.sup.10
cells/m.sup.2, preferably in the range of at least 10.sup.7 to
10.sup.9 cells/m.sup.2. The clones may be administered by a single
infusion, or by multiple infusions over a range of time. However,
since different individuals are expected to vary in responsiveness,
the type and amount of cells infused, as well as the number of
infusions and the time range over which multiple infusions are
given are determined by the attending physician, and can be
determined by routine examination. The generation of sufficient
levels of T lymphocytes (including cytotoxic T lymphocytes and/or
helper T lymphocytes) is readily achievable using the rapid
expansion method of the present invention, as exemplified
herein.
[0121] It has also been observed that T cells expanded using REM
exhibited very high levels of transduction using vectors such as
retroviral vectors which will be of great use in the contexts of
cellular immunotherapy and gene therapy using lymphocytes.
[0122] The examples in Riddell et al., supra, exemplify the basic
REM protocol (i.e. hp-REM), and also help to illustrate the general
applications of REM technology to the preparation and use of
expanded T cell populations and, in that regard, exemplify
techniques and principles that can also be applied in the context
of modified-REM.
[0123] The examples below illustrate exemplary modifications of the
REM technology according to the present invention (i.e.
modified-REM), to enable a reduction or elimination of the PBMC
and/or EBV-LCL feeder cells that are characteristic of the hp-REM
protocol.
[0124] All of the examples presented below are provided as a
further guide to the practitioner of ordinary skill in the art, and
are not to be construed as limiting the invention in any way.
EXAMPLES
Example 1
[0125] The Contribution of Monocyte Fc-.gamma. Receptors in Rapid
Expansion
[0126] The PBMC feeder cells, which are used in large excess to
drive hp-REM, are a heterogeneous population of cells including B
lymphocytes, T lymphocytes, monocytes, macrophages, and
granulocytes, and Natural Killer ("NK") cells.
[0127] One of the activities believed to be supplied by PBMCs in
the hp-REM protocol is the provision of Fc-.gamma. receptors which
can bind to the Fc portion of IgG antibody molecules. In
particular, it is believed that T cell activation in the hp-REM
protocol can be mediated by binding of Fc-.gamma. receptors
("Fc.gamma.R") on monocytes within the PBMC population to the Fc
portion of anti-CD3 antibody (e.g. OKT3), which can thereby be
"presented" by the monocytes to T cells within the population to be
expanded. Following such activation, T cells are believed to be
capable of initiating an "autocrine" growth stimulatory cycle in
which activated T cells both secrete growth-stimulatory cytokines
and also increase the expression of cell surface receptors for such
cytokines. Supplying Fc-.gamma. receptors, or otherwise effectively
presenting anti-CD3 antibody, is thus believed to function in the
initiation and promotion of T cell expansion.
[0128] Confirming and quantifying the contribution of monocyte
Fc-.gamma. receptors in the hp-REM protocol can be accomplished by
depleting monocytes from the PBMC feeder cells.
[0129] Peripheral blood mononuclear cells can be obtained from any
of a variety of sources, as described above. For the following
examples, buffy coat layers (derived from healthy human donors)
were obtained from a Red Cross blood bank. PBMCs were isolated
using a Ficoll gradient, washed and stored in cell culture media
(at 4 degrees Celsius) using standard techniques as referred to
above.
[0130] A variety of techniques can be used for separating out
various cell types from a mixed population such as PBMCs. By way of
illustration, depletion of the monocyte/macrophage population was
performed using sephadex G-10 chromatography (see, e.g., Section
3.6 in "Current Protocols in Immunology" (Wiley Interscience,
1992)).
[0131] Monocyte depletion was monitored by flow cytometry following
staining with FITC-conjugated anti-CD14 monoclonal antibody
(available from, e.g., PharmaGen). CD14 expression before depletion
was about 7.4% of total cells. Following depletion, it was about
1.5%.
[0132] In order to assess the impact of depleting monocytes on the
ability of PBMCs to promote rapid expansion, a standard hp-REM
protocol was used and monocyte-depleted PBMCs were compared with
non-depleted PBMCs.
[0133] For purposes of illustration, a CTL line (designated "27EB")
was prepared by procedures analogous to those described above.
Briefly, PBMCs were obtained from an individual blood sample and
were cultured with EBV-LCL derived from the same individual. After
two weeks, CD8+ CTLs were isolated by "panning" with a flask coated
with anti-CD8 antibodies (e.g. the AIS-CD8+"CELLector flask" from
Applied Immune Sciences).
[0134] For all of these illustrative examples, PBMCs were
gamma-irradiated at 3600 rads (using a Cs-137 source) and EBV-LCL
were irradiated at 10,000 rads.
[0135] Cultures were generally maintained as described above for
hp-REM except that 10% fetal calf serum was used in place of human
serum; and IL-2 was used at 25 units/ml and was generally first
added on "day 0" (as opposed to 1 day after culture initiation),
and then at 3-5 day intervals (generally, on day 5, and then again
on day 8), as otherwise described above for the hp-REM protocol.
OKT3 was generally used at about 10 ng/ml. Cells were typically
harvested and quantified after 14 days of culture.
[0136] For this example, cultures were established with
5.times.10.sup.4 CTL ("27EB", as described above), 5.times.10.sup.6
irradiated EBV-LCL (i.e. a 100:1 excess over CTLs) (prepared and
irradiated as described above), 10 ng/ml OKT3, 25 U/ml IL-2 and
2.5.times.10.sup.7 irradiated PBMC (i.e. a 500:1 excess over CTLs)
(either monocyte-depleted or nondepleted, prepared and irradiated
as described above). Typical control cultures would include, for
example, cultures without any added CTL. After 14 days of culture,
cells were harvested and quantified.
[0137] In the case of the standard hp-REM protocol using
nondepleted PBMCs, approximately 4.86.times.10.sup.7 T cells were
recovered, representing an expansion of approximately 882-fold.
[0138] As shown in TABLE 1, when monocyte-depleted PBMCs were used
instead, only about 2.7.times.10.sup.7 T cells were recovered,
indicating that the expansion rate had dropped to about 55% of the
control rate.
2TABLE 1 Method of T Cell % Control T PBMC Cells depletion Recovery
cell Expansion Nondepleted PBMC none 4.86 .times. 10.sup.7 100%
Monocyte-depleted Sephadex column 2.70 .times. 10.sup.7 55%
PBMC
[0139] The above results provided further indication that monocytes
within the PBMC population apparently contribute significantly to
the ability of PBMCs to bring about the rapid expansion of T cells.
In the following example, the ability to provide Fc.gamma.R
activity or its equivalent from a source other than PBMCs as a
means for reducing the dependence of REM on large excesses of PBMC
feeder cells.
Example 2
[0140] Replacement of Monocyte Fc-.gamma. Receptor Activity in
Modified-REM
[0141] As discussed above, Fc-.gamma. receptors found on monocytes
are believed to be responsible for a significant portion of the
stimulatory activity supplied by PBMCs in the presence of
antibodies such as anti-CD3 antibody (e.g. OKT3).
[0142] Having identified a stimulatory component supplied by the
heterogeneous PBMC population, it is possible to reduce the
dependence on PBMCs themselves by providing that activity (or its
equivalent) from another source. Preferably, for use in
modified-REM as described herein, the identified stimulatory
activity will be provided by a mammalian cell line or as a
non-cellular additive to the REM culture. Illustrative examples of
both are provided below.
[0143] a. Use of Mammalian Cell Lines in Modified-REM
[0144] Cell lines expressing one of more identified T cell
stimulatory activities provided by PBMCs (or LCL) can be
effectively used to reduce the dependence of REM on such PBMC (or
LCL) feeder cells. Where such a cell line is to be incorporated
into the protocol, it is preferable, as described above, that the
cell line not be a potential source of adventitious agents such as
viruses. Accordingly, the supplemental cell line used is preferably
not an EBV-transformed cell line (such as EBV-LCL). Also, for the
rapid expansion of human T cells, it is generally preferable to use
a cell line derived from a higher mammal, especially a primate,
most preferably a human.
[0145] Mammalian cell lines expressing Fc-.gamma. receptors have
been described in the literature and can be obtained from a variety
of sources. For example, a number of human tumor lines have been
demonstrated to express Fc-.gamma. receptors (see, e.g., R. J.
Looney et al., J. Immunol. 136: 1641 (1986) (describing K562, an
erythroleukemia cell line); S. J. Collins at el. 1977. Nature 270:
347 (1977) (describing HL60, a promyelocytic cell line); C. Lozzio
and B. Lozzio, Blood 45: 321 (1975) (describing U937, a histiocytic
lymphoma cell line); and G. R. Crabtree et al., Cancer Res. 38:
4268 (1979).
[0146] Cell lines expressing Fc-.gamma. receptors can also be
readily prepared using standard molecular biological techniques. By
way of illustration, Fc.gamma.R-positive cell lines can be obtained
by immortalizing cells that already express Fc-.gamma. receptors
using any of a variety of well-known techniques for transforming
mammalian cells. Alternatively, an existing cell line such as a
human cell line can be genetically modified to express Fc-.gamma.
receptors by introducing genes encoding Fc-.gamma. receptors into
the cells. Thus, a cell line of choice, such as a human cell line
already expressing a stimulatory component such as a cytokine or a
cell adhesion-accessory molecule (both of which are discussed
below), can be further modified by introduction of genes encoding
an Fc.gamma.R.
[0147] By way of illustration, human monocytes apparently express
two distinct Fc-.gamma. receptor types ("Fc.gamma.RI" and
"Fc.gamma.RII") which differ in their affinity for IgG antibody
binding, (see, e.g., Ravetch and Kinet Annu. Review of Immunol. 9:
457-492, 1991). In particular, Fc.gamma.RI is generally a high
affinity receptor (K.sub.a=10.sup.8-10.sup.9) while Fc.gamma.RII is
generally a lower affinity receptor (K.sub.a-10.sup.7). The genes
for both Fc.gamma.RI and Fc.gamma.RII have been identified and
cloned. Previous studies have demonstrated that fibroblasts
expressing the Fc.gamma.RII receptor following gene transfer could
effectively restore anti-CD3-dependent proliferation of
monocyte-depleted T lymphocyte cultures (see, e.g., Peltz et al.,
J. Immunol. 141: 1891 (1988)). Thus, a cell line genetically
modified to express Fc.gamma.R should be capable of supplying a
significant portion of the stimulatory activity supplied by PBMCs
in the hp-REM protocol. Use of such a cell line, potentially in
conjunction with other components such as cytokines or
adhesion-accessory molecules as described below, would thereby
enable a decrease in the number of PBMCs required for rapid
expansion.
[0148] b. Use of Non-Cellular Additives for Modified-REM
[0149] In addition to providing T cell stimulatory components by
way of a cell line, such as described above, it will be possible to
provide a number of components (or their functional equivalents) as
non-cellular additives to the modified-REM culture medium. Thus, as
a different alternative to the Fc.gamma.R activity apparently
contributed by PBMC monocytes, it will be possible to provide a
substitute or structural equivalent for Fc.gamma.R activity. For
example, an alternative means for achieving "presentation" of
antibodies such as anti-CD3 antibodies to T cells is to conjugate
such antibodies to beads (such as sephadex beads or magnetic
beads).
[0150] In order to assess the ability of such bead-conjugated
antibodies to substitute for soluble antibodies (which are
presumably presented via Fc.gamma.R), we conducted experiments to
determine whether anti-CD3-conjugated magnetic beads can
effectively replace soluble anti-CD3 monoclonal antibodies in the
REM protocol.
[0151] Anti-CD3-conjugated magnetic beads ("BioMag anti-CD3") were
obtained from Perceptive Diagnostics. The particles used were
approximately 1 .mu.m in size and had covalently attached anti-CD3
monoclonal antibodies loaded at approximately 20 .mu.g
antibody/1.times.10.sup.7 beads. A range of beads was chosen to
approximate the number of antigen presenting cells ("APCs")
estimated to be present within the PBMC population used in
hp-REM.
[0152] For quantifying the actual impact on hp-REM, T cell
expansion cultures containing 5.times.10.sup.4 CTL,
5.times.10.sup.6 irradiated EBV-LCL, 2.5.times.10.sup.7 irradiated
allogeneic PBMC and 25 U/ml IL-2 were established, essentially as
described above in Example 1. Either 10 ng/ml of soluble anti-CD3
antibody (OKT3) or various quantities of anti-CD3-conjugated beads
were added as the T cell activation reagent. Cell cultures were
expanded and T cells counted, essentially as described for Example
1.
[0153] The results are shown in TABLE 2. While T cell expansion
using anti-CD3-conjugated beads was somewhat less than with soluble
OKT3 (in the range of about 80%), the results suggest that
antibody-coated beads would be capable of inducing substantial
levels of T cell activation/proliferation within a modified REM
protocol. More detailed quantification of the relative role of
anti-CD3 presentation as compared to other activities potentially
provided by APCs within the PBMC population can be readily obtained
by assessing the expansion rates obtainable with anti-CD3 beads
using APC-depleted cultures, either in the presence or absence of
various cytokines or other soluble stimulatory factors (which are
described in more detail below).
3TABLE 2 Anti-CD3 source T Cell Recovery T Cell Expansion Soluble
anti-CD3 Ab (OKT3) 6.52 .times. 10.sup.7 1304-fold 1 .times.
10.sup.7 BioMag anti-CD3 5.36 .times. 10.sup.7 1071-fold 5 .times.
10.sup.6 BioMag anti-CD3 5.0 .times. 10.sup.7 1000-fold 2.5 .times.
10.sup.6 BioMag anti-CD3 5.25 .times. 10.sup.7 1051-fold
[0154] Comparisons of the relative efficiency of providing various
PBMC replacement components, as described herein, can be readily
achieved by standard titration analyses in which the various
components are added back at varying concentrations to a
PBMC-limited REM culture (i.e. a culture in which PBMCs are
included at a sub-optimal level). By way of illustration,
experiments described below assessed the impact of adding various
combinations of exogenous cytokines to sub-optimized hp-REM
cultures in which the PBMC population had been reduced to one-half
or one-quarter of an optimal starting level. Analogous assays can
be readily performed for other components such as cells expressing
Fc.gamma.R or adhesion-accessory components, anti-CD3-conjugated
beads, and/or other soluble stimulatory factors (such as monoclonal
antibodies directed to T cell surface components), as described in
more detail below.
Example 3
[0155] The Contribution of B Cells in Rapid Expansion
[0156] In order to quantify the contribution of B lymphocytes to
the stimulus supplied by PBMCs, we examined the relative ability of
B-cell-depleted PBMC populations to support REM.
[0157] Isolation of cells such as B cells (or other cells referred
to herein) can be conveniently achieved using antibodies directed
to a cell surface marker known to be present on the cells to be
depleted. A variety of such markers are well known, including the
various "CD" or "cluster of differentiation" markers; and
antibodies to many such markers are readily obtainable. Also, for
many such markers, beads conjugated with the antibodies are readily
available and can greatly facilitate cell separation.
[0158] By way of illustration, CD19 is a well-known cell surface
marker for B lymphocytes. Magnetic beads that had been conjugated
with anti-CD19 antibodies were obtained from Dynal, and were used
to deplete a PBMC population of B cells, following standard
procedures as described by the manufacturer.
[0159] Depletion was evaluated by fluorescence activated cell
sorting ("FACS") after CD19 staining. The PBMC population was
estimated to contain approximately 12% B cells prior to depletion
and less than 1% B cells after depletion.
[0160] For testing the impact of B cell depletion on hp-REM, T cell
expansion cultures containing 5.times.10.sup.4 CTL,
5.times.10.sup.6 irradiated EBV-LCL, 2.5.times.10.sup.7 irradiated
allogeneic PBMC (B-cell-depleted or nondepleted), 10 ng/ml OKT3 and
25 U/ml IL-2 were established; and, after 14 days, T cells were
harvested and quantified as described above. The results, shown in
TABLE 3, suggested that B cells also contribute to the stimulating
activity supplied by PBMCs.
4TABLE 3 T Cell % Control T PBMC Cells Method of Depletion Recovery
Cell Expansion Nondepleted PBMC none 4.28 .times. 10.sup.7 100%
B-cell-depleted Anti-CD19 9.8 .times. 10.sup.6 23% PBMC Magnetic
Beads
[0161] The decreased levels of T cell expansion in this and the
preceding experiments suggests a role for monocytes and B cells as
antigen presenting cells ("APCs") in the hp-REM protocol. (The
inability to inhibit T cell expansion to the levels observed in
Example 1 to the levels observed in this experiment may be a result
of differences in cell depletion by the various methods used and/or
the presence of small numbers of APC undetected by the assays used.
It should also be noted that monocyte depletion as measured in
Example 1 only reduced the monocyte population from about 7.5% to
about 1.5%.)
[0162] There are a number of other well-known techniques that can
be used to deplete various cell types from the PBMC population and
that can therefore be used to provide additional confirmation and
quantification of the results described herein. Thus, for example,
nylon wool can be used to remove both monocytes and B cells (as
well as any fibroblasts) from the PBMC population. The replacement
of various APC activities in modified-REM is further described in
the following example.
Example 4
[0163] Replacement of Various APC Activities in Modified-REM
[0164] The results obtained in the B-cell-depletion and
monocyte-depletion experiments, described above, indicated that
putative APCs in the PBMC population appear to contribute to the
stimulus supplied by PBMCs. As described in Examples 1-2, the role
of Fc.gamma.R activity in presentation of anti-CD3 antibody is
expected to account for some portion of the activity provided by
putative APCs. Such Fc.gamma.R activity can be supplied by another
(non-PBMC) source, e.g. a cell line expressing Fc.gamma.R or
anti-CD3-conjugated beads, as also described above.
[0165] It is believed that APCs within the PBMC population also
contribute adhesion-accessory molecules and stimulatory cytokines
that would be expected to further enhance the
activation/proliferation process. (Furthermore, as described below,
T lymphocytes within the PBMC population are also expected to
produce stimulatory cytokines as a result of activation via the
anti-CD3 antibody.) The roles of such adhesion-accessory molecules
and cytokines are described in more detail in the examples
below.
Example 5
[0166] The Contribution of Cytokines in REM
[0167] Although anti-CD3 antibody (e.g. OKT3) is used to activate
and induce the proliferation of T cell clones for their in vitro
expansion, the .gamma.-irradiated feeder PBMC population also
contains a substantial population of T lymphocytes that are
believed to be activatable by the anti-CD3 antibody. While such
irradiated feeder cells are incapable of dividing, their activation
via anti-CD3 antibody is believed to result in the secretion of
multiple cytokines which can provide additional
lympho-proliferative signals. For example, in addition to IL-2,
anti-CD3 activation of T cells is believed to result in the
secretion of other stimulatory cytokines including IL-1.alpha. and
.beta., IL-6, IL-8, GM-CSF, IFN-.alpha. and TNF.alpha. and 0 (du
Moulin et. al. 1994. Cytotechnology 15: 365). It is believed that
the secretion of one or more of those cytokines can contribute
substantially to the proliferative stimulus provided by PBMCs
within the hp-REM protocol. Of the numerous other cytokines that
have been characterized, a number of these are known to stimulate
the growth of T cells, including, for example, IL-7 and IL-15.
Others can be readily screened for their ability to enhance T cell
proliferation and for their relative ability to reduce the
dependence of REM on large numbers of PBMCs, as described
herein.
[0168] By way of illustration, we analyzed the ability of a number
of exogenously-supplied cytokines to reconstitute T cell expansion
in REM cultures in which the numbers of PBMC feeder cells had been
reduced to sub-optimal levels, in order to quantify the potential
role of such cytokines in promoting REM.
[0169] Following procedures essentially analogous to those
described above, cultures containing 5.times.10.sup.4 CTL,
5.times.10.sup.6 EBV-LCL, 10 ng/ml OKT3 and 25 U/ml IL-2 were
established with either 2.5.times.10.sup.7 irradiated PBMC (100%
control), 1.25.times.10.sup.7 irradiated PBMC (50%), or
6.12.times.10.sup.6 irradiated PBMC (25%).
[0170] It is believed that a number of cytokines can act
synergistically with IL-2 to promote T cell proliferation. In this
illustrative experiment, the following exogenous cytokines were
added to the cultures either alone or in various combinations as
described: IL-1 (40 U/ml), IL-4 (200 U/ml), IL-6 (500 U/ml) and
IL-12 (20 U/ml).
[0171] The results, shown in TABLE 4, confirmed that such cytokines
can substantially enhance T cell expansion when PBMC populations
are reduced to sub-optimal levels. It is not unexpected that
expansion levels were not returned to that observed with the
optimal number of PBMC feeders, because the PBMC population is
believed to supply additional stimulatory activities as described
herein. The data suggest that replacement of IL-4 with IL-12 in a
cytokine cocktail may further enhance proliferation. The
properties, sources, and DNA and protein sequences of many such
cytokines are described in cytokine reference books such as "The
Cytokine Facts Book" by R. Callard et al., supra. To take a single
example for purposes of illustration, IL-12 is known to be a
heterodimeric cytokine comprising two peptide chains (p35 and p40)
that induces IFN.gamma. production by T lymphocytes and
co-stimulates the proliferation of peripheral blood lymphocytes.
IL-12 also stimulates proliferation and differentiation of TH1 T
lymphocytes, and is known to be produced by B cells,
monocytes/macrophages, and B lymphoblastoid cells. The complete
amino acid sequences for both the p35 and p40 chains are known and
available on Genbank (Accession numbers provided in Callard). The
IL-12 receptor has also been characterized (id.).
[0172] Additional cytokines and cocktails thereof can readily be
tested in an analogous manner; and a comparison of stimulatory
cocktails can then be made using even lower levels of PBMCs.
5TABLE 4 Percent Added Cytokine(s) PBMC Cell Recovery Expansion
Control IL-2 2.5 .times. 10.sup.7 4.4 .times. 10.sup.7 882-fold
100% IL-2 1.25 .times. 10.sup.7 2.8 .times. 10.sup.7 564-fold 64%
IL-2 6.12 .times. 10.sup.6 1.8 .times. 10.sup.7 360-fold 41% IL-2 +
IL-1 1.25 .times. 10.sup.7 3.2 .times. 10.sup.7 648-fold 73% IL-2 +
IL-1 6.12 .times. 10.sup.6 2.4 .times. 10.sup.7 486-fold 55% IL-2 +
IL-4 1.25 .times. 10.sup.7 2.0 .times. 10.sup.7 402-fold 46% IL-2 +
IL-4 6.12 .times. 10.sup.6 1.5 .times. 10.sup.7 295-fold 33% IL-2 +
IL-6 1.25 .times. 10.sup.7 3.2 .times. 10.sup.7 636-fold 72% IL-2 +
IL-6 6.12 .times. 10.sup.6 3.0 .times. 10.sup.7 606-fold 69% IL-2 +
IL-12 1.25 .times. 10.sup.7 4.65 .times. 10.sup.7 930-fold 105%
IL-2 + IL-12 6.12 .times. 10.sup.6 2.88 .times. 10.sup.7 558-fold
63% IL-2 + IL-1 + 1.25 .times. 10.sup.7 3.8 .times. 10.sup.7
768-fold 87% IL-4 + IL-6 IL-2 + IL-1 + 6.12 .times. 10.sup.6 3.1
.times. 10.sup.7 618-fold 70% IL-4 + IL-6
[0173] Further evidence that soluble components of the feeder cell
supernatant can provide an effective stimulus for low-PBMC REM was
obtained by reducing the PBMC population to sub-optimal levels and
using a REM supernatant to provide soluble stimulatory signals.
[0174] Briefly, a standard hp-REM protocol was performed as
described above, using an antigen-specific CTL clone and performing
a 48-hour REM expansion with PBMC (500:1), EBV-LCL (100:1),
anti-CD3-antibody (10 ng/ml), and recombinant human IL-2 (25
units/ml). After 48 hours, the cells were harvested and the
supernatant (`REM supernatant`) was examined as a source of soluble
stimulatory factors in a REM expansion in which PBMC were reduced
to sub-optimal levels (i.e. 1/2, 1/4 or 1/8 of optimal or
"SOP").
[0175] The results, as shown in TABLE 5, confirm that such soluble
factors can provide an effective stimulatory signal in the context
of low-PBMC REM. In particular, a large proportion of the reduction
in fold proliferation levels observed when PBMC are reduced can be
overcome by using the REM supernatant in place of the standard
medium. In addition, the more the PBMC were reduced (i.e. to 1/8 of
optimum), the greater was the observed effect from using the REM
supernatant (1022-fold average expansion using the REM supernatant
versus 359-fold expansion without). Such supernatants and/or their
components such as individual cytokines or "cocktails" thereof can
thus be used to reduce the need for conducting REM with large
excesses of feeder cells such as PBMCs).
6 TABLE 5 Avg. Fold Std. Medium PBMC Proliferation Dev. SOP MEDIUM
SOP 1255 .+-.160 1/2 SOP 1178 .+-.64 1/4 SOP 996 .+-.23 1/8 SOP 359
.+-.29 48 HR. REM SUP. SOP 1253 .+-.144 1/2 SOP 1218 .+-.73 1/4 SOP
1178 .+-.89 1/8 SOP 1022 .+-.77
Example 6
[0176] Replacement of Cytokine Activity in Modified-REM
[0177] As described above, a large number of cytokines have been
described and are widely available, including a number of cytokines
that are known to stimulate T lymphocytes. As will be apparent to
those of skill in the art, such cytokines (whether or not they were
previously known to stimulate T cells) can be readily tested for
their ability to augment rapid expansion using methods such as
those above. In addition, for any of the rapid expansion techniques
described herein, the resulting expanded T cells can be monitored
for the maintenance of various desired characteristics, using
methods such as those illustrated above for hp-REM.
[0178] Cytokines to be used in modified-REM can be introduced to
the target T cells in any of several ways as illustrated herein.
Thus, for example, one or more cytokines can be added to the
medium, as exemplified above. Alternatively, or in addition,
cytokines can also be supplied by cells secreting the cytokines
into the REM medium. Thus, by way of illustration, a mammalian cell
line known to secrete a particular cytokine or combination of
cytokines can be used. Alternatively, a mammalian cell line that
does not already secrete a particular cytokine (or that secretes it
at suboptimal levels) can be readily modified by introducing a gene
encoding the desired cytokine. As is well known, the gene can be
placed under the control of any of a variety of promoters (as
alternatives to its original promoter) so that expression of the
cytokine can be controlled to maximize its effectiveness. The
entire sequences for a large number of cytokines are known and
encoding DNA is often available. Many such sequences are published
in nucleic acid and/or protein databases (such as GenEMBL, GENBANK
or Swissprot); see, e.g., the Cytokine Facts Book, R. E. Callard et
al., Academic Press, 1994). Also, as described above, such
additional mammalian cell lines can be modified to provide several
T cell stimulatory activities at once.
Example 7
[0179] The Role of Accessory-Adhesion Molecules in Rapid
Expansion
[0180] As discussed above, APCs such as monocytes and B cells also
provide other T cell co-stimulatory signals which serve to enhance
T cell activation/proliferation. Thus, while T cell activation
involves the specific recognition of MHC-bound antigenic peptides
on the surface of APCs (which interact with the T cell receptor/CD3
complex), a number of antigen-non-specific receptor:ligand
interactions between APCs and T cells can further enhance T cell
activation/proliferation. In particular, APCs express ligands for a
variety of receptors on T cells, and it appears that T cell
activation/proliferation is the result of a combination of signals
delivered through the T cell receptor and other signaling
molecules. A number of such receptor:ligand interactions have
already been identified and, for a number of those, inhibition of
the receptor:ligand interactions have been reported to inhibit T
cell proliferation and cytokine secretion. By way of illustration,
a number of receptor:ligand pairs that are considered likely to
play a role in T cell activation/proliferation are listed in TABLE
6 below.
7 TABLE 6 Receptor (T cell) Ligand (APC) CD4 Class II MHC CD8 Class
I MHC CD11a (LFA-1) CD54 (ICAM-1) and ICAM 2 & 3 CD2 CD58
(LFA-3) CD5 CD72 CD49d (VLA-4) fibronectin (FN) CD27 ligand to CD27
CD28 CD80 (B7.1) and CD86 (B7.2) CD44 hyaluronate
[0181] While many of these molecules have been reported to function
in adhesion (enhancing cell:cell and/or cell:substrate
interactions), many have also been shown to deliver T cell
co-stimulatory signals such as enhancing intracellular calcium and
the activation of PI and PKC (see, e.g., Geppert et al. 1990.
Immunol. Reviews 117: 5-66).
[0182] The interactions of such adhesion-accessory molecules as
described above have been shown to positively enhance activation of
resting T lymphocytes. Antibodies which bind these accessory
molecules have been shown, under specific conditions, to provide T
cell activation signals (see, e.g., the references cited below).
Also, the addition of purified accessory molecule ligands ICAM-1
and LFA-3 (ligands for CD11a and CD2 respectively) to purified T
cells being stimulated with anti-CD3 monoclonal antibody has been
shown to provide co-stimulatory signals for T cell activation and
proliferation (see, e.g., Semnani et al. 1994. J. Exp. Med. 180:
2125).
[0183] Thus, various antibodies directed against CD4 and CD8 are
capable of either inhibiting T cell activation (see, e.g., I. Bank
and L. Chess. 1985. J. Exp. Med. 162: 1194; G. A. van Seventer.
1986. Eur. J. Immunol. 16: 1363) or synergizing with anti-CD3 mAb
to induce T cell proliferation (see, e.g., F. Emmrich et al. 1986.
PNAS 83: 8298; T. Owens et al. 1987. PNAS 84: 9209; K. Saizawa et
al. 1987. Nature 328: 260). As is well known by those of skill in
the art, a collection of antibodies raised against a particular
antigen would be expected to contain antibodies binding to a
variety of different sites on the antigen.
[0184] A number of studies have shown that antibodies to other
adhesion-accessory molecules are capable of augmenting T cell
stimulation/proliferation. By way of illustration, see, e.g., J. A.
Ledbetter et al. 1985. J. Immunol 135: 2331 (antibodies directed to
CD5 and CD28 augment anti-CD3-induced T cell proliferation); P. J.
Martin et al. 1986. J. Immunol. 136: 3282 (antibodies to CD28
augment anti-CD3-induced T cell proliferation); R. Galandrini et
al. 1993. J. Immunol. 150: 4225, and Y. Shimizu. 1989. J. Immunol
143: 2457 (antibodies directed against CD44 augment anti-CD3
induced T cell proliferation); S. C. Meur et al. 1984. Cell 36: 897
(antibodies directed against the T11.2 and T11.3 epitopes of CD2
stimulate T cell proliferation); R. van Lier. 1987. J. Immunol.
139: 1589 (antibodies directed against CD27 augment
anti-CD3-induced T cell proliferation); Bossy et al. 1995. Eur. J.
Immunol. 25: 459 (antibodies to CD50 (ICAM-3) augment
anti-CD3-induced T cell proliferation); M. C. Wacholtz et al. 1989.
J. Exp. Med. 170: 431 (antibodies directed to LFA-1 augment
anti-CD3-induced proliferation when the two antibodies are
crosslinked on the T cell surface); G. A. van Seventer et. al.
1990. J. Immunol. 144: 4579 (purified ICAM-1 immobilized on plastic
with anti-CD3 mAb co-stimulates T cell proliferation via the LFA-1
molecule); Y. Shimizu et al. 1990. J. Immunol 145: 59 (purified
fibronectin on plastic with anti-CD3 mAb co-stimulates T cell
proliferation, and antibodies to VLA4 and VLA5 inhibited this
activity indicating the role of VLA4 and VLA5 as co-stimulatory T
cell receptors); N. K. Damle et al. 1992. J. Immunol. 148: 1985
(soluble ICAM-1, B7-1, LFA-3 and VCAM augment anti-CD3 induced T
cell proliferation).
[0185] Quantification of the relative contribution of such
adhesion-accessory factors within the REM protocol can be readily
accomplished using deletion techniques and titration experiments in
PBMC-limited hp-REM assays analogous to those illustrated above for
the combinations of various cytokines.
Example 8
[0186] Replacement of Adhesion-Accessory Molecule Activity in
Modified-REM
[0187] In an analogous manner to the modifications described above,
and perhaps in combination with such modifications, the REM
protocol can thus be modified to include a characterized cell line
expressing high levels of these receptor ligands (obtained by,
e.g., gene modification of a cell line of choice or by the
identification of established cell lines already expressing such
molecules). It is also possible to utilize antibodies directed
against accessory molecules known to induce signal transduction
and/or to use purified accessory ligand molecules as means of
substituting for the corresponding activity provided by the PBMC
feeder cells, thereby enabling a reduction in the number of PBMCs
required to drive REM.
Example 9
[0188] Replacement of Additional Stimulatory Activities Provided by
EBV-LCL
[0189] While EBV-LCL do not appear to be sufficient for achieving
maximal T cell expansion, they are capable of augmenting expansion
in the hp-REM protocol. Analysis of EBV-LCL has indicated that they
express adhesion molecules such as LFA-1, ICAM-1, and LFA-3, as
well as Fc.gamma.R. In addition, EBV-LCL secrete IL-1 (Liu et al.
Cell Immunol. 108: 64-75, 1987) and IL-12 (Kobayashi et al., 1989.
J. Exp. Med. 170: 827), both of which are also secreted by
APCs.
[0190] As described above, it is believed that such components can
be readily supplied by other sources--thereby reducing the need for
the large numbers of PBMC and/or EBV-LCL feeder cells
characteristic of hp-REM.
Example 10
[0191] The Use of anti-CD21 Antibody in Modified REM
[0192] CD21 is an accessory molecule expressed on mature B
lymphocytes and, at low levels, on T lymphocytes. We examined the
ability of a molecule that binds to CD21 to provide a stimulatory
signal in the context of modified REM.
[0193] In a first set of experiments, we used plate-bound anti-CD21
antibody to examine the ability to provide a stimulatory signal in
modified REM in which the EBV-LCL feeder population was completely
eliminated. Two different antigen-specific CTL clones ("R7" which
is alloantigen-specific, and "11E2" which is EBV-specific) were
tested in a modified REM procedure in which EBV-LCL were
eliminated, but other components were maintained as described above
(PBMC at 500:1, IL-2 at 25 units/ml). Anti-CD21 antibodies are
available from commercial sources. We used the anti-CD21 antibody
available from Pharmingen. Anti-CD3 antibody was also used, and was
bound to plates, as with anti-CD21. Cultures were expanded over a
two week standard REM cycle, essentially as described above.
[0194] The data, as shown in TABLE 7, revealed that the inclusion
of anti-CD21 antibody resulted in a large increase in the fold
proliferation obtainable without the use of EBV-LCL (to 650% of
control and 408% of control for R7 and 1 E2, respectively).
[0195] A second set of experiments, performed using soluble
anti-CD21 antibody, provided additional confirmatory data. In
particular, a range of anti-CD21 concentrations was used in REM as
above, except that both anti-CD21 and anti-CD3 were supplied as
soluble antibodies (anti-CD21 at concentrations ranging from 0
ng/ml to 1.75 ng/ml; anti-CD3 at 10 ng/ml).
[0196] As shown in TABLE 8, the removal of all EBV-LCL feeder cells
from the cultures resulted in a substantial reduction in the
average fold proliferation (to 10% of control and 14% of control
for R7 and 11E2, respectively). The addition of even small amounts
of anti-CD21 antibody to the culture media resulted in a large
increase in fold proliferation (to 72% of control and 57% of
control for R7 and 11E2, respectively).
[0197] While anti-CD21 antibody provides a conveninet method for
enhancing the stimulatory signal, it is also possible to stimulate
CD21 in other ways. For example, in addition to anti-CD21 antibody,
other molecules that can be used to bind to CD21 include C3d, C3dg,
iC3b and gp350/220 of EBV (see, e.g., W. Timens et al., pages
516-518 in "Leucocyte Typing V, White Cell Differentiation
Antigens," Schlossman, S. F., et al. (eds.), Oxford University
Press, Oxford, 1995). Also, as described above, while such T cell
stimulatory components can be provided as soluble factors in the
modified REM medium, they can also be provided by a cell line
included in the medium (e.g., a cell line that secretes or presents
a molecule that binds to CD21).
8TABLE 7 Fold % Clone Specificity Stimulation Proliferation Control
R7 Alloantigen anti-CD3 96 100% anti-CD3 + anti-CD21 624 650% 11E2
EBV anti-CD3 48 100% anti-CD3 + anti-CD21 196 408%
[0198]
9 TABLE 8 Fold % Clone Condition Proliferation Control R7 SOP REM
900 100% 1.75 ng/ml anti-CD21 228 25% 1.25 ng/ml anti-CD21 156 17%
0.625 ng/ml anti-CD21 516 57% 0.325 ng/ml anti-CD21 372 41% 0 ng/ml
anti-CD21 90 10% 11E2 SOP REM 420 100% 1.75 ng/ml anti-CD21 96 24%
1.25 ng/ml anti-CD21 192 48% 0.625 ng/ml anti-CD21 288 72% 0.325
ng/ml anti-CD21 132 33% 0 ng/ml anti-CD21 60 14%
* * * * *