U.S. patent application number 11/870776 was filed with the patent office on 2008-06-05 for adoptive transfer of cd8 + t cell clones derived from central memory cells.
Invention is credited to Carolina Berger, Michael C. Jensen, Stanley R. Riddell.
Application Number | 20080131415 11/870776 |
Document ID | / |
Family ID | 39468213 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131415 |
Kind Code |
A1 |
Riddell; Stanley R. ; et
al. |
June 5, 2008 |
ADOPTIVE TRANSFER OF CD8 + T CELL CLONES DERIVED FROM CENTRAL
MEMORY CELLS
Abstract
The present invention provides a method of carrying out adoptive
immunotherapy in a primate subject in need thereof by administering
the subject a cytotoxic T lymphocytes (CTL) preparation in a
treatment-effective amount. The method comprises administering as
the CTL preparation a preparation consisting essentially of an in
vitro expanded primate CTL population, the CTL population enriched
prior to expansion for central memory T lymphocytes, and depleted
prior to expansion of effector memory T lymphocytes. In some
embodiments, the method may further comprise concurrently
administering Interleukin-15 to the subject in an amount effective
to increase the proliferation of the central memory T cells in the
subject. Pharmaceutical formulations produced by the method, and
methods of using the same, are also described.
Inventors: |
Riddell; Stanley R.;
(Sammamish, WA) ; Berger; Carolina; (Seattle,
WA) ; Jensen; Michael C.; (Sierra Madre, CA) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
39468213 |
Appl. No.: |
11/870776 |
Filed: |
October 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60867880 |
Nov 30, 2006 |
|
|
|
Current U.S.
Class: |
424/93.71 ;
435/372.3 |
Current CPC
Class: |
A61K 2039/5158 20130101;
C12N 15/85 20130101; A61K 35/17 20130101; C12N 5/0636 20130101;
C12N 2501/2315 20130101; A61K 39/0011 20130101; C12N 5/0638
20130101 |
Class at
Publication: |
424/93.71 ;
435/372.3 |
International
Class: |
A61K 35/00 20060101
A61K035/00; C12N 5/06 20060101 C12N005/06 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with Government support under Grant
Numbers R01CA114536, R01AI053193, CA18029, U01HL66947, and
M01RR00037 from the National Institutes of Health. The US
Government has certain rights to this invention.
Claims
1. In a method of carrying out adoptive immunotherapy in a primate
subject in need thereof by administering said subject a cytotoxic T
lymphocytes (CTL) preparation in a treatment-effective amount, the
improvement comprising: administering as said CTL preparation a
preparation consisting essentially of an in vitro expanded primate
CTL population, said CTL population enriched prior to expansion for
central memory T lymphocytes, and depleted prior to expansion of
effector memory T lymphocytes.
2. The method of claim 1, wherein said CTL preparation is produced
by the process of: (a) collecting a first CTL population from a
donor; (b) separating a CTL subpopulation enriched for CD62L.sup.+
central memory T lymphocytes and depleted of CD62L.sup.- effector
memory T lymphocytes to produce a central memory-enriched CTL
subpopulation; (c) expanding said central memory-enriched CTL
subpopulation in vitro in a culture medium; and then (d) collecting
cells from said culture medium to produce said CTL preparation.
3. The method of claim 2, wherein said separating step is carried
out by: (i) contacting said first CTL population to anti-CD62L
antibody, wherein said antibody is immobilized on a solid support,
so that central memory cells bind to said support; then (ii)
separating said support from said CTL population with central
memory cells bound thereto; (iii) and then separating said central
memory cells from said solid support to produce said central memory
enriched CTL subpopulation.
4. The method of claim 1, further comprising the step of
concurrently administering Interleukin-15 to said subject in an
amount effective to increase the proliferation of said central
memory T cells in said subject.
5. The method of claim 1, wherein said subject is a human
subject.
6. The method of claim 1, wherein said subject is afflicted with
cancer.
7. The method of claim 1, wherein said subject is afflicted with an
infectious disease.
8. The method of claim 1, wherein said subject is
immunodeficient.
9. The method of claim 1, wherein said central memory-enriched T
cells are modified in vitro with at least one gene that targets
cancer cells.
10. A pharmaceutical formulation consisting essentially of an in
vitro expanded primate cytotoxic T lymphocyte (CTL) population,
said CTL population enriched prior to expansion for central memory
T lymphocytes, and depleted prior to expansion of effector memory T
lymphocytes.
11. The formulation of claim 10, wherein said CTL population is
produced by the process of: (a) collecting a first CTL population
from a donor; (b) separating a CTL subpopulation enriched for
CD62L.sup.+ central memory T lymphocytes and depleted of
CD62L.sup.- effector memory T lymphocytes to produce a central
memory-enriched CTL subpopulation; (c) expanding said central
memory-enriched CTL subpopulation in vitro in a culture medium; and
then (d) collecting cells from said culture medium to produce said
CTL preparation.
12. The formulation of claim 11, wherein said separating step is
carried out by: (i) contacting said first CTL population to
anti-CD62L antibody, wherein said antibody is immobilized on a
solid support, so that central memory cells bind to said support;
then (ii) separating said support from said CTL population with
central memory cells bound thereto; (iii) and then separating said
central memory cells from said solid support to produce said
central memory enriched CTL subpopulation.
13. The formulation of claim 10, wherein said central
memory-enriched T cells are modified in vitro with at least one
gene that targets cancer cells.
14. A method of making a cytotoxic T lymphocyte (CTL) preparation
useful for adoptive immunotherapy; said method comprising the steps
of: (a) collecting a first CTL population from a donor; (b)
separating a CTL subpopulation enriched for CD62L.sup.+ central
memory T lymphocytes and depleted of CD62L.sup.- effector memory T
lymphocytes to produce a central memory-enriched CTL subpopulation;
(c) expanding said central memory enriched CTL subpopulation in
vitro in a culture medium; and then (d) collecting cells from said
culture medium to produce said CTL preparation.
15. The method of claim 14, wherein said expanding step comprises
administering Interleukin-15 to said central memory subpopulation
in vitro.
16. The method of claim 14, wherein said central memory-enriched T
cells are modified in vitro with at least one gene that targets
cancer cells.
17. An in vitro expanded primate cytotoxic T lymphocyte (CTL)
preparation produced by the process of claim 14.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/867,880, filed Nov. 30, 2007, the
disclosure of which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention concerns methods and compositions for
carrying out adoptive immunotherapy.
BACKGROUND OF THE INVENTION
[0004] Studies in rodents have demonstrated that adoptive
immunotherapy with antigen specific T cells is effective for cancer
and infections, and there is evidence this modality has therapeutic
activity in humans.sup.1-8. For clinical applications, it is
necessary to isolate T cells of a desired antigen specificity or to
engineer T cells to express receptors that target infected or
transformed cells, and then expand these cells in culture.sup.9-14.
The transfer of T cell clones is appealing because it enables
control of specificity and function, and facilitates evaluation of
in vivo persistence, toxicity and efficacy. Additionally, in the
setting of allogeneic stem cell transplantation, the administration
to recipients of T cell clones from the donor that target pathogens
or malignant cells can avoid graft-versus-host disease that occurs
with infusion of unselected donor T cells.sup.3,4,15. However, it
is apparent from clinical studies that the efficacy of cultured T
cells, particularly cloned CD8.sup.+ T cells, is frequently limited
by their failure to persist after adoptive transfer.sup.16,17.
[0005] The pool of lymphocytes from which CD8.sup.+ T cells for
adoptive immunotherapy can be derived contains naive and
long-lived, antigen experienced memory T cells (T.sub.M). T.sub.M
can be divided further into subsets of central memory (T.sub.CM)
and effector memory (T.sub.EM) cells that differ in phenotype,
homing properties and function's. CD8.sup.+ T.sub.CM express CD62L
and CCR7, which promote migration into lymph nodes, and proliferate
rapidly if re-exposed to antigen. CD8.sup.+ T.sub.EM lack CD62L
enabling migration to peripheral tissues, and exhibit immediate
effector function.sup.19.
[0006] In response to antigen stimulation, CD8.sup.+ T.sub.CM and
T.sub.EM both differentiate into cytolytic effector T cells
(T.sub.E) that express a high level of granzymes and perforin, but
are short-lived.sup.20. Thus, the poor survival of T cells in
clinical immunotherapy trials may simply result from their
differentiation during in vitro culture to T.sub.E that are
destined to die.sup.17,21,22.
SUMMARY OF THE INVENTION
[0007] A first aspect of the present invention is a method of
carrying out adoptive immunotherapy in a primate subject in need
thereof by administering the subject a cytotoxic T lymphocytes
(CTL) preparation in a treatment-effective amount. The method
comprises administering as the CTL preparation a preparation
consisting essentially of an in vitro expanded (e.g., grown in
vitro for 1, 2, or 3 days up to 3 or 4 weeks or more) primate CTL
population, the CTL population enriched prior to expansion for
central memory T lymphocytes, and depleted prior to expansion of
effector memory T lymphocytes. In some embodiments, the method may
further comprise concurrently administering Interleukin-15 to the
subject in an amount effective to increase the proliferation of the
central memory T cells in the subject.
[0008] A further aspect of the invention is a pharmaceutical
formulation comprising, consisting essentially of or consisting of
an in vitro expanded primate cytotoxic T lymphocyte (CTL)
population, the CTL population enriched prior to expansion for
central memory T lymphocytes, and depleted prior to expansion of
effector memory T lymphocytes.
[0009] A further aspect of the invention is the use of a
formulation as described herein for the preparation of a medicament
for carrying out a method as described herein (e.g., treating
cancer or an infectious disease in a human, or primate,
subject).
[0010] In some embodiments the CTL preparation (or population) is
produced by the process of: (a) collecting a first CTL population
from a donor; (b) separating a CTL subpopulation enriched for
CD62L.sup.+ central memory T lymphocytes and depleted of
CD62L.sup.- effector memory T lymphocytes to produce a central
memory-enriched CTL subpopulation; (c) expanding the central
memory-enriched CTL subpopulation in vitro in a culture medium
(e.g., for 1, 2, or 3 days up to 3 or 4 weeks or more); and then
(d) collecting cells from the culture medium to produce the CTL
preparation. The separating step "b" can in some embodiments be
carried out by: (i) contacting the first CTL population to
anti-CD62L antibody, wherein the antibody is immobilized on a solid
support, so tht central memory cells bind to the support; then (ii)
separating the support from the CTL population with central memory
cells bound thereto; (iii) and then separating the central memory
cells from the solid support to produce the central memory enriched
CTL subpopulation. In some embodiments, the expanding step "c"
further comprises administering Interleukin-15 to the central
memory subpopulation in vitro.
[0011] In some embodiments, the central memory-enriched T cells are
modified in vitro with at least one gene that targets (e.g.,
specifically binds to) cancer cells or other pathogenic cells in
the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. Isolation and genetic modification of CMV-specific
CD8.sup.+ T cell clones from CD62L.sup.- and CD62L.sup.+ T cell
subsets for adoptive transfer.
[0013] FIG. 1a. CMV IE-specific memory T cells are present in both
CD62L.sup.+ and CD62L.sup.- subsets of CD8.sup.+ peripheral blood
lymphocytes. PBMC were sorted into CD62L-CD8.sup.+ and
CD62L.sup.+CD8.sup.+ fractions after staining with anti-CD8 and
anti-CD62L antibodies (upper panels). The CD62L.sup.- and
CD62L.sup.+ fractions were stimulated for 6 hours with autologous
CD40L-activated B cells pulsed with medium alone or with CMV-IE
peptide. Brefeldin A was added for the final 4 hours and CD8.sup.+
T cells that produced IFN-.gamma. were detected by staining for
intracellular IFN-.gamma.. The lower left panels show IFN-.gamma.
production by the CD62L.sup.-CD8.sup.+ subset and the lower right
panels show IFN-.gamma. production by the CD62L.sup.+CD8.sup.+
subset. Data from macaque 02269 is shown and is representative of
data obtained in four consecutive macaques.
[0014] FIG. 1b. Strategy for the isolation of CMV-specific
CD8.sup.+ T cell clones from CD62L.sup.-T.sub.EM and CD62L.sup.+
T.sub.CM subsets. Aliquots of PBMC were obtained from each macaque
and sorted into CD62L.sup.-CD8.sup.+ and CD62L.sup.+CD8.sup.-
fractions after staining with anti-CD8 and anti-CD62L antibodies.
The sorted T cells were cultured with autologous monocytes pulsed
with the CMV IE peptide and after 1 week of stimulation, T cells
from the cultures were cloned by limiting dilution. T cell clones
were screened to identify those that lysed autologous
peptide-pulsed target cells, and then transduced with a retroviral
vector that encoded a cell surface .DELTA.CD19 or CD20 molecule.
Individual T cell clones were expanded in vitro to
>5.times.10.sup.9 cells by stimulation with anti-CD3 and
anti-CD28 antibodies in cultures supplemented with
.gamma.-irradiated human PBMC and EBV-LCL prior to adoptive
transfer.
[0015] FIG. 1c. Retroviral vector constructs encoding for macaque
cell surface marker genes. Abbreviations: MPSV-LTR,
myeloproliferative sarcoma virus retroviral long terminal repeat;
.PSI.+, extended packaging signal; PRE, woodchuck hepatitis virus
posttanscriptional regulatory element; .DELTA.CD19, truncated
macaque CD19 cDNA encoding the extracellular and transmembrane
domains, and 4 aa of the cytosolic tail; CD20, full-length macaque
CD20 cDNA.
[0016] (d) Immunomagnetic bead selection of .DELTA.CD19 and
CD20-modified CD8.sup.+ T cell clones. Transduced T cells were
enriched for .DELTA.CD19 or CD20 expressing cells on day 8 after
transduction using a two-step immunomagnetic selection with
anti-CD19 or anti-CD20 antibodies and rat anti-mouse IgG coupled
microbeads. Aliquots of unmodified (left panels) and .DELTA.CD19 or
CD20-modified T cells (right panels) were removed after selection,
co-stained with anti-CD8 and anti-CD19 or anti-CD20 antibodies, and
analyzed by flow cytometry to assess purity. The percentages of
CD8.sup.+ T cells positive for the transgene are indicated.
[0017] FIG. 2. CMV-specific CD8.sup.+ T cell clones derived from
T.sub.CM and T.sub.EM subsets exhibit an effector phenotype, and
comparable avidity and proliferation in vitro.
[0018] FIG. 2a. Individual T.sub.CM and T.sub.EM-derived macaque
CMV-specific CD8.sup.+ T cell clones were examined by flow
cytometry for expression of CD62L, CCR7, CD28, CD127, granzyme B
and perforin (bold line) and stained with isotype control
antibodies (dotted line). Inset values represent the mean
fluorescence intensity (MFI). The data is shown for T cell clones
that were adoptively transferred to macaque 02269 and is
representative of the data for all of the T cell clones used in
adoptive transfer experiments in the three macaques.
[0019] FIG. 2b. Cytotoxic activity of each pair of T.sub.EM (filled
triangle) and T.sub.CM (filled square) -derived CMV-specific
CD8.sup.+ T cell clones used in adoptive transfer was examined in a
4-hour chromium release assay at an effector to target ratio of
20:1 using autologous target cells pulsed with the CMV IE peptide
at various concentrations. The sequences of the IE-1 peptides were
KKGDIKDRV (02269) and EEHVKLFFK (A99171), the sequence of the IE-2
peptide was ATTRSLEYK (02258).
[0020] FIG. 2c. In vitro growth of CMV-specific CD8.sup.+ T cell
clones. The T.sub.EM (filled triangle) and T.sub.CM (filled square)
derived T cell clones used for adoptive transfer were stimulated
with anti-CD3 and anti-CD28 in the presence of .gamma.-irradiated
feeder cells and IL-2 (50 U/ml). Cell growth over 14 days of
culture was measured by counting viable cells using trypan blue
exclusion.
[0021] FIG. 2d. Telomere length in CMV-specific T cell clones
derived from CD62L.sup.+ and CD62L.sup.- subsets. The median
telomere length of duplicate samples was measured by automated
flow-FISH in peripheral blood T lymphocytes (shaded squares), in
the infused T cell clones (iT.sub.EM filled squares, iT.sub.CM open
squares), and in each of two additional randomly selected T.sub.EM
(left hatched squares ) and T.sub.CM-derived (right hatched squares
) T cell clones from each macaque.
[0022] FIG. 3. Persistence and migration of T.sub.EM and
T.sub.CM-derived CD8.sup.+ T.sub.E clones in peripheral blood, bone
marrow, and lymph nodes following adoptive transfer.
[0023] FIGS. 3a-b. In vivo persistence and migration of
.DELTA.CD19-modified T.sub.EM-derived (a) and T.sub.CMderived (b) T
cell clones in macaque 02269. The T cell clones were transferred in
separate infusions given intravenously more than 10 weeks apart at
a cell dose of 3.times.10.sup.8/kg. Samples of PBMC were collected
at intervals for 6-8 weeks after each infusion. Bone marrow and
lymph node samples were collected before infusion and fourteen days
after each T cell infusion. Samples were stained with
fluorochrome-conjugated anti-CD3, CD8, and CD19 antibodies,
respectively. The frequency of transferred CD19.sup.+ T cells was
determined by flow cytometry with gating on CD3.sup.+CD8.sup.+
cells. Left panels show the percentage of CD8.sup.+ T cells that
expressed CD19 in blood, bone marrow and lymph nodes prior to and
at intervals after infusion of the T.sub.EM-derived (a) and
T.sub.CM-derived (b) T cell clones. Right panels show the absolute
numbers of CD19.sup.+CD3.sup.+CD8.sup.+ T cells per .mu.l of blood.
This was determined by calculating the absolute number of CD3+CD8+
T cells per .mu.l of blood at the indicated days (% of
CD3.sup.+CD8.sup.+ T cells in an aliquot of mononuclear
cells.times.mononuclear cell count per .mu.l blood/100).
Subsequently, the number of CD19.sup.+CD3.sup.+CD8.sup.+ T cells
was derived (% CD19.sup.+CD3.sup.+CD8.sup.+ T cells x absolute
CD3.sup.+CD8.sup.+ count per .mu.l blood/100).
[0024] FIGS. 3c-d. In vivo persistence and migration of adoptively
transferred .DELTA.CD19-modified T.sub.CM derived and CD20-modified
T.sub.EM-derived T cell clones in macaque 02258. CMV-specific
CD8.sup.+ T cell clones were adoptively transferred in separate
intravenous infusions at a cell dose of 6.times.10.sup.8/kg.
Aliquots of blood, bone marrow, and lymph nodes were obtained
before and at the indicated times after each T cell infusion and
analyzed by flow cytometry using specific antibodies to detect the
transferred CD19.sup.+CD8.sup.+ or CD20.sup.+CD8.sup.+ T cells,
respectively. Left panels show the percentage of CD8.sup.+ T cells
that expressed .DELTA.CD19 (c) or CD20 (d) in blood, bone marrow
and lymph nodes at intervals after each infusion. Right panels show
the absolute number of CD3+CD8+T cells per .mu.l of blood that
expressed .DELTA.CD19 (c) or CD20 (d) at intervals after
infusion.
[0025] FIG. 4. T.sub.CM-derived T.sub.E clones undergo less
apoptosis after adoptive transfer compared with T.sub.EM-derived
T.sub.E clones, and are rescued from cell death in vitro by
IL-15.
[0026] FIG. 4a. Expression of bcl-x1 and bcl-2 by T.sub.EM-derived
(open squares) and T.sub.CM-derived (filled squares) CD8.sup.+ T
cell clones adoptively transferred to macaques 02269, 02258, and
A99171. The T cells were analyzed at the end of a 14-day
stimulation cycle by intracellular staining with bcl-x1 and bcl-2
specific antibodies. The mean fluorescence intensity of staining is
shown on the y axis.
[0027] FIG. 4b. Apoptosis of T.sub.EM-derived and T.sub.CM-derived
CD8.sup.+T.sub.E clones in vivo. A .DELTA.CD19-modified T cell
clone derived from the CD62L T.sub.EM fraction was adoptively
transferred at a dose of 6.times.10.sup.8/kg to macaque A99171,
followed 5 weeks later by the infusion of the same cell dose of a
.DELTA.CD19-modified T cell clone derived from the CD62L.sup.+
T.sub.CM fraction. Samples of PBMC were obtained on day 1 after
transfer and the proportion of .DELTA.CD19-expressing CD8.sup.+ T
cells that bound Annexin V and/or stained positive for PI was
determined by flow cytometry after staining with anti-CD8 and CD19
antibodies, and with Annexin V and PI. PBMC were analyzed directly
and after 24 hours of culture in CTL media. The analysis was
performed after gating on CD8.sup.+CD19.sup.- T cells (open
squares) and on CD8.sup.+CD19.sup.+ T cells (filled squares),
respectively. The hatched bars show the proportion of T cells in
each of the cell products prior to infusion that bound Annexin V or
stained positive for PI.
[0028] FIG. 4c. Persistence and migration of T.sub.EM-derived and
T.sub.CM-derived T.sup.E clones in macaque A99171. Peripheral
blood, bone marrow, and lymph node cells obtained at intervals
after each T cell infusion were analyzed by flow cytometry to
detect transferred CD19.sup.+CD8.sup.+ T cells. The samples were
analyzed after gating on CD3.sup.+CD8.sup.+ cells.
[0029] FIG. 4d. IL-15 supports the in vitro survival of
T.sub.CM-derived but not T.sub.EM-derived CD8+ T.sub.E clones.
Aliquots of the CMV-specific T.sub.EM-derived and T.sub.CM-derived
CD8+ T cell clones that were used for adoptive transfer to macaques
02269, 02258, and A99171 respectively, were plated in wells
containing medium alone (open triangles) or medium supplemented
with IL-2 (16.6 U/ml; 1 ng/ml) (filled diamonds) or IL-15 (1 ng/ml)
(filled squares), respectively. The number of viable cells was
determined at the indicated days (up to 34 days) by counting cells
using trypan dye exclusion.
[0030] FIG. 4e. CD8.sup.+ T.sub.CM-derived clones express higher
levels of IL-15 receptor (IL-15R) chains. Expression of
IL-15R.alpha., IL-2R.beta., and IL-2R.gamma. on aliquots of
CMV-specific T.sub.CM-derived (bold line) and T.sub.EM-derived
(black line) T cell clones was measured by flow cytometry on day
13-14 after stimulation. Staining with isotope control antibody is
shown by the dotted line. The data is shown for the T cell clones
used for the adoptive transfer experiments in macaque 02269, and is
representative of the data obtained for the T cell clones
administered to macaques 02258 and A99171, respectively.
[0031] FIG. 5. Adoptively transferred T.sub.CM-derived T.sub.E
cells reacquire markers of T.sub.CM in vivo and persist in memory
cell niches.
[0032] FIG. 5a. Expression of CD62L on T.sub.EM-derived (upper
panels) or T.sub.CM-derived (lower panels) T.sub.E clones after
adoptive transfer. Samples of PBMC were obtained before and on day
3 after the infusion of T.sub.CM and T.sub.EM-derived T cell
clones, and analyzed by flow cytometry after staining with
fluorochrome-conjugated anti-CD3, CD8, CD62L, and either CD19 or
CD20 antibodies. Cells were gated to identify CD3.sup.+CD8.sup.+ T
cells and the percentage of T cells that expressed the CD19 or CD20
marker gene and CD62L is shown in the upper right quadrant of each
panel.
[0033] FIGS. 5b-e. A major fraction of CD8.sup.+ T cells that
persist after adoptive transfer acquire phenotypic markers of
T.sub.CM and reside in lymph nodes. Aliquots of PBMC, lymph nodes
(LN), and bone marrow (BM) were obtained from macaque 02258 at day
14 and day 56 after the infusion of the .DELTA.CD19-modified
T.sub.CM-derived CMV-specific T cell clone. The expression of
phenotypic markers of T.sub.CM including CD62L (b), CCR7 (c), CD28
(d), and CD127 (e) on the subset of transferred CD19.sup.+ T cells
was determined by flow cytometry after gating on CD3.sup.+CD8.sup.+
cells.
[0034] FIG. 6. Adoptively transferred CD8.sup.+ T cells that
persist in vivo exhibit functional properties of memory T
cells.
[0035] FIG. 6a. Adoptively transferred CD8.sup.+ T cells that
persist in vivo produce IFN-.gamma. after antigen stimulation. PBMC
were obtained before the T cell infusion (Pre) and 21 days after
(Post) the infusion of a .DELTA.CD19-modified T.sub.CM-derived
CMV-specific CD8.sup.+ T cell clone. Aliquots of PBMC were
stimulated with medium alone, PMA/ionomycin, or the CMV LE peptide
recognized by the transferred T cell clone. IFN-.gamma. production
by CD8.sup.+ T cells was examined by cytokine flow cytometry after
staining with anti-CD3, CD8, CD19, and IFN-.gamma. antibodies. The
analysis was performed after gating on CD3.sup.+CD8.sup.+ T cells.
The data is shown for macaque 02269 and is representative of a
separate experiment in macaque 02258.
[0036] FIG. 6b. Adoptively transferred T cells that re-express
CD62L lack direct cytotoxicity but differentiate into cytotoxic
cells after TCR stimulation. Left panel: PBMC obtained 14-70 days
after the infusion of a .DELTA.CD19-modified T.sub.CM-derived
CD8.sup.+ T cell clone were pooled and sorted into
CD19.sup.+CD62L.sup.-CD8.sup.+ and CD19.sup.+CD62L.sup.+CD8.sup.+
fractions that were at least 80% pure. The sorted cells were
examined for recognition of autologous target cells alone (open
squares) or pulsed with CMV IE peptide (closed squares) in a 4-hour
chromium release assay at an E/T ratio of 5:1. The lysis of target
cells by the in vitro cultured T.sub.CM-derived CD19-modified
CD8.sup.+ T cell clone served as a positive control. Right panel:
Aliquots of the sorted CD62L.sup.-.DELTA.CD19.sup.+CD8.sup.+ and
CD62L.sup.+.DELTA.CD19.sup.+CD8.sup.+ T cells were stimulated in
vitro using anti-CD3 and CD28 antibodies, in the presence of
.gamma.-irradiated feeder cells and IL-2 (50 U/ml). After 14 days
of culture, the cultures were assayed for recognition of peptide
pulsed target cells in a 4-hour chromium release assay (E/T ratio
of 5:1). The data is shown for macaque 02258 and is representative
of that observed with macaque 02269.
[0037] FIG. 6c. Adoptively transferred T cells that reacquire CD62L
in vivo exhibit more rapid proliferation than those that remain
CD62L.sup.-. Aliquots of the sorted
.DELTA.CD19.sup.+CD62L.sup.+CD8.sup.+ and
.DELTA.CD19.sup.+CD62L.sup.-CD8.sup.+ T cells from macaque 02269
were labeled with CFSE (upper panels) and stimulated in vitro with
either CMV IE peptide-pulsed autologous CD40L activated B cells or
medium alone. After 5 days, dilution of CFSE was assessed by flow
cytometry after gating on .DELTA.CD19.sup.+CD3.sup.+CD8.sup.+
cells. The M3 gate identifies cells that have undergone more than
five divisions.
[0038] FIG. 6d. Adoptively transferred T cells can be driven to
expand in vivo by the infusion of TAPC. Autologous T cells obtained
and cryopreserved from macaque A99171 prior to any T cell infusions
were thawed and expanded by stimulation with anti-CD3 and anti-CD28
antibodies, and IL-2. After expansion, the T cells were pulsed with
the CMV IE peptide recognized by the adoptively transferred
.DELTA.CD19-modified T.sub.CM clone. Eight weeks after the T cell
infusion when a stable level of CD19.sup.+ T cells was established
in vivo, a dose of 1.times.10.sup.7 T-APC/kg was administered
intravenously. The absolute number of CD3.sup.+CD8.sup.- T cells
and CD19.sup.+CD8.sup.+ T cells/.mu.l of blood was measured on
samples obtained prior to administration of T-APC, and on day +3,
+5 and +7 after the infusion of T-APC. The number of endogenous
IE-specific CD8.sup.+ T cells was measured at the same time points
by cytokine flow cytometry after gating on
CD19.sup.-CD3.sup.+CD8.sup.+ cells. The data shows the
fold-increase in the absolute numbers of CD3.sup.+CD8.sup.+ T cells
(open squares), CD19.sup.+CD8.sup.+ T cells (shaded squares), and
CD19.sup.- IE-specific CD8.sup.+ T cells (filled squares) at day
+3, +5 and +7 after the infusion of T-APC.
[0039] FIG. 6e. T-APC pulsed with IE peptide are lysed by
IE-specific CD8.sup.+ T cells. T-APC generated from macaque A99171
either pulsed with IE peptide (filled squares) or with media alone
(open squares) were labeled with .sup.51Cr and used as targets for
an aliquot of the autologous IEspecific CD8.sup.+ T cell clone that
was adoptively transferred to macaque A99171.
[0040] The present invention is explained in greater detail below.
The disclosures of all United States patent references cited herein
are incorporated by reference herein in their entirety.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] "T cells" or "T lymphocytes" as used herein may be from any
mammalian, preferably primate, species, including monkeys, dogs,
and humans. In some embodiments the T cells are allogenic (from the
same species but different donor) as the recipient subject; in some
embodiments the T cells are autologous (the donor and the recipient
are the same); in some embodiments the T cells are syngeneic (the
donor and the recipients are different but are identical
twins).
[0042] Cytotoxic T lymphocyte (CTL) as used herein refers to a T
lymphocyte that expresses CD8 on the surface thereof (i.e., a
CD8.sup.+ T cell). In some embodiments such cells are preferably
"memory" T cells (T.sub.M cells) that are antigen-experienced.
"Central memory" T cell (or "T.sub.CM") as used herein refers to a
CTL that expresses CD62L on the surface thereof (i.e.,
CD62L.sup.+CD8.sup.+ cells).
[0043] "Effector memory" T cell (or "T.sub.EM") as used herein
refers to a CTL that does not express CD62L on the surface thereof
(i.e., CD62L.sup.-CD8.sup.+ cells).
[0044] "Enriched" and "depleted" as used herein to describe amounts
of cell types in a mixture refers to the subjecting of the mixture
of the cells to a process or step which results in an increase in
the number of the "enriched" type and a decrease in the number of
the "depleted" cells. Thus, depending upon the source of the
original population of cells subjected to the enriching process, a
mixture or composition may contain 60, 70, 80, 90, 85, or 99
percent or more (in number or count) of the "enriched" cells and
40, 30, 20, 10, 5 or 1 percent or less (in number or count) of the
"depleted" cells.
[0045] Interleukin-15 is a known and described in, for example,
U.S. Pat. No. 6,344,192.
I. In Vitro Expansion
[0046] T lymphocytes can be collected in accordance with known
techniques and enriched or depleted by known techniques such as
affinity binding to antibodies such as flow cytometry and/or
affinity binding. After enrichment and/or depletion steps, in vitro
expansion of the desired T lymphocytes can be carried out in
accordance with known techniques (including but not limited to
those described in U.S. Pat. No. 6,040,177 to Riddell et al.), or
variations thereof that will be apparent to those skilled in the
art.
[0047] For example, the desired T cell population or subpopulation
may be expanded by adding an initial T lymphocyte population to a
culture medium in vitro, and then adding to the culture medium
feeder cells, such as non-dividing peripheral blood mononuclear
cells (PBMC), (e.g., such that the resulting population of cells
contains at least about 5, 10, 20, or 40 or more PBMC feeder cells
for each T lymphocyte in the initial population to be expanded);
and incubating the culture (e.g. for a time sufficient to expand
the numbers of T cells). The order of additional of the T cells and
feeder cells to the culture media can be reversed if desired. The
culture can typically 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.
[0048] The T lymphocytes expanded are typically cytotoxic T
lymphocytes (CTL) that are specific for an antigen present on a
human tumor or a pathogen.
[0049] The non-dividing feeder cells can comprise gamma-irradiated
PBMC feeder cells. In some embodiments, the PBMC are irradiated
with gamma rays in the range of about 3000 to 3600 rads.
[0050] Optionally, the expansion method may further comprise the
step of adding non-dividing EBV-transformed lymphoblastoid cells
(LCL) as feeder cells. LCL can be irradiated with gamma rays in the
range of about 6000 to 10,000 rads. The LCL feeder cells may be
provided in any suitable amount, such as a ratio of LCL feeder
cells to initial T lymphocytes of at least about 10:1.
[0051] Optionally, the expansion method may further comprise the
step of adding anti-CD3 monoclonal antibody to the culture medium
(e.g., at a concentration of at least about 0.5 ng/ml). Optionally,
the expansion method may further comprise the step of adding IL-2
and/or IL-15 to the culture medium (e.g., wherein the concentration
of IL-2 is at least about 10 units/ml).
[0052] In some embodiments it may be desired to introduce
functional genes into the T cells to be used in immunotherapy in
accordance with the present invention. 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 PCT/US91/08442 and PCT/US94/05601 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. This can be carried out in accordance with known
techniques (see, e.g., U.S. Pat. No. 6,040,177 to Riddell et al. at
columns 14-17) or variations thereof that will be apparent to those
skilled in the art based upon the present disclosure.
[0053] 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, adenoviruses, adeno-associated virus (AAV), and
retroviruses. Thus, gene transfer and expression methods are
numerous but essentially function to introduce and express genetic
material in mammalian cells. Several of the above techniques have
been used to transduce hematopoietic or lymphoid cells, including
calcium phosphate transfection, protoplast fusion, electroporation,
and infection with recombinant adenovirus, adeno-associated virus
and retrovirus vectors. Primary T lymphocytes have been
successfully transduced by electroporation and by retroviral
infection
[0054] 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.
[0055] 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-I TK) gene (Wigler et
al., Cell 11:223, 1977) which confers ganciclovir sensitivity; the
cellular hypoxanthine phosphribosyltransferase (HPRT)gene, the
cellular adenine phosphoribosyltransferase (APRT) gene, bacterial
cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA.
89:33 (1992)).
[0056] In some embodiments it may be 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 daminase gene (ADA), and the multi-drug
resistance (MDR) gene.
[0057] 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.
[0058] 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.
[0059] A variety of methods can be employed for transducing T
lymphocytes, as is well known in the art. For example, retroviral
transductions can be carried out as follows: on day 1 after
stimulation using REM as described herein, provide the cells with
20-30 units/ml IL-2; on day 3, replace one half of the medium with
retroviral supernatant prepared according to standard methods and
then supplement the cultures with 5 ug/ml polybrene and 20-30
units/ml IL-2; on day 4, wash the cells and place them in fresh
culture medium supplemented with 20-30 units/ml IL-2; on day 5,
repeat the exposure to retrovirus; on day 6, place the cells in
selective medium (containing, e.g., an antibiotic corresponding to
an antiobiotic resistance gene provided in the retroviral vector)
supplemented with 30 units/ml IL-2; on day 13, separate viable
cells from dead cells using Ficoll Hypaque density gradient
separation and then subclone the viable cells.
II. Compositions and Methods
[0060] Subjects that can be treated by the present invention are,
in general, human and other primate subjects, such as monkeys and
apes for veterinary medicine purposes. The subjects can be male or
female and can be any suitable age, including infant, juvenile,
adolescent, adult, and geriatric subjects.
[0061] Subjects that can be treated include subjects afflicted with
cancer, including but not limited to colon, lung, liver, breast,
prostate, ovarian, skin (including melanoma), bone, and brain
cancer, etc. In some embodiments the tumor associated antigens are
known, such as melanoma, breast cancer, squamous cell carcinoma,
colon cancer, leukemia, myeloma, prostate cancer, etc. (in these
embodiments memory T cells can be isolated or engineered by
introducing the T cell receptor genes). In other embodiments the
tumor associated proteins can be targeted with genetically modified
T cells expressing an engineered immunoreceptor. Examples include
but are not limited to B cell lymphoma, breast cancer, prostate
cancer, and leukemia.
[0062] Subjects that can be treated also include subjects afflicted
with, or at risk of developing, an infectious disease, including
but not limited to viral, retroviral, bacterial, and protozoal
infections, etc.
[0063] Subjects that can be treated include immunodeficient
patients afflicted with a viral infection, including but not
limited to Cytomegalovirus (CMV), Epstein-Barr virus (EBV),
adenovirus, BK polyomavirus infections in transplant patients,
etc.
[0064] Cells prepared as described above can be utilized in methods
and compositions for adoptive immunotherapy in accordance with
known techniques, or variations thereof that will be apparent to
those skilled in the art based on the instant disclosure. See,
e.g., US Patent Application Publication No. 2003/0170238 to
Gruenberg et al; see also U.S. Pat. No. 4,690,915 to Rosenberg.
[0065] In some embodiments, the cells are formulated by first
harvesting them from their culture medium, and then washing and
concentrating the cells in a medium and container system suitable
for administration (a "pharmaceutically acceptable" carrier) in a
treatment-effective amount. Suitable infusion medium can be any
isotonic medium formulation, typically normal saline, Normosol R
(Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water
or Ringer's lactate can be utilized. The infusion medium can be
supplemented with human serum albumen.
[0066] A treatment-effective amount of cells in the composition is
at least 10.sup.9, typically greater than 10.sup.9, at least
10.sup.10 cells, and generally more than 10.sup.10. The number of
cells will depend upon the ultimate use for which the composition
is intended as will the type of cells included therein. For
example, if cells that are specific for a particular antigen are
desired, then the population will contain greater than 70%,
generally greater than 80%, 85% and 90-95% of such cells. For uses
provided herein, the cells are generally in a volume of a liter or
less, can be 500 mls or less, even 250 mls or 100 mls or less.
Hence the density of the desired cells is typically greater than
10.sup.6 cells/ml and generally is greater than 10.sup.7 cells/ml,
generally 10.sup.8 cells/ml or greater. The clinically relevant
number of immune cells can be apportioned into multiple infusions
that cumulatively equal or exceed 10.sup.9, 10.sup.10 or 10.sup.11
cells.
[0067] In some embodiments, the lymphocytes of the invention may be
used to confer immunity to 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, the 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. See, e.g., U.S. Pat. No. 6,040,177 to Riddell
et al. at column 17.
[0068] The present invention is illustrated further in the examples
set forth below.
Experimental
[0069] In the normal host, T cell memory persists for life
indicating the capacity for selfrenewal in addition to
differentiation.sup.23. This quality of T cell memory has been
suggested to reside in the CD62L.sup.+ T.sub.CM subset, although a
unique subset of T cells that self-renew and differentiate has been
identified in mice.sup.18,23,24. We sought to determine if T.sub.E
clones differentiated in vitro from T.sub.CM or T.sub.EM subsets
might differ intrinsically in their potential to persist after
adoptive transfer. Here, we show in a non-human primate model
relevant for human translation, that antigen-specific T.sub.E
clones derived from T.sub.CM but not T.sub.EM precursors can
persist, migrate to lymph nodes and bone marrow, reacquire
phenotypic properties of T.sub.CM and T.sub.EM, and respond to
antigen challenge.
Methods
[0070] Animals and experimental design. Adult macaques (Macaca
nemestrina) were housed at the University of Washington Regional
Primate Research Center, under American Association for
Accreditation of Laboratory Animal Care approved conditions. The
Institutional Animal Care and Use Committee approved the
experimental protocols.
[0071] Healthy macaques were selected for this study if they had
evidence of prior CMV infection as determined by a positive
lymphoproliferative response to CMV antigen and a CD8.sup.+ T cell
response to CMV IE-1 or IE-2 peptides.sup.25. CMV-specific
CD8.sup.+ T cell clones were isolated from CD62L.sup.+CD8.sup.+ and
CD62L.sup.-CD8.sup.+ T cells obtained by cell sorting of PBMC from
each macaque, modified by retroviral gene transfer, expanded in
vitro and infused intravenously at cell doses of
3-6.times.10.sup.8/kg. Blood samples were obtained by venipuncture
at intervals after each infusion and bone marrow from the posterior
iliac crest and a lymph node from the inguinal region were obtained
under anaesthesia.
[0072] Accredited clinical laboratories performed CBC and serum
chemistry on blood obtained before and at intervals after each T
cell infusion. The macaques were followed for at least 7 weeks
after each T cell infusion.
[0073] Cytokine flow cytometry for detection of CMV-specific
CD8.sup.+ T cells. PBMC were isolated from peripheral blood by
Ficoll-Hypaque gradient separation. Multiparameter flow cytometry
was used to detect CD8.sup.+ T cells in PBMC that expressed
intracellular IFN-.gamma. after stimulation with pools of 15-mer
synthetic peptides with an 11 amino acid (aa) overlap that spanned
the 558 aa sequence of the rhCMV IE1 protein (GenBank accession
number: M93360), or with an IE-2 peptide kindly provided by Dr. L.
Picker (Oregon Health Sciences University).sup.25. The peptides
that comprised the panel were synthesized using standard FMOC
chemistry (NMI) and arranged in an analytic grid composed of 24
pools, each containing 11-12 peptides. Aliquots of PBMC were
stimulated for 6 hours at 37.degree. C. with the peptide pools (5
.mu.g/ml) or medium alone in the presence of 1 .mu.g/ml monoclonal
anti-CD28 and anti-CD49d antibodies. Stimulation with PMA (10
ng/ml; Sigma) and ionomycin (1 .mu.g/ml; Sigma) was performed as a
positive control. After two hours, Brefeldin A (10 .mu.g/ml; Sigma)
was added. The cells were first stained with phycoerythrin
(PE)-labeled anti-CD8.beta. (Immunotech Coulter) and peridinin
chlorophyll protein (Percp)-Cy5.5-labeled anti-CD4, then
permeabilized using Cytofix/Cytoperm Permeabilization Solution (BD
Biosciences (BD)), and stained with a fluorescein isothiocyanate
(FITC)-labeled anti-IFN-.gamma. antibody (BD). All analyses were
performed on a FACSCalibur and the data analyzed using CellQuest
Software (BD). Immunogenic CMV peptides were identified by the
responses of CD8.sup.+ T cells to intersecting peptides in the grid
and the assays were repeated with each individual peptide and
derivative 9-mer peptides to confirm and map specificity.
[0074] To determine if CD8.sup.+ T cells for CMV IE peptides were
present in the T.sub.CM and T.sub.EM subsets, PBMC were stained
with fluorochrome conjugated anti-CD8 and anti-CD62L monoclonal
antibodies, and CD62L.sup.+CD8.sup.+ T cells and
CD62L.sup.-CD8.sup.+ T cells were sorted using a Vantage BD cell
sorter. The sorted cells were assayed by cytokine flow cytometry
after stimulation with autologous B cells that were activated by
culture on NIH 3T3 cells modified to express human CD40L, and then
pulsed with individual IE peptides or with media alone. The
CD40L-activated B cells were cocultured with the purified
CD62L.sup.+CD8.sup.+ and CD62L.sup.-CD8.sup.+ T cells for 6 hours
prior to the addition of Brefeldin A for an additional 4 hours,
followed by permeabilization and staining for intracellular
IFN-.gamma..
[0075] Retroviral vectors. A truncated macaque CD19 gene encoding
for the extracellular and transmembrane domain (.DELTA.CD19) and
four aa of the cytoplasmic tail to abrogate signaling, and the full
length CD20 gene were amplified by RT-PCR from cDNA generated from
macaque PBMC and cloned into pcDNA3.1 vector (Invitrogen). The
.DELTA.CD19 and CD20 genes were subcloned into the retroviral
plasmid pMP71GFP.sub.pre (W. Uckert, Max-Delbruck-Center, Berlin,
Germany) after removing the GFP gene.sup.43. Retrovirus supernatant
was produced in the packaging cell line Phoenix Galv (G. Nolan,
Stanford University, USA) after transfection using Fugene G
according to the manufacturer's instruction (Roche
Diagnostics).
[0076] Culture and genetic modification of CMV-specific CD8.sup.+ T
cell clones for adoptive transfer. CD62L.sup.-CD8.sup.+ and
CD62L.sup.-CD8.sup.+ T cell fractions were resuspended in RPMI 1640
supplemented with 25 mM HEPES, 10% human AB serum, 25 .mu.M
2-mercaptoethanol, and 4 mM L-glutamine (T-cell media), and
co-cultured with autologous monocytes pulsed with CMV IE-1 or IE-2
peptides (1 .mu.g/ml). IL-2 (10 U/ml; Chiron Corporation) was added
on day 3 of the culture. On day 7, T cell clones were generated by
plating 0.3 T cells per well in 96-well round bottom plates with
1.times.10.sup.5 .gamma.-irradiated autologous PBMC pulsed with CMV
IE-1 or IE-2 peptides (0.5 .mu.g/ml) and 1.times.10.sup.4
irradiated B-lymphoblastoid cells (LCL) as feeder cells in the
presence of 50 U/ml IL-2.
[0077] After 14 days, an aliquot from cloning wells with visible
growth was tested in a chromium release assay for recognition of
autologous target cells either pulsed with CMV peptide or with
medium alone. T cell clones that only lysed peptide pulsed target
cells were expanded by stimulation with monoclonal anti-CD3 and
anti-CD28 antibodies in the presence of human .gamma.-irradiated
PBMC and EBV transformed LCL, and IL-2 (50 U/ml) as
described.sup.44,45. On day 2 after stimulation, the cells were
pelleted, resuspended in .DELTA.CD19 or CD20 retroviral supernatant
with IL-2 (50 U/ml) and polybrene (5 .mu.g/ml), centrifuged at 1000
g for 1 hour at 32.degree. C., and incubated overnight. The cells
were then washed and cultured in CTL medium containing IL-2. On day
6 after retroviral transduction, T cells that expressed the
.DELTA.CD19 or CD20 transgene were selected with immunomagnetic
beads. Briefly, the transduced T cells were incubated with
monoclonal anti-CD19 (Immunotech Coulter) or anti-CD20 antibody
(BD), washed, incubated with rat anti-mouse IgG-coupled magnetic
beads (Miltenyi Biotec), and selected using the MidiMACS device.
The selected cells were then cultured for an additional 6 days and
cryopreserved in aliquots that could be thawed subsequently for
adoptive transfer experiments. The clonality of the infused T cell
clones was confirmed by analysis of TCR V.beta. gene
rearrangements.
[0078] To assess cell viability in vitro, aliquots of the T cell
clones at the end of the 14-day stimulation cycle and immediately
prior to adoptive transfer were plated in 12-well tissue culture
plates at 2-4. 10.sup.6 cells/well in T-cell media alone, media
with IL-2 (1 ng/ml or 16 U/ml; Chiron), IL-15 (1 ng/ml; R&D
Systems), or IL-7 (10 ng/ml; R&D).
[0079] The viability of T cells was assessed every 3-4 days by
trypan blue dye exclusion. In some experiments, aliquots of the T
cell clones were cultured with media containing IL19 21 (30 ng/ml)
(R&D) or cocultured with autologous PBMC and mature
monocytederived dendritic cells.sup.46.
[0080] Generation of T-APC. To generate T-APC, we expanded aliquots
of autologous polyclonal T cells by stimulation with anti-CD3 and
anti-CD28 monoclonal antibodies and IL-2 (50 U/ml). At the end of a
14 day stimulation cycle, the T cells were harvested, pulsed for 30
min with 1 .mu.g/ml of the cognate CMV IE peptide, washed twice in
media, suspended in normal saline and administered to the animal by
intravenous infusion at a cell dose of 1.times.10.sup.7/kg. An
aliquot of the autologous T cells before and after pulsing with the
CMV IE peptide were labeled with .sup.51Cr and tested as targets
for the CMV-specific T cell clone.
[0081] Cytotoxicity assays. Cytotoxic responses of CMV-specific T
cells were examined as described.sup.44,45,47. Briefly, autologous
.sup.51Cr-labeled T cells were pulsed overnight with peptide
antigen at various concentrations or medium alone and used in a
chromium release assay for recognition by CD8.sup.+ T cell
clones.
[0082] Flow cytometry. PBMC and T cell clones were analyzed by flow
cytometry after staining with fluorochrome-conjugated monoclonal
anti-CD3 (SP34), CD4, CD8, CD27, CD28, CD45RA, CD62L, CCR7, CD122
(IL2-R.beta.), CD132 (IL2-R-.gamma.) (BD), CD19, CD80, CD45RO, and
CD127 (Immunotech Coulter), and IL15R((R&D) antibodies. For
intracellular staining, cells were suspended in
CYTOFIX/CYTOPERM.TM. Permeabilization Solution (BD) according to
the manufacturer's instruction and stained with monoclonal
antibodies to granzyme B (BD), perforin (Kamiya Biomedical), bcl-2
(BD), and bcl-x1 (Southern Biotech). Isotype-matched irrelevant
control monoclonal antibodies served as controls (BD). In some
experiments, samples of PBMC were labeled with anti-CD8-FITC and
anti-CD19 allophycocyanin (APC) and stained with Annexin V-PE and
PI according to the manufacturer's instruction. All analyses were
performed on a FACSCalibur and the data analyzed using CellQuest
Software (BD).
[0083] CFSE-labeling of T cells. CD19.sup.+CD62L.sup.+CD8.sup.+ and
CD19.sup.+CD62L.sup.-CD8.sup.+ T cells were flow sorted from pooled
PBMC samples obtained after adoptive transfer and labeled with
0.625 .mu.M CFSE (Molecular Probes) in PBS for 10 minutes at
37.degree. C. under constant agitation, followed by blocking with
20% FCS medium. CFSE-labeled T cells were plated at
1.times.10.sup.5 cells/well in a 96-well plate with autologous
CD40L activated B cells (2.5.times.10.sup.4 cells/well) pulsed with
200 ng/ml CMV IE peptide. CFSE dilution was analyzed by flow
cytometry after five days.
[0084] Fluorescent Probe PCR. PCR amplifications and analyses were
performed using a quantitative real-time PCR assay (Perkin-Elmer
Applied Biosystems).sup.45,48, DNA (0.3-1 .mu.g) was amplified in
duplicate using PCR primers and TaqMan probes (Synthegen) designed
to detect a unique MP71CD20.sub.pre sequence using a
fluorescent-tagged probe encompassing the junction of the CD20 gene
and the retroviral vector pMP71.sub.pre.
[0085] Standards consisted of DNA derived from the infused CD20+ T
cells. Aliquots of preinfusion PBMC served as negative control.
[0086] Telomere length analysis. The average length of telomere
repeats in individual lymphocytes was measured by automated
flow-FISH as described.sup.49,50.. Cells, which were previously
frozen in 10% (v/v) DMSO 20% (v/v) human serum, were thawed and
processed in microtiter plates for automated flow-FISH. Cells were
hybridized with or without 0.3 .mu.g/ml telomere specific
FITC-conjugated (CCCTAA).sub.3 PNA probe, washed and counterstained
with 0.01 .mu.g/ml LDS 751 (Exciton Chemical). To convert the
fluorescence measured in sample cells hybridized with the
FITC-labeled telomere PNA probe into kilobases (kb) of telomere
repeats, fixed bovine thymocytes with known telomere length as an
internal control were processed simultaneously with each
sample.sup.50. FITC-labeled fluorescent beads were used to correct
for daily shifts in the linearity of the flow cytometer and
fluctuations in the laser intensity and alignment. Flow cytometric
data collection was performed on a FACSCalibur.TM. apparatus and
the data analysis was performed using a CellQuestPro.TM. data
analysis system (BD).
Results
[0087] Characterization of Cytomegalovirus (CMV)-specific CD8.sup.+
T cell clones derived from CD62L.sup.+ T.sub.CM and CD62L.sup.-
T.sub.EM subsets. Immunocompetent Macaca nemestrina with latent CMV
infection were used in this study. To identify CMV-specific
CD8.sup.+ T cells, we stimulated aliquots of peripheral blood
mononuclear cells (PBMC) from four macaques with rhesus CMV
immediate early (IE)-1 or IE-2 peptides and analyzed
interferon-gamma (IFN-.gamma.) production by flow cytometry.sup.25.
After identification of an immunogenic CMV epitope for each
macaque, we evaluated whether specific CD8.sup.+ T cells were
present in CD62L+and CD62L.sup.- T cell fractions, purified from
PBMC and containing T.sub.CM and T.sub.EM respectively. Stimulation
of CD62L.sup.+ and CD62L.sup.- T cells with CMV peptides identified
CD8.sup.+ T cells that produced IFN-.gamma. in both subsets (FIG.
1a). The majority of CD62L.sup.+ T cells were positive for CCR7 and
CD28, and expressed absent or low levels of granzyme B. The
CD62L.sup.- T cells were mostly negative for CD28 and CCR7, and
positive for granzyme B (not shown).
[0088] We next generated CD8.sup.+CMV-specific T cell clones for
adoptive transfer from purified CD62L.sup.+CD8.sup.+ (T.sub.CM) and
CD62L.sup.-CD8.sup.+ (T.sub.EM) cells of three macaques (FIG. 1b).
Cytolytic CD8.sup.+CMV-specific T cell clones were obtained from
both T.sub.M subsets in all macaques. The cloning efficiency was
13.6-23.2% for cultures initiated from T.sub.CM, and 0.4-10.4% for
cultures initiated from T.sub.EM. Individual T cell clones were
transduced with a retroviral vector encoding either a truncated
cell surface CD19 (.DELTA.CD19) or full-length CD20 molecule to
permit tracking the cells in vivo, and transduced T cells were
selected with immunomagnetic beads (FIGS. 1c, d). A pair of
T.sub.CM and T.sub.EM-derived clones was randomly selected from
each of the three macaques, and expanded in culture to
>5.times.10.sup.9 cells over a total culture duration of 49 days
before adoptive transfer. Independent of their derivation from
CD62L.sup.+ or CD62L.sup.- cells, all of the CMV-specific T cell
clones had differentiated to T.sub.E and were negative for CD62L,
CCR7, CD28, and CD127, and positive for granzyme B and perforin
(FIG. 2a). The T cell clones of each pair recognized the same CMV
peptide, had comparable avidity, displayed nearly identical growth
after T cell receptor (TCR) stimulation, and had similar telomere
lengths (FIGS. 2b-d).
[0089] In vivo persistence of CD8.sup.+ T.sub.E clones derived from
CD62L.sup.+ T.sub.CM or CD62L.sup.- T.sub.EM. We administered the
autologous gene-modified T cells intravenously and measured their
frequency in the blood, lymph node, and bone marrow at intervals
after infusion. A .DELTA.CD19-modified T cell clone derived from
T.sub.EM was transferred to macaque 02269 at a dose of
3.times.10.sup.8 T cells/kg, which is approximately 5-10% of the
macaque total body lymphocyte pool.sup.26. CD19.sup.+CD8.sup.+ T
cells were easily detected in the blood one day after the infusion
at a frequency of 1.2% of CD8.sup.+ cells or 10 cells/.mu.l of
blood. The CD19.sup.+T cells in the blood peaked at 3.7% of
CD8.sup.+ T cells (40 cells/.mu.l) on day three after the infusion.
However, the T cells were not detected in blood obtained at day
five or multiple times up to forty-two days after infusion, and
were not present in bone marrow or lymph node samples obtained
fourteen days after the infusion (FIG. 3a). We transferred the same
dose of a .DELTA.CD19-modified CMV-specific T cell clone derived
from the T.sub.CM subset to this macaque. On day one post infusion,
the frequency of CD19.sup.+CD8.sup.+ T cells in the blood was 2.2%
of CD8.sup.+ T cells (10 cells/.mu.l). In contrast to the
T.sub.EM-derived clone, the T.sub.CM-derived cells persisted in the
blood for greater than fifty-six days after infusion at .about.0.2%
of CD8.sup.+ T cells (3-6 cells/.mu.l), and comprised 1.2% and 0.6%
of CD8.sup.+ T cells in bone marrow and lymph node samples
respectively, obtained fourteen days after the infusion (FIG. 3b).
In a second macaque, we infused a .DELTA.CD19-modified
T.sub.CM-derived clone first at a higher cell dose of
6.times.10.sup.8/kg. With this dose, transferred cells were
detected in the blood at a frequency of 26.3% of CD8.sup.+ T cells
(228 cells/.mu.l) on day one and 46.3% of CD8.sup.+ T cells (734
cells/.mu.l) on day three. The transferred T.sub.CM-derived clone
was present in the bone marrow and lymph node obtained on day
fourteen at 4.7% and 0.7% of CD8.sup.+ T cells, respectively (FIG.
3c). The frequency of the transferred cells in the blood declined
gradually over twenty-eight days to a stable level of 7-10
cells/.mu.l that persisted for greater than eleven months after
infusion (FIG. 3c). We administered the same dose of a
T.sub.EM-derived clone transduced to express CD20 to enable the
cells to be tracked and distinguished from the previously
transferred .DELTA.CD19-modified T cells. The frequency of
CD20.sup.+CD8.sup.+ T cells in the blood one day after the infusion
was 16.3% of CD8.sup.+ T cells (103 cells/.mu.l), but the
transferred cells disappeared from the blood by day five and were
not detected in the bone marrow and lymph node obtained fourteen
days after the infusion (FIG. 3d). The disappearance of
CD20-modified T cells was confirmed by PCR for vector sequences on
DNA isolated from peripheral blood, bone marrow and lymph node
mononuclear cells obtained on day 14 (data not shown).
[0090] CD8.sup.+ T.sub.E cells derived from T.sub.EM undergo rapid
apoptosis after adoptive transfer and respond poorly to IL-15. The
failure of T.sub.E clones derived from T.sub.EM to persist in
blood, marrow or lymph nodes after adoptive transfer could be due
to cell death in vivo and/or migration to other tissue sites.
T.sub.EM-derived clones expressed lower levels of the
anti-apoptotic proteins bclx1 and bcl-2 than T.sub.CM-derived
clones, suggesting they may be more susceptible to apoptosis (FIG.
4a). Therefore, we infused a .DELTA.CD19-modified T.sub.EM-derived
T cell clone to the third macaque and measured the proportion of
transferred cells in PBMC that were positive for propidium iodide
(PI) and Annexin V binding.sup.27. Approximately 40% of the
CD19.sup.+CD8.sup.+ T cells in PBMC were PI and/or Annexin V
positive when analyzed directly on day one, and 45% were positive
after culturing the PBMC for 24 hours (FIG. 4b).
[0091] Consistent with the prior animals, the T.sub.EM-derived
clone was only detected in the blood for three days, and was not
present in the day fourteen bone marrow or lymph node samples (FIG.
4c). We then infused a .DELTA.CD19-modified T.sub.CM-derived T cell
clone into the same animal and analyzed the proportion of apoptotic
cells. Less than 20% of the transferred T.sub.CM cells were PI
and/or Annexin V positive in PBMC analyzed directly, and this
fraction decreased to 12% after 24 hours of culture (FIG. 4b). The
T.sub.CM-derived clone migrated to the bone marrow and lymph nodes
(FIG. 4c), and persisted in the blood for greater than
seventy-seven days as observed previously (data not shown). We
reasoned that the selective survival of T.sub.CM-derived T cells in
vivo might be due to responsiveness to homeostatic cytokines such
as IL-15 that maintain endogenous CD8.sup.+ memory T
cells.sup.28-30, and cultured aliquots of the T.sub.CM and
T.sub.EM-derived clones in media containing low doses of IL-15,
IL-2, or IL-7. Both T.sub.EM and T.sub.CM-derived T.sub.E.sub.E
clones lacked expression of IL-7R.alpha. (FIG. 2a) and died rapidly
in vitro when cultured in IL-7 or in media alone (FIG. 4d). Culture
in IL-15 or IL-2 also did not improve the viability of
T.sub.EM-derived clones (FIG. 4d). By contrast, T.sub.CM-derived
clones were rescued from cell death for over 30 days in IL-15 and
exhibited improved survival in IL-2 (FIG. 4d). The responsiveness
of T.sub.CM-derived clones to IL-15 correlated with higher levels
of cell surface IL-15R.alpha., IL-2R.beta..RTM., and IL-2R.gamma.
compared with T.sub.EM-derived clones (FIG. 4e).
[0092] Adoptively transferred CD8.sup.+ T.sub.E clones derived from
T.sub.CM acquire a memory phenotype in vivo. The migration of
T.sub.CM-derived CD8.sup.+ T.sub.E clones to lymph nodes suggested
that CD62L might be re-expressed on transferred cells in vivo.
CD62L was not detected on the T cell clones at the time of adoptive
transfer (FIG. 2a), or after culture in the absence of antigen
stimulation with cytokines (IL-2, IL-7, IL-15, IL-21), autologous
PBMC, or monocyte-derived dendritic cells (data not shown).
However, in all three macaques, CD19.sup.+CD62L.sup.+CD8.sup.+
cells were observed in the blood as early as three days after the
infusion of the T.sub.CM-derived clones and were detected in the
blood, bone marrow and lymph node samples obtained on day 14 (FIG.
5a, b). The proportion of transferred T cells that were CD62L.sup.+
in the lymph node was greater than in blood or marrow (FIG.
5b).
[0093] CD62L was not expressed during the brief period the
T.sub.EM-derived clone persisted in the blood in any of the three
macaques (FIG. 5a). We next examined whether the T cells that
persisted in vivo after adoptive transfer acquired other phenotypic
markers of memory T cells that were absent on the infused T.sub.E.
Fourteen days after cell transfer, a subset of the
CD19.sup.+CD8.sup.+ T cells in blood and lymph node expressed CCR7,
CD28, and CD127 (FIGS. 5c-e). We repeated this analysis on blood,
bone marrow, and lymph node samples obtained two months after the
infusion of the T.sub.CM-derived clone in macaque 02258. At this
later time, the infused CD19.sup.+T cells comprised 1.4% of the
CD8.sup.+ T cells in the lymph node and these T cells were
uniformly positive for T.sub.CM markers including CD62L, CCR7,
CD28, and CD127 (FIGS. 5b-e). Transferred T cells were present at
lower levels in the blood and bone marrow, and contained both
CD62L.sup.+ and CD62L.sup.- fractions. The transferred CD19.sup.+T
cells that persisted long-term in the blood and expressed CD62L,
also expressed CR7, CD28 and CD127 (not shown). Thus, despite
differentiation to T.sub.E during expansion of a single T.sub.CM
cell to more than 5.times.10.sup.9 cells, a significant fraction of
the transferred T cell clones were able to persist long-term,
compete with endogenous T cells for anatomical niches of T.sub.CM,
and re-express phenotypic markers of T.sub.CM.
[0094] Memory T cells established by adoptive transfer exhibit
functional properties of both T.sub.CM and T.sub.EM. An objective
of T cell immunotherapy is to establish a stable pool of memory T
cells that can respond to antigen by producing cytokines and
differentiating into cytolytic effector cells. The transferred
CD19.sup.+T.sub.CM-derived cells that persisted in the blood
produced IFN-.gamma. after peptide stimulation and their frequency
was comparable to endogenous CD19.sup.- CMV-specific CD8.sup.+ T
cells (FIG. 6a). The cytolytic function of these T cells was
examined by sorting T cells from the blood into
CD19.sup.+CD62L.sup.+CD8.sup.+ and CD19.sup.+CD62L.sup.-CD8.sup.+
fractions. For these experiments, it was necessary to pool PBMC
samples from multiple time points to obtain sufficient cells for
the assays. The CD19.sup.+CD62L.sup.- T cells demonstrated
cytolytic activity nearly equivalent to that of the T cell clone
and expressed granzyme B, while the CD19.sup.+CD62L.sup.+ T cells
lacked direct cytolytic activity and had low granzyme B expression
(FIG. 6b). In vitro stimulation of the CD19.sup.+CD62L.sup.+ T
cells with anti-CD3 and anti-CD28 monoclonal antibodies generated
T.sub.E cells with cytolytic activity, demonstrating these cells
could be re-induced to differentiate to cytolytic T.sub.E (FIG.
6b). We also labeled the sorted CD19.sup.+CD62L.sup.+ and
CD19.sup.+CD62L.sup.- T cell fractions with CFSE and monitored
proliferation five days after antigen stimulation in vitro.
Seventy-four percent of CD62L.sup.+ cells underwent 5 or more cell
divisions, but only 13% of CD62L.sup.- cells underwent 5 or more
divisions (FIG. 6c). Thus, the adoptive transfer of a T.sub.E clone
derived from the T.sub.CM pool established distinct populations of
cells in vivo with the functional properties of T.sub.CM and
T.sub.EM.
[0095] We have previously shown in human studies that the infusion
of T cells that express a foreign antigen can prime and boost
endogenous antigen-specific memory T cell responses.sup.31. Thus,
to determine if T.sub.M established by adoptive transfer could
respond to antigen stimulation in vivo, we infused a small dose
(1.times.10.sup.7/kg) of autologous T cells pulsed with CMV IE
peptide (T-APC) into macaque A99171 two months after administering
the .DELTA.CD19-modified T.sub.CM-derived T cells. The peptide
pulsed T-APC were efficiently lysed by the CMV-specific T cell
clone in vitro (FIG. 6e). Within 7 days of the infusion of T-APC,
there was a 4 to 5-fold increase in the absolute numbers of both
.DELTA.CD19-modified and endogenous IE-specific T cells in the
blood (FIG. 6d). Thus, the memory T cells established by adoptive
transfer were as capable as endogenous memory T cells of expanding
in response to antigen in vivo.
Discussion
[0096] Poor survival of adoptively transferred T cells that target
infected or cancerous cells has correlated with lack of therapeutic
efficacy in clinical trials.sup.14,16,16. The persistence and
efficacy of cultured T cells can sometimes be improved by depletion
of host lymphocytes prior to cell transfer to eliminate regulatory
cells and competition for pro-survival cytokines, and by the
administration of IL-2 after cell transfer.sup.16,17. However,
these interventions are not uniformly successful in improving cell
persistence suggesting that intrinsic properties of T cells
isolated for adoptive therapy may be critical for establishing
durable immunity. Here, we used gene marking to show that
antigen-specific CD8.sup.+ T.sub.E clones derived in vitro from the
T.sub.CM but not from the T.sub.EM subset, persist longterm in
vivo, occupy memory T cell niches, and reacquire phenotypic and
functional properties of T.sub.CM after adoptive transfer.
[0097] After encountering antigen in vivo, T cells undergo
proliferation and programmed differentiation evoked by signals from
the TCR, co-stimulatory and adhesion molecules, and cytokine
receptors.sup.18,20,32. These events result in the generation of
large numbers of T.sub.E that die as antigen is cleared, and a
smaller pool of phenotypically distinct T.sub.CM and T.sub.EM that
persist for life and respond to antigen re-exposure by
differentiating into T.sub.E. Data from both murine and human
studies support a linear differentiation model in which T.sub.CM
give rise to both T.sub.EM and T.sub.E, although it remains
possible that T.sub.CM and T.sub.EM represent separate
lineages.sup.18,33-35. The capacity of T cell memory to be
maintained for life suggests that at least some T.sub.M must be
capable of both self-renewal and differentiation.sup.23.
[0098] The results presented here suggest that isolating T.sub.CM
for gene insertion may provide a tumor-reactive T cell population
with an improved capacity to persist after adoptive transfer.
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[0149] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
* * * * *