U.S. patent application number 13/523828 was filed with the patent office on 2013-01-17 for isolation and/or preservation of dendritic cells for prostate cancer immunotherapy.
This patent application is currently assigned to NORTHWEST BIOTHERAPEUTICS, INC.. The applicant listed for this patent is Alton L. Boynton, Gerald P. Murphy, Benjamin A. Tjoa. Invention is credited to Alton L. Boynton, Gerald P. Murphy, Benjamin A. Tjoa.
Application Number | 20130017600 13/523828 |
Document ID | / |
Family ID | 24025874 |
Filed Date | 2013-01-17 |
United States Patent
Application |
20130017600 |
Kind Code |
A1 |
Murphy; Gerald P. ; et
al. |
January 17, 2013 |
ISOLATION AND/OR PRESERVATION OF DENDRITIC CELLS FOR PROSTATE
CANCER IMMUNOTHERAPY
Abstract
Methods and compositions for use of human dendritic cells to
activate T cells for immunotherapeutic responses against primary
and metastatic prostate cancer are disclosed. In one embodiment,
human dendritic cells, after exposure to a prostate cancer antigen
or specific antigenic peptide, are administered to a prostate
cancer patient to activate the relevant T cell responses in vivo.
In an alternate embodiment, human dendritic cells are exposed to a
prostate cancer antigen or specific antigenic peptide in vitro and
incubated or cultured with primed or unprimed T cells to activate
the relevant T cell responses in vitro. The activated T cells are
then administered to a prostate cancer patient. Methods and
compositions for human dendritic cells with extended life span and
cryopreserved dendritic cells are disclosed.
Inventors: |
Murphy; Gerald P.; (Seattle,
WA) ; Boynton; Alton L.; (Redmond, WA) ; Tjoa;
Benjamin A.; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Murphy; Gerald P.
Boynton; Alton L.
Tjoa; Benjamin A. |
Seattle
Redmond
Seattle |
WA
WA
WA |
US
US
US |
|
|
Assignee: |
NORTHWEST BIOTHERAPEUTICS,
INC.
Bethesda
MD
|
Family ID: |
24025874 |
Appl. No.: |
13/523828 |
Filed: |
June 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09016737 |
Jan 30, 1998 |
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13523828 |
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PCT/US1996/012389 |
Jul 29, 1996 |
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09016737 |
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08509254 |
Jul 31, 1995 |
5788963 |
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PCT/US1996/012389 |
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Current U.S.
Class: |
435/372 |
Current CPC
Class: |
A61K 2039/5154 20130101;
A61K 39/001195 20180801; A61K 2039/5158 20130101; A61P 35/00
20180101; A61P 13/08 20180101; A61P 37/04 20180101; C07K 14/705
20130101; C07K 7/06 20130101; A61K 39/0011 20130101 |
Class at
Publication: |
435/372 |
International
Class: |
C12N 5/0784 20100101
C12N005/0784 |
Claims
1. A composition comprising an isolated cell population having
human dendritic cells, wherein said cell population has been
cultured in the presence of granulocyte-macrophage colony
stimulating factor (GM-CSF), interleukin 4 (IL-4), and exposed in
vitro to a soluble prostate antigen, the cell population having an
increased ability to activate T cells specific to the prostate
antigen as compared to a similar isolated cell population cultured
in the presence of granulocyte-macrophage colony stimulating factor
(GM-CSF), and interleukin 4 (IL-4) that has not been exposed in
vitro to the prostate antigen.
2. The composition according to claim 1, in which the prostate
antigen is a lysate of LNCaP cells, a membrane preparation of LNCaP
cells, a lysate of prostate tumor cells from a prostate cancer
patient, a membrane preparation of prostate tumor cells from a
prostate cancer patient, isolated prostate specific membrane
antigen (PSMA), purified prostate specific membrane antigen (PSMA),
a peptide having the amino acid sequence LLHETDSAV (SEQ ID NO: 1),
a peptide having the amino acid sequence ALFDIESKV (SEQ ID NO: 2),
a peptide having the amino acid sequence XL(or M)XXXXXV(or L) (SEQ
ID NO: 3), where X represents any amino acid, purified prostate
specific antigen (PSA), or a purified prostate mucin antigen
recognized by monoclonal antibody PD41.
3. The composition according to claim 1, in which the prostate
antigen is selected from the group consisting of: TABLE-US-00004
WLCAGALVL; (SEQ. ID. NO. 4) VLAGGFFLL; (SEQ. ID. NO. 5) ELAHYDVIA;
(SEQ. ID. NO. 6) NLNGAGDPL; (SEQ. ID. NO. 7) TLRVDCTPL; (SEQ. ID.
NO. 8) VLRMMNDQL; (SEQ. ID. NO. 9) PMFKYHLTV; (SEQ. ID. NO. 10)
NMKAFLDEL; (SEQ. ID. NO. 11) LMYSLVHNL; (SEQ. ID. NO. 12)
MMNDQLMFL; (SEQ. ID. NO. 13) EGDLVYVNY; (SEQ. ID. NO. 14)
AGDPLTPGY; (SEQ. ID. NO. 15) RVDCTPLMY; (SEQ. ID. NO. 16)
LFEPPPPGY; (SEQ. ID. NO. 17) TYELVEKFY; (SEQ. ID. NO. 18)
AGESFPGIY; (SEQ. ID. NO. 19) WGEVKRQIY; (SEQ. ID. NO. 20)
IVRSFGTLKKE; (SEQ. ID. NO. 21) DELKAENIKKF; (SEQ. ID. NO. 22)
KSLYESWTKKS; (SEQ. ID. NO. 23) AYINADSSI; (SEQ. ID. NO. 24)
KYADKIYSI; (SEQ. ID. NO. 25) GYYDAQKLL; (SEQ. ID. NO. 26)
TYSVSFDSL; (SEQ. ID. NO. 27) NYARTEDFF; (SEQ. ID. NO. 28)
LYSDPADYF; (SEQ. ID. NO. 29) LPSIPVHPI; (SEQ. ID. NO. 30)
SPSPEFSGM; (SEQ. ID. NO. 31) VLVHPQWUL; (SEQ. ID. NO. 32)
KLQCVDLHV; (SEQ. ID. NO. 33) ALPERPSLY; (SEQ. ID. NO. 34)
JVGGWECEK; (SEQ. ID. NO. 35) QVHPQKVTK; (SEQ. ID. NO. 36)
VVHYRKWIK; (SEQ. ID. NO. 37) CYASGWGSI. (SEQ. ID. NO. 38)
4. The composition according to claim 1, in which the dendritic
cells are extended life span dendritic cells.
5. The composition according to claim 1, in which the prostate
tissue antigen is: TABLE-US-00005 WLCAGALVL; (SEQ. ID. NO. 4)
VLAGGFFLL; (SEQ. ID. NO. 5) ELAHYDVLL; (SEQ. ID. NO. 6) NLNGAGDPL;
(SEQ. ID. NO. 7) TLRVDCTPL; (SEQ. ID. NO. 8) VLRMMNDQL; (SEQ. ID.
NO. 9) PMFKYHLTV; (SEQ. ID. NO. 10) NMKAFLDEL; (SEQ. ID. NO. 11)
LMYSLVHNL; (SEQ. ID. NO. 12) MMNDQLMFL; (SEQ. ID. NO. 13)
EGDLVYVNY; (SEQ. ID. NO. 14) AGDPLTPGY; (SEQ. ID. NO. 15)
RVDCTPLMY; (SEQ. ID. NO. 16) LFEPPPPGY; (SEQ. ID. NO. 17)
TYELVEKFY; (SEQ. ID. NO. 18) AGESFPGIY; (SEQ. ID. NO. 19)
WGEVKRQIY; (SEQ. ID. NO. 20) IVRSFGTLKKE; (SEQ. ID. NO. 21)
DELKAENIKKF; (SEQ. ID. NO. 22) KSLYESWTKKS; (SEQ. ID. NO. 23)
AYINADSSI; (SEQ. ID. NO. 24) KYADKIYSI; (SEQ. ID. NO. 25)
GYYDAQKLL; (SEQ. ID. NO. 26) TYSVSFDSL; (SEQ. ID. NO. 27)
NYARTEDFF; (SEQ. ID. NO. 28) LYSDPADYF; (SEQ. ID. NO. 29)
LPSIPVHPI; (SEQ. ID. NO. 30) SPSPEFSGM; (SEQ. ID, NO. 31)
VLVHPQWUL; (SEQ. ID. NO. 32) KLQCVDLHV; (SEQ. ID. NO. 33)
ALPERPSLY; (SEQ. ID. NO. 34) JVGGWECEK; (SEQ. ID. NO. 35)
QVHPQKVTK; (SEQ. ID. NO. 36) VVHYRKWIK; (SEQ. ID. NO. 37) or
CYASGWGSI. (SEQ. ID. NO. 38)
6. The composition according to claim 1, in which the dendritic
cells have been cryopreserved prior to exposure in vitro to the
prostate antigen, wherein said dendritic cells retain the ability
to take up and present antigen.
7. The composition according to claim 6, in which the prostate
antigen is a lysate of LNCaP cells, a membrane preparation of LNCaP
cells, a lysate of prostate tumor cells from a prostate cancer
patient, a membrane preparation of prostate tumor cells from a
prostate cancer patient, isolated prostate specific membrane
antigen (PSMA), purified prostate specific membrane antigen (PSMA),
a peptide having the amino acid sequence LLHETDSAV (SEQ. ID. NO.
1), a peptide having the amino acid sequence ALFDIESKV (SEQ. ID.
NO. 2), a peptide having the amino acid sequence XL(or M)XXXXXV(or
L) (SEQ. ID. NO. 3), where X represents any amino acid, purified
prostate specific antigen (PSA), or a purified prostate mucin
antigen recognized by monoclonal antibody PD41.
8. The composition according to claim 6, in which the dendritic
cells are extended life dendritic cells.
9. The composition according to claim 1, wherein said dendritic
cells can activate 2 to 3 fold more T cells specific to the
prostate antigen as compared to an isolated cell population
cultured in the presence of granulocyte-macrophage colony
stimulating factor (GM-CSF) and interleukin 4 (IL-4) that has not
been exposed in vitro to the prostate antigen.
10. The composition according to claim 1, wherein the human
dendritic cells are immature dendritic cells.
11. The composition according to claim 1, wherein the T cells are
CD4.sup.+.
12. The composition according to claim 1, wherein the T cells are
CD8.sup.+.
13. The composition according to claim 1, wherein the dendritic
cells are isolated from a prostate cancer patient.
14. The composition according to claim 1, wherein the dendritic
cells are isolated from a normal individual.
15. The composition according to claim 14, wherein the dendritic
cells are HLA-matched for a recipient.
Description
1. FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods of
isolating and/or preserving and using human dendritic cells for
immunotherapy for cancer. In particular, the invention relates to
methods for the use of dendritic cells for the activation and
expansion of large numbers of prostate antigen specific T cells for
use in adoptive cellular immunotherapy against prostate cancer, as
well as methods for the use of prostate antigen pulsed dendritic
cells as vaccines and/or immunotherapeutics to slow or inhibit the
growth of primary or metastatic prostate cancer.
2. BACKGROUND OF THE INVENTION
2.1. Prostate Cancer
[0002] Prostate cancer is the second leading cause of death from
cancer among men. In fact, prostate cancer is the most common
(noncutaneous) cancer diagnosed in the American male and is
steadily increasing, as a result of the increasing population of
older men as well as greater awareness and earlier diagnosis of the
disease. See, Wright et al., 1995, Urol. Oncol. 1:18-28. In 1995,
it is projected that over 244,000 men will be diagnosed with
prostate cancer in this year. There will be 40,400 deaths. The life
time risk for men, to suffer prostate cancer is now 1 in 8 for
Caucasians, and an estimated 1 in 7 for African Americans. High
risk groups are those with a positive family history or African
Americans. Over a lifetime, above 25% of the men diagnosed with
prostate cancer will die of the disease. Wingo et al., 1995, CA
Cancer J. Clin. 45(2):8-30. Moreover, many patients who do not die
of prostate cancer require treatment to ameliorate symptoms such as
pain, bleeding, and urinary obstruction. Thus, prostate cancer is
also a major cause of suffering and of health care expenditures.
Catalona, W. J., 1994, New Eng. J. Med. 331:996-1004.
[0003] Cytotoxic chemotherapy is largely ineffective in treating
prostate cancer. A combination of agents is no more effective than
a single agent, and the addition of chemotherapy to hormonal
therapy does not improve survival. Eisenberger, M. A., 1988,
Chemotherapy for prostate carcinoma. In: Wittes, R. E., ed.
Consensus Development Conference on the Management of Clinically
Localized Prostate Cancer. NCI monographs No. 7 Washington D.C.:
Government Printing Office: 151-153 (NIH publication no. 88-3005).
Accordingly, there is a great demand for improved prostate cancer
therapeutics.
2.2 Cancer Immunotherapy
[0004] It is well established that the immune system can function
to kill tumor cells, including both primary and metastatic cancer
cells. Indeed, evidence that the immune system recognizes the
presence of neoplastic cancerous cells is supported by the
existence of infiltrating lymphocytes in tumor tissues (Haskill et
al., 1978, Contemp. Top. Immunobiol. 8:107-170; Vose and Moore,
1985, Semin. Hematol. 22:27-40). Yet, for reasons that have not
been completely clear, despite the presence of immune cells, tumors
often prevail and not only survive but metastasize to distant sites
with unrestricted growth.
[0005] Recent advances in the understanding of T cell activation
and recognition of target cells have begun to permit some progress
in development of T cell mediated cancer immunotherapy (Schwartz,
1992, Cell 71:1065-1068; Pardoll, 1992, Curr. Opin. Immunol.
4:619-623).
[0006] In its most general form, the generation of an immune
response begins with the sensitization of helper (T.sub.H,
CD4.sup.+) and cytotoxic (CD8.sup.+) T cell subsets through their
interaction with antigen presenting cells (APC) that express major
histocompatibility (MHC)-class I or class II molecules associated
with antigenic fragments (i.e., specific amino acid sequences
derived from the antigen which bind to MHC I and MHC II for
presentation on the cell surface). The sensitized or primed
CD4.sup.+ T cells produce lymphokines that participate in the
activation of B cells as well as various T cell subsets. The
sensitized CD8.sup.+ T cells increase in numbers in response to
lymphokines and are capable of destroying any cells that express
the specific antigenic fragments associated with matching
MHC-encoded class I molecules. Thus, in the course of a cancerous
tumor, CTL eradicate cells expressing cancer associated or cancer
specific antigens, thereby limiting the progression of tumor spread
and disease development.
[0007] Various methods for immunotherapy of a number of particular
cancers have been suggested; however, to date no one has developed
any therapeutic method that successfully elicits an effective
immunotherapeutic response against primary or metastatic prostate
cancer.
2.3 Dendritic Cells for Antigen Presentation
[0008] Antigen presenting cells (APC) are particularly important in
eliciting an effective immune response.
[0009] By definition, APC not only can present antigens to T cells
with antigen-specific receptors, but can provide all the signals
necessary for T cell activation. Such signals are incompletely
defined, but probably involve a variety of cell surface molecules
as well as cytokines or growth factors. Further, the factors
necessary for the activation of naive or unprimed T cells may be
different from those required for the re-activation of previously
primed memory T cells. The ability of APC to both present antigens
and deliver signals for T cell activation is commonly referred to
as an accessory cell function. Although monocytes and B cells have
been shown to be competent APC, their antigen presenting capacities
in vitro appear to be limited to the re-activation of previously
sensitized T cells. Hence, they are not capable of directly
activating functionally naive or unprimed T cell populations.
[0010] The term "dendritic cells" refers to a diverse population of
morphologically similar cell types found in a variety of lymphoid
and non-lymphoid tissues (Steinman, 1991, Ann. Rev. Immunol.
9:271-296). These cells include lymphoid DC of the spleen,
Langerhans cells of the epidermis, and veiled cells in the blood
circulation. Although they are collectively classified as a group
based on their morphology, high levels of surface MHC-class II
expression, and absence of certain other surface markers expressed
on T cells, B cells, monocytes, and natural killer cells, it is
presently not known whether they derive from a common precursor or
can all function as APC in the same manner. It should be noted that
a putative novel antigenic marker of human DC recognized by
monoclonal antibody CMRF-44 has been reported (Hock et al., 1994
Immunol. 83(4):P573-581).
[0011] Recent studies have described methods for the isolation and
expansion of human DC's, including, from human peripheral blood.
[Macatonia et al., 1991, Immunol. 74:399-406; O'Doherty et al.,
1993, J. Exp. Med. 178:1067-1078 (isolation); and Markowicz et al.,
1990, J. Clin. Invest. 85:955-961; Romani et al., 1994, J. Exp.
Med. 180:83-93; Sallusto et al., 1994, J. Exp. Med. 179:1109-1118;
Berhard et al., 1995, Cancer Res. 55:1099-1104 (expansion)]. PCT
Publication WO 94/02156 describes a method for isolating human DC's
to present antigens to induce antigen specific T cell-mediated
responses. Adoptive cellular immunotherapy and use of the DC's
against infectious diseases and cancer are mentioned.
[0012] Citation or identification of any reference in Section 2 (or
any other section) of this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
3. SUMMARY OF THE INVENTION
[0013] The present invention provides methods, and compositions,
for use of dendritic cells to activate T cells for
immunotherapeutic responses against primary or metastatic prostate
cancer. The DC's obtained from human donors, after exposure to a
prostate cancer antigen or antigenic fragment, are administered to
a prostate cancer patient to activate the relevant T cell responses
in vivo. Alternatively, the DC's are exposed to a prostate cancer
antigen in vitro and incubated with primed or unprimed T cells to
activate the relevant T cell responses in vitro. The activated T
cells are then administered to a prostate cancer patient. In either
case, the DC's are advantageously used to elicit an
immunotherapeutic growth inhibiting response against a primary or
metastatic prostate tumor.
[0014] In one embodiment, the invention provides a method for
producing a cancer growth inhibiting response, which comprises
administering, to a prostate cancer patient in need thereof, an
effective amount of activated T cells, in which the T cells were
activated in vitro by exposure to human dendritic cells exposed to
a prostate cancer antigen. In another embodiment, the invention
provides a method for producing a cancer growth inhibiting
response, which comprises administering, to a prostate cancer
patient in need thereof, an effective amount of human dendritic
cells, exposed in vitro to a prostate cancer antigen, such that
after administration the human dendritic cells elicit an immune
response or augment an existing immune response against the
prostate cancer.
[0015] Prostate cancer antigens useful for the methods and
compositions of the invention include but are not limited to: a
lysate of LNCaP cells, a membrane preparation of LNCaP cells, a
lysate of prostate tumor cells of a prostate cancer patient, a
membrane preparation of prostate tumor cells of a prostate cancer
patient, purified prostate specific membrane antigen (PSMA a/k/a
PSM), a peptide having the amino acid sequence LLHETDSAV (SEQ. ID.
NO. 1), a peptide having the amino acid sequence ALFDIESKV (SEQ.
ID. NO. 2), a peptide having the amino acid sequence XL(or
M)XXXXXXV(or L) (SEQ. ID. NO. 3) where X represents any amino acid,
purified prostate specific antigen (PSA), and a purified prostate
mucin antigen recognized by monoclonal antibody PD41.
[0016] The present invention, further provides compositions
comprising isolated human dendritic cells exposed to a prostate
cancer antigen(s), as well as cryopreserved isolated human
dendritic cells and extended life span human dendritic cells which
are useful for eliciting immunotherapeutic responses against
primary and/or metastatic prostate cancer.
4. BRIEF DESCRIPTION OF THE FIGURES
[0017] The present invention may be more fully understood by
reference to the following detailed description of the invention,
examples of specific embodiments of the invention and the appended
figures in which:
[0018] FIG. 1 represents histograms of results of flow cytometric
analyses of cultured dendritic cells which illustrate the expressed
cell surface antigens. The cells were cultured in the presence of
GM-CSF and IL-4. The (topmost) upper left histogram represents
background fluorescence staining using the secondary (2.degree.)
antibody, i.e., goat anti-mouse Ig, in the absence of any primary
(1.degree.) antibody. The rest of the histograms represent
fluorescence staining in the presence of 2.degree. antibody and
each of the enumerated 1.degree. antibodies, respectively,
anti-CD1a, anti-CD3, anti-CD4, anti-CD11c, anti-CD14, anti-CD19,
anti-B7/BB1 and anti-HLA-DR antibodies. See text Section 6 for
details.
[0019] FIG. 2 graphically illustrates in vitro activation of T
cells by tetanus toxoid (TT) presented by autologous DC's. See text
Section 6 for details of the assays conducted in triplicate. The
extent of T cell proliferation, represented by .sup.3HTdR
incorporated (cpm), is presented along the y axis. Three different
culture conditions for the T cells are represented along the x
axis: tetanus toxoid alone (+TT); dendritic cells alone (+DC); and
tetanus toxoid with dendritic cells (+DC-TT). Individual standard
deviations are shown.
[0020] FIG. 3 graphically illustrates in vitro activation of T
cells from four prostate cancer patients by presentation of
prostate cancer antigen by autologous dendritic cells. Data from
each individual patient is shown by different bar graph patterns as
indicated. The extent of T cell proliferation, represented by
.sup.3H-Thymidine eHTdR) incorporated (cpm), is presented along the
y axis. Three different culture conditions for the T cells are
represented along the x axis: a prostate cancer antigen alone
(+LNCaP lysate); dendritic cells alone (+DC); and a prostate cancer
antigen with dendritic cells (+DC+LNCaP lysate). See text Section
6.4. for details.
[0021] FIG. 4 is a histogram illustrating fluorescence flow
cytometric analysis of the population of T cells proliferated in
response to autologous DC presentation of LNCaP lysate as antigen.
Background fluorescence represented by experiments with 2.degree.
antibody (goat anti-mouse Ig) alone is shown by the dotted line
histogram. Fluorescence obtained in experiments using the 2.degree.
antibody with the 1.degree. antibody (anti-CD8) antibody are shown
by the solid line histogram. See text Section 6 for details.
[0022] FIG. 5 graphically illustrates cytolytic activity of T cells
stimulated, in vitro, in response to autologous DC presentation of
LNCaP lysate as antigen. Average percent specific lysis of target
cells is represented (y axis) as a function of (x axis) Effector
(activated T cells): Target Cells (autologous DC's presenting LNCaP
or autologous DC's alone ). Experiments were performed in
triplicate and the values presented represent the average; standard
error of the mean for all experiments was <10%. See text Section
6.4 for details
[0023] FIG. 6 illustrates in vitro activation of T cells (PBMC) by
presentation of prostate cancer antigen by previously frozen
autologous dendritic cells. The prostate cancer antigen used was
purified prostate specific membrane antigen (PSMA). The extent of T
cell proliferation, represented by .sup.3H-TdR incorporated is
shown along the y axis. Values are expressed as mean
cpm.+-.standard deviation (S.D.). Each experiment was conducted in
4 replicates. Three different culture conditions for the T cells
are represented along the x axis: T cells plus dendritic cells
(PBMC+DC); T cells plus PSMA alone (PBMC+PSMA); and T cells plus
dendritic cells plus PSMA (PBMC+DC+PSMA). See text Section 7.1 for
details.
[0024] FIG. 7 illustrates in vitro activation of T cells by
presentation of antigens by previously frozen autologous extended
life span dendritic cells. The extent of T cell proliferation,
represented by .sup.3H-TdR incorporated is shown along the x axis.
Values are expressed as mean value.+-.S.D. after subtraction of
average background (PBMC+medium only). Five different cultures of T
cells are represented along the y axis: T cells plus extended life
span dendritic cells plus tetanus toxoid (+EBV-cells+TT); T cells
plus extended life span dendritic cells plus prostate antigen
(+EBV-cells+LNCaP); T cells plus extended life span dendritic cells
plus no antigen (+EBV-cells); T cells plus tetanus toxoid alone
(+TT); and T cells plus prostate antigen alone (+LNCaP). See text
Section 8 for details.
[0025] FIG. 8 illustrates that a peptide having amino acid sequence
LLHETDSAV (comprising a portion of the amino acid sequence of PSMA)
can stimulate proliferation of a mixed population of T cells (PBMC)
obtained from peripheral blood of a prostate cancer patient. The
extent of T cell proliferation, represented by .sup.3HTdR
incorporation (cpm), is presented along the y axis. See text
Section 9 for details.
[0026] FIG. 9 (A-C) is a histogram illustrating fluorescence flow
cytometric analysis of the population of T cells proliferated in
response to the peptide LLHETDSAV as illustrated in FIG. 8. FIG. 9A
illustrates background fluorescence obtained in experiments using
2.degree. antibody only. FIG. 9B illustrates bound fluorescence
obtained using the 2.degree. antibody and anti-CD4 as the 1.degree.
antibody. FIG. 9C illustrates bound fluorescence obtained using
2.degree. antibody and anti-CD8 as the 1.degree. antibody. See text
Section 9 for details.
[0027] FIG. 10 illustrates that a peptide having amino acid
sequence ALFDIESKV can stimulate T cells (PBMC) obtained from
purified blood of prostate cancer patients. The extent of T cell
proliferation, represented by .sup.3HTdR incorporation (cpm), is
greater in HLA-A2(+) cells. See text Section 9 for details.
[0028] FIG. 11 (A-E) present results of a clinical trial
demonstrating that administration of dendritic cells exposed to a
peptide having amino acid sequence LLHETDSAV (PSM-P1) or amino acid
sequence ALFDIESKV (PSM-P2) induced an enhanced immune response in
late stage prostate cancer patients as assessed by ratio of T cell
proliferation pre- and post-administration of the DC's.
[0029] The ratio is designated "stimulation ratio". FIG. 11(A)
illustrates results with patients infused with peptide PSM-P1 alone
(Group I); FIG. 11(B), with patients infused with peptide PSM-P2
alone (Group II); FIG. 11(C), with patients infused with autologous
dendritic cells alone (Group III); FIG. 11(D), with patients
infused with dendritic cells and PSM-P1 (Group IV); and FIG. 11(E),
with patients infused with dendritic cells and PSM-P2 (Group V).
See text Section 10 for details.
5. DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides methods, and compositions,
for use of dendritic cells to activate T cells for
immunotherapeutic responses against primary or metastatic prostate
cancer. The DC's obtained from human donors, once exposed to a
prostate cancer antigen or specific antigenic peptide, are
administered to a prostate cancer patient to activate the relevant
T cell responses in vivo. Alternatively, the DC's are exposed to a
prostate cancer antigen or specific antigenic peptide in vitro and
incubated with primed or unprimed T cells to activate the relevant
T cell responses in vitro. The activated T cells are then
administered to a prostate cancer patient. In either case, the DC's
are advantageously used to elicit an immunotherapeutic growth
inhibiting response against a primary or metastatic prostate
tumor.
[0031] Solely for ease of explanation, the description of the
invention is divided into the following sections: (1) methods for
obtaining or isolating dendritic cells, including DC's with
extended lifespan or cryopreserved DC's; (2) prostate specific
antigens for presentation by DC's; and (3) applications or methods
of use of DC's to stimulate T cells against prostate cancer in
vitro and in vivo.
5.1. Isolation of Dendritic Cells
[0032] Human DC's are obtained from any tissue where they reside
including non-lymphoid tissues such as the epidermis of the skin
(Langerhans cells) and lymphoid tissues such as the spleen, bone
marrow, lymph nodes and thymus as well as the circulatory system
including blood (blood DC's) and lymph (veiled cells). Human
peripheral blood is an easily accessible ready source of human DC's
and is used as a source according to a preferred embodiment of the
invention. Cord blood is another source of human DC's and in cases
where a male is born into a family known to be at high risk for
prostate cancer, cord blood can be used as a source of DC's which
can be cryopreserved for later use, if needed.
[0033] Because DC's occur in low numbers in any tissues in which
they reside, including human peripheral blood, DC's must be
enriched or isolated for use. Any of a number of procedures
entailing repetitive density gradient separation, positive
selection, negative selection or a combination thereof are used to
obtain enriched populations or isolated DC's. Examples of such
methods for isolating DC's from human peripheral blood include:
O'Doherty et al, 1993, J. Exp. Med. 178:1067-1078; Young and
Steinman, 1990, J. Exp. Med. 171:1315-1332; Freudenthal and
Steinman, 1990, PNAS USA 87:7698-7702; Macatonia et al., 1989,
Immunol. 67:285-289; and Markowicz and Engleman, 1990, J. Clin.
Invest. 85:955-961. A method for isolating DC's from human
peripheral blood which avoids exposure of the cells to sheep red
blood cells and/or fetal calf serum is described in PCT Publication
WO94/02156. An example of a method for isolating DC's from lymphoid
tissue is described in Macatonia et al., 1989, J. Exp. Med.
169:1255-1264.
[0034] Once the DC's are obtained, they are cultured in appropriate
culture medium to expand the cell population and/or maintain the
DC's in a state for optimal antigen uptake, processing and
presentation. Particularly advantageous for maintenance of the
proper state of "maturity" of DC's in in vitro culture is the
presence of both granulocyte/macrophage colony stimulating factor
(GM-CSF) and interleukin 4 (IL-4). Preferred is a combination of
GM-CSF: IL-4 in concentration of about 500 units/ml of each. A
recent study reveals optimal antigen presentation by "immature" vs.
mature DC (Koch et al., J. Immunol. 155:93-100). Immature DC's may
be preferred according to certain embodiments of the present
invention.
[0035] As illustrated in the examples, infra, in Section 6, human
DC's were isolated from peripheral blood of prostate cancer
patients, and after about 7 days in culture, about 20-50 fold
higher numbers of DC's competent and able to activate prostate
antigen specific T cells were recovered compared to those directly
isolated from peripheral blood.
[0036] According to a preferred embodiment of the invention, DC's
are obtained from a prostate cancer patient to be treated. The DC's
are used to activate autologous T cells of the patient, either in
vitro or in vivo, for cancer immunotherapy and/or tumor growth
inhibition.
[0037] According to an alternate embodiment, DC's are obtained from
a healthy individual known not to be suffering from prostate
cancer. The relevant HLA antigens (both class I and II, e.g.,
HLA-A, B, C and DR) on the individual's PBMC's are identified and
DC's which match the prostate cancer patient, in terms of HLA
antigens, are isolated and expanded as described above. For
example, in certain instances, a late stage prostate cancer patient
who has been treated with radiation and/or chemotherapy agents
often are not able to provide sufficient or efficient DC's. Thus,
DC's from healthy HLA-matched individuals, such as siblings, can be
obtained and expanded using any of the methods described above and
incubated in vitro with a prostate cancer antigen to elicit
activated T cells for immunotherapy and/or tumor growth inhibition
in the HLA-matched prostate cancer patient.
[0038] According to another embodiment of the invention, "extended
life span dendritic cells" are used. Human cells have a finite life
span in vitro usually limited to approximately 50-70 population
doublings before undergoing apoptosis. As used herein, the term
"extended life span dendritic cells" is intended to mean DC's that
have been genetically modified so that they can be expanded in in
vitro cell culture medium for an extended period of time, including
but not limited to at least about 100 additional population
doublings. Extended life span DC's are obtained, for example, by
EBV-transformation of DC's obtained from peripheral blood of
prostate cancer patients, or by insertion into DC's, using
techniques known to those skilled in the art, of a specific cell
cycle regulatory gene including but not limited to a gene which
encodes cyclin A, B, D or E or retinoblastoma protein.
[0039] As illustrated in the examples, infra, in Section 8,
extended life span DC's have been obtained by EBV transformation of
a population of isolated DC's. A number of clones have been
obtained which express the relevant DC characteristic antigens and
continue to grow and divide well after normal isolated DC's do
not.
[0040] According to yet another embodiment of the invention, DC's
can be preserved, e.g., by cryopreservation either before exposure
or following exposure to a prostate cancer antigen.
[0041] Cryopreservation agents which can be used include but are
not limited to dimethyl sulfoxide (DSO) (Lovelock and Bishop, 1959,
Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205),
glycerol, polyvinylpyrrolidone (Rinfret, 1960, nn. N.Y. Acad. Sci.
85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature
196:548), albumin, dextran, sucrose, ethylene glycol, i-erythritol,
D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157),
D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al.,
1960, J. Appl. Physiol. 15520), amino acids (Phan The Tran and
Bender, 1960, Exp. Cell Res. 20:651), methanol, acetamide, glycerol
monoacetate (Lovelock, 1954, Biochem. J. 56:265), and inorganic
salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med.
104:388; Phan The Tran and Bender, 1961, in Radiobiology,
Proceedings of the Third Australian Conference on Radiobiology,
Ilbery, P. L. T., ed., Butterworth, London, p. 59).
[0042] A controlled slow cooling rate is critical. Different
cryoprotective agents (Rapatz et al., 1968, Cryobiology 5(1):18-25)
and different cell types have different optimal cooling rates (see,
e.g., Rowe and Rinfret, 1962, Blood 20:636; Rowe, 1966, Cryobiology
3(1):12-18; Lewis et al., 1967, Transfusion 7(1):17-32; and Mazur,
1970, Science 168939-949 for effects of cooling velocity on
survival of marrow-stem cells and on their transplantation
potential). The heat of fusion phase where water turns to ice
should be minimal. The cooling procedure can be carried out by use
of, e.g., a programmable freezing device or a methanol bath
procedure.
[0043] Programmable freezing apparatuses allow determination of
optimal cooling rates and facilitate standard reproducible cooling.
Programmable controlled-rate freezers such as Cryomed or Planar
permit tuning of the freezing regimen to the desired cooling rate
curve.
[0044] After thorough freezing, cells can be rapidly transferred to
a long-term cryogenic storage vessel. In a preferred embodiment,
samples can be cryogenically stored in liquid nitrogen
(-196.degree. C.) or its vapor (-165.degree. C.). Such storage is
greatly facilitated by the availability of highly efficient liquid
nitrogen refrigerators, which resemble large Thermos containers
with an extremely low vacuum and internal super insulation, such
that heat leakage and nitrogen losses are kept to an absolute
minimum.
[0045] Considerations and procedures for the manipulation,
cryopreservation, and long term storage of hematopoietic stem
cells, particularly from bone marrow or peripheral blood, is
largely applicable to the DC's of the invention. Such a discussion
can be found, for example, in the following references,
incorporated by reference herein: Gorin, 1986, Clinics in
Haematology 15(1) 19-48; Bone-Marrow Conservation, Culture and
Transplantation, Proceedings of a Panel, Moscow, Jul. 22-26, 1968,
International Atomic Energy Agency, Vienna, pp. 107-186.
[0046] Other methods of cryopreservation of viable cells, or
modifications thereof, are available and envisioned for use (e.g.,
cold metal-mirror techniques; Livesey and Linner, 1987, Nature
327:255; Linner et al., 1986, J. Histochem. Cytochem.
34(9):1123-1135; see also U.S. Pat. No. 4,199,022 by Senken et al.,
U.S. Pat. No. 3,753,357 by Schwartz, U.S. Pat. No. 4,559,298 by
Fahy.
[0047] Frozen cells are preferably thawed quickly (e.g., in a water
bath maintained at 37-41.degree. C.) and chilled immediately upon
thawing.
[0048] It may be desirable to treat the cells in order to prevent
cellular clumping upon thawing. To prevent clumping, various
procedures can be used, including but not limited to the addition
before and/or after freezing of Dnase (Spitzer et al., 1980, Cancer
45:3075-3085), low molecular weight dextran and citrate,
hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20:17-24),
etc.
[0049] The cryoprotective agent, if toxic in humans, should be
removed prior to therapeutic use of the thawed DC's.
[0050] One way in which to remove the cryoprotective agent is by
dilution to an insignificant concentration.
[0051] Once frozen DC's have been thawed and recovered, they are
used to activate T cells as described above with respect to
non-frozen DC's.
[0052] As illustrated in the examples infra, in Section 8, extended
life span DC's have been cryopreserved, thawed and used to present
antigen to activate T cells in vitro.
5.2 Prostate Specific Antigens for Presentation by Dendritic
Cells
[0053] A number of antigens or antigenic compositions are useful,
according to the present invention, for presentation by the DC's to
activate T cells for prostate immunotherapeutics.
[0054] According to one embodiment, a lysate of LNCaP, a prostate
cancer cell line, first described by Horoszewicz et al., 1980,
Prog. Clin. Biol. Res. 37:115-132; 1983, Cancer Res. 43:1809-1818,
is used as antigen presented by the DC's. A crude cell lysate
obtained simply by repetitive freezing and thawing of LNCaP cells
can be used as antigen. An illustrative example of the use of this
antigen is presented in Section 6, infra. Alternatively, a membrane
preparation of LNCaP cells, as described by Horoszewicz et al.,
1987, Anticancer Res. 7:927-936, can be used.
[0055] According to another embodiment, a prostate tumor cell
lysate recovered from surgical specimens can be used as antigen.
For example, a sample of a cancer patient's own tumor, obtained at
biopsy or at surgical resection, can be used to provide a cell
lysate for antigen. Alternatively, a membrane preparation of tumor
cells of a prostate cancer patient can be used as antigen.
[0056] According to another embodiment, purified prostate specific
membrane antigen (PSMA, also known as CYPP antigen and as PSM
antigen), which specifically reacts with monoclonal antibody
7E11-C.5 [(see generally Horoszewicz et al., 1987, supra, U.S. Pat.
No. 5,162,504; Feng et al., 1991, Proc. Am. Assoc. Cancer Res.
32:(Abs. 1418):238)] can be used as antigen. Cloning of the gene
encoding the PSMA antigen has been described by Israeli et al.,
1994, Cancer Res. 54:1807-1811. Expression of the cloned gene,
e.g., in yeast cells, in a baculovirus expression system or in a
mammalian cell culture expression system, will provide a ready
source of the PSMA antigen for use according to the present
invention.
[0057] According to another embodiment, an antigenic peptide having
the amino acid sequence LLHETDSAV (SEQ. ID. NO. 1) (designated
"PSM-P1") which corresponds to amino acid residues 4-12 PSMA can be
used as antigen. According to another embodiment, an antigenic
peptide having the amino acid sequence ALFDIESKV (SEQ. ID. NO. 2)
(designated "PSM-P2") which corresponds to amino acid residues
711-719 of PSMA can be used as antigen.
[0058] According to another embodiment, an antigenic peptide having
an amino acid sequence XL(or M)XXXXXXV(or L) (SEQ. ID. NO. 3)
(designated "PSM-X") where X represents any amino acid residue can
be used as antigen. This peptide resembles the HLA-A0201 binding
motif, i.e., a binding motif of 9-10 amino acids with "anchor
residues", leucine and valine found in HLA-A2+ patients. Grey et
al., 1995, Cancer Surveys 22:37-49. This peptide is preferably used
as antigen for HLA-A2+ patients. HLA-A2+ is expressed by a large
proportion of patients. [See, Central Data Analysis Committee
"Allele Frequencies", Section 6.3, Tsuji, K. et al. (eds.), Tokyo
University Press, pp. 1066-1077].
[0059] According to another embodiment, an antigenic peptide
selected from the peptides listed in Table 1A (below) can be used
as antigen. The peptides listed in Table 1A have amino acid
sequences corresponding to fragments of PSM and have been matched
to a binding motif of a specific haplotype. According to a
preferred embodiment, the peptides are selected to be presented by
DC's to activate T cells of a patient which matched the haplotype
indicated for each peptide in Table 1A.
TABLE-US-00001 TABLE 1A PSM Peptides* Initial Amino Amino SEQ Acid
Acid ID ID No. Haplotype Residue** Sequence NO. PSM-P3 A2 20
WLCAGALVL 4 PSM-P4 A2 27 VLAGGFFLL 5 PSM-P5 A2 109 ELAHYDVLL 6
PSM-P6 A2 260 NLNGAGDPL 7 PSM-P7 A2 461 TLRVDCTPL 8 PSM-P8 A2 660
VLRMMNDQL 9 PSM-P9 A2 568 PMFKYHLTV 10 PSM-P10 A2 57 NMKAFLDEL 11
PSM-P11 A2 469 LMYSLVHNL 12 PSM-P12 A2 663 MMNDQLMFL 13 PSM-P16 A1
171 EGDLVYVNY 14 PSM-P17 A1 264 AGDPLTPGY 15 PSM-P18 A1 463
RVDCTPLMY 16 PSM-P19 A1 143 LFEPPPPGY 17 PSM-P20 A1 558 TYELVEKFY
18 PSM-P21 A1 701 AGESFPGIY 19 PSM-P22 A1 725 WGEVKRQIY 20 PSM-P26
A11 398 IVRSFGTLKKE 21 PSM-P27 A11 63 DELKAENIKKF 22 PSM-P28 A11
491 KSLYESWTKKS 23 PSM-P36 A24 448 AYINADSSI 24 PSM-P37 A24 606
KYADKIYSI 25 PSM-P38 A24 298 GYYDAQKLL 26 PSM-P39 A24 624 TYSVSFDSL
27 PSM-P40 A24 178 NYARTEDFF 28 PSM-P41 A24 227 LYSDPADYF 29
PSM-P46 B3501 289 LPSIPVHPI 30 PSM-P47 B3501 501 SPSPEFSGM 31 *"PSM
peptides" refers to peptides having an amino acid sequence
corresponding to a fragment of PSMA (a/k/a PSM). **"Initial Amino
Acid Residue" refers to the residue number of the amino acid of PSM
to which the first amino acid of the peptide corresponds.
[0060] According to yet another embodiment, prostate specific
antigen (PSA) (see Papsidero et al., 1980, Cancer Res.
40:2428-2432; McCormack et al., 1995, Urology 45(5):729-744) can be
used as antigen.
[0061] According to another embodiment, an antigenic peptide
selected from the peptides listed in Table 1B (below) can be used
as antigen. The peptides listed in Table 1B have amino acid
sequences corresponding to fragments by PSA and [PLEASE CONFIRM]
have been matched to a binding motif of a specific haplotype as
indicated in Table 1B. According to a preferred embodiment, the
peptide are presented by DC's to activate T cells of patients which
match the haplotype indicated for each peptide in Table 1B.
TABLE-US-00002 TABLE 1B PSA Peptides* Initial Amino Amino SEQ Iden.
Acid Acid ID No. Haplotype Residue** Sequence NO. PSA-P1 A2 53
VLVHPQWVL 32 PSA-P2 A2 171 KLQCVDLHV 33 PSA-P11 A1 235 ALPERPSLY 34
PSA-P21 A11 25 IVGGWECEK 35 PSA-P22 A11 185 QVHPQKVTK 36 PSA-P23
A11 245 VVHYRKWIK 37 PSA-P31 A24 152 CYASGWGSI 38 *"PSA peptides"
refers to peptides having an amino acid sequence corresponding to a
fragment of PSA. **"Initial Amino Acid Residue" refers to the
residue number of the amino acid of PSA to which the first amino of
the peptide corresponds.
[0062] According to still another embodiment, a prostate mucin
antigen, recognized by monoclonal antibody PD41, described by
Wright (U.S. Pat. No. 5,227,471 and No. 5,314,996; Beckett et al.,
1991, Cancer Res. 51:1326-1333) can be used as antigen.
Alternatively, a crude lysate of prostate tumor cells which bind to
the antibody produced by the hybridoma cell line (ATCC HB 11094)
and which express the PD41 mucin antigen could be used as
antigen.
[0063] According to the present invention, DC's can be exposed to a
desired prostate cancer antigen or antigenic composition by
incubating the DC's with the antigen in in vitro culture medium. In
one mode, the antigen, in aqueous soluble or aqueous suspension
form, is added to cell culture medium at the same time as the DC's
and T cells to be stimulated are added. See Section 6, infra, for
an illustrative example of this method. As demonstrated in Section
6, the DC's advantageously took up antigen for successful
presentation to T cells. In another mode, antigens are introduced
to the cytosol of the DC's by alternate methods, including but not
limited to osmotic lysis of pinocytic vesicles and the use of pH
sensitive liposomes, etc. See, generally, Okada et al., 1982,
"Introduction of Macromolecules Into Cultured mammalian Cells by
Osmotic Lysis of Pinocytic Vesicles", Cell 29:33; Poste et al.,
1976, "Lipid Vesicles as Carriers for Introducing Biologically
Active Materials Into Cells", Methods Cell Biol. 14:33; Reddy et
al., 1991, "pH Sensitive Liposomes Provide an Efficient Means of
Sensitizing Target Cells to Class I Restricted CTL Recognition of a
Soluble Protein", J. Immunol. Methods 141:157.
5.3 Applications or Methods of Use
5.3.1 Use of Dendritic Cells to Present Prostate Antigen In
Vitro
[0064] As mentioned above, according to one embodiment of the
invention, isolated human DC's, exposed to a prostate specific
antigen by any of the methods described above in Section 5.2, are
used to activate T cells in vitro against prostate cancer. The DC's
can be used immediately after exposure to antigen to stimulate T
cells. Alternatively, the DC's can be maintained in the presence of
a combination of GM-CS and IL-4 prior to simultaneous exposure to
antigen and T cells.
[0065] T cells or a subset of T cells can be obtained from various
lymphoid tissues for use as responder cells. Such tissues include
but are not limited to spleens, lymph nodes, and peripheral blood.
The cells can be co-cultured with DC exposed to antigen as a mixed
T cell population or as a purified T cell subset.
[0066] For example, it may be desired to culture purified CD8.sup.+
T cells with antigen exposed DC's to elicit prostate specific CTL.
In addition, early elimination of CD4.sup.+ T cells may prevent the
overgrowth of CD4.sup.+ cells in a mixed culture of both CD8.sup.+
and CD4.sup.+ T cells. T cell purification may be achieved by
positive, or negative selection, including but not limited to, the
use of antibodies directed to CD2, CD3, CD4, CD5, and CD8.
[0067] On the other hand, it may be desired to use a mixed
population of CD4.sup.+ and CD8.sup.+ T cells to elicit a prostate
specific response encompassing both a cytotoxic and T.sub.H immune
response.
[0068] According to a preferred embodiment, the T cells are
obtained from the same prostate cancer patient from which the DC's
were obtained. After stimulation or activation in vitro, the
autologous T cells are administered to the patient to provoke and
afford an immunoresponse which slows or inhibits prostate tumor
growth.
[0069] For example, T cells are administered, by intravenous
infusion, at doses of about 10.sup.8-10.sup.9 cells/m.sup.2 of body
surface area (see, Ridell et al., 1992, Science 257: 238-241).
Infusion can be repeated at desired intervals, for example,
monthly. Recipients are monitored during and after T cell infusions
for any evidence of adverse effects.
[0070] According to another embodiment, the T cells are obtained
from a prostate cancer patient and the DC's which are used to
stimulate the cells are obtained from an HLA-matched healthy donor.
According to yet another embodiment, both the T cells and the DC's
are obtained from an HLA-matched healthy donor, e.g., a sibling of
the prostate cancer patient. This embodiment may be particularly
advantageous, for example, when the patient is a late stage
prostate cancer patient who has been treated with radiation and/or
chemotherapy agents and may not be able to provide sufficient or
efficient DC's. The T cells after stimulation, are administered as
described above.
5.3.2 Use of Dendritic Cells to Present Prostate Antigens In
Vivo
[0071] According to another embodiment of the invention, DC's
isolated from a prostate cancer patient are cultured, exposed in
vitro to a prostate cancer antigen and after expansion and/or
cryopreservation are administered back to the patient to stimulate
an immune response, including T cell activation, against the
patient's cancer cells in vivo. Using this approach with the
patient's own dendritic cells provides the following advantages:
(1) no foreign DNA is utilized; (2) infection of cells for purposes
of cDNA expression using various viral vectors are eliminated; (3)
antigen is presented to dendritic cells in the form of soluble
protein which will be taken into the dendritic cells and processed
for MHC/peptide presentation of the cell surface; (4) dendritic
cells express B7's on their surface alleviating the necessity to
transfect this cDNA into dendritic cells; (5) the use of endogenous
B7's on dendritic cell surface eliminates the need to provide T
cells with 11-2 or other cytokines either in the form of the
cytokine itself or transfection of the cDNA into specific cells;
(6) all procedures are carried out using the patient's own
cells.
[0072] In practice, DC's obtained as described above in Section
5.1, are exposed in vitro to a prostate cancer antigen, washed and
administered to elicit an immune response or to augment an
existing, albeit weak, response. As such, the DC's constitute an
anti-prostate cancer vaccine and/or immunotherapeutic agent. DC's
presenting a prostate specific antigen are administered, via
intravenous infusion, at a dose of about 10.sup.6-10.sup.8 cells.
The immune response of the patient can be monitored. Infusion can
be repeated at desired intervals based upon the patient's immune
response.
[0073] The following examples are presented for purposes of
illustration only and are not intended to limit the scope of the
invention in any way.
6. Example
Use of Dendritic Cells to Stimulate Prostate Specific T Cells
[0074] The following example demonstrates that human dendritic
cells, obtained from prostate cancer patients, were able to elicit
the proliferation of both helper and cytolytic T cells.
6.1. Materials and Methods
6.1.1. Cell Lines and Reagents
[0075] LNCaP, a prostate cancer cell line, Horoszewicz et al.,
1983, LNCaP Model of Human Prostatic Carcinoma, Cancer Research,
43:1809-1818, (CRL 1740, ATCC, Rockville, Md.), was maintained in
culture in RPMI 1640. Granulocyte/macrophage colony stimulating
factor (GM-CSF), recombinant human interleukin 2 (IL2) and
interleukin 4 (IL4) were generous gifts from Amgen (Thousand Oaks,
Calif.). Monoclonal antibodies Leu-6 (anti-CD1a), Leu-4 (anti-CD3),
Leu-3a (anti-CD4), Leu-2a (anti-CD8), Leu-M3 (anti-CD14),
anti-HLA-DR (MHC Class II), and BB1 (anti-B7/BB1) were purchased
from Becton-Dickinson, San Jose, Calif. Monoclonal antibodies
S125-C1 (anti-CD19) and 3.9 (anti-CD11c) were purchased from Sigma,
St. Louis, Mo.
6.1.2. Prostate Cancer Patients
[0076] Patients with a histologic confirmation of prostatic cancer
were selected for this study which included a signed informed
consent. Fifty cc of heparinized peripheral blood were drawn every
2 weeks during the period of observation which continues. Details
regarding clinical stage, hematologic status, and other relevant
treatments are recorded in Table 2. The American Urological System
of staging was employed, i.e., B.sub.2=tumor confined to the
prostate in both lobes, C.sub.2=large locally invasive tumor,
D.sub.1=positive pelvic lymph node, D.sub.2=metastatic disease.
TABLE-US-00003 TABLE 2 Clinical Profiles of Dendritic Cell Donors
Average PBMC Clinical Bone Marrow Hematocrit Platelets yield#
.times. DC Patient Age Stage Hormonal Status Status (Vol %)
<100,000/mm.sup.3 1000 yield* 1 64 D.sub.1 Hormone Refractory,
Intact 45 0 1000 8-12% Leupron + Flutamide 2 71 D.sub.2 Hormone
Refractory, Impaired 34 + 100 4-10% Post-Orchiectomy Ext.
Radiation, 2 .times. Sr.sup.89 3 68 C.sub.2 Hormone Refractory,
Ext. Radiation 46 0 1000 7-11% Post-Orchiectomy 4 65 D.sub.2
Hormone Refractory, Ext. radiation 33 0 1000 10-14%
Post-Orchiectomy 5 75 B.sub.2 Non-Treated Intact 47 0 1000 10-13% 6
70 D.sub.2 Hormone Refractory, Impaired, 25 + 100 2-8%
Post-Orchiectomy Ext. Radiation, Multiple Sites, 1 .times.
Sr.sup.89 7 80 D.sub.2 Hormone Refractory, Ext. Radiation, 29 +
1000 5-10% Post-Orchiectomy Multiple Sites 8 69 D.sub.1 Hormone
Refractory, Intact 45 0 1000 8-11% Post-Orchiectomy R.sub.x
Flutamide 9 62 D.sub.2 Hormone Refractory, Impaired, 33 0 300 6-9%
Post-Orchiectomy Ext. Radiation, R.sub.x Emcyt Multiple Sites, 1
.times. Sr.sup.89 10 85 B.sub.2 Intact Intact 46 0 900 9-13% #The
number of PBMC isolated from 1 ml of blood *The number of DC
cultured for 7 days/the starting number of PBMC .times. 100%. The
average volume of blood drawn every session = 50 ml
[0077] Most of these patients are in clinical stages D.sub.1 or
D.sub.2, hormone refractory prostatic adenocarcinoma, and have
undergone radiation therapy. Seven patients have undergone
orchiectomy, among whom three have undergone Sr.sup.89 therapy
(patients 2, 6, and 9). Table 2 shows that peripheral blood
mononuclear cells (PBMC) yields from these three patients were
considerably lower (1-3.times.10.sup.5/cc) than those who were not
given Sr.sup.89 therapy (10.sup.6/cc).
6.1.3. Isolation of Dendritic Cells
[0078] Peripheral blood was drawn from prostate cancer patients and
was subjected to Lymphoprep (GIBCO-BRL, Gaithersburg, Md.) density
gradient centrifugation. The peripheral blood mononuclear cells
(PBMC) isolated were plated in 24 well plates (10.sup.6-10.sup.7
cells/well) and were incubated in a humidified incubator
(37.degree. C., 5% Co.sub.2) for 90 minutes.
[0079] Non-adherent cells were removed with the supernatant and the
wells are washed gently with warm (37.degree. C.) OPTIMEM medium
(GIBCO-BRL, Gaithersburg, Md.) and 5% FCS. Dendritic cell
propagation medium (DCPM: OPTIMEM supplemented with 5% FCS, 500
units/ml GM-CSF and 500 units/ml IL-4) was added to the adherent
cells (1 ml/well). These DC cells were cultured for 4-6 days before
subculture 1:3 in DCPM.
[0080] The purity and identity of the isolated DC was confirmed by
incubation with monoclonal antibodies anti-CD1a, -CD3, -CD4, -CD8,
-CD11c, -CD14, -CD19, --HLA-DR and -B7/BB1 for 30 minutes on ice
followed by a fluorescein-isothiocyanate labeled goat-anti-mouse Ig
antibody for 30 minutes on ice. Fluorescence binding was analyzed
using a FACS SCAN flow cytometer (Becton Dickinson, San Jose,
Calif.). In other words, the cultured DC cells were harvested by
pipetting (leaving behind highly adherent, bound macrophages) and
were subjected to flow cytometer analyses for surface expression of
different protein markers for cells of hematopoietic origin to
confirm their DC identity. B7/BB1.sup.+, CD11c.sup.+, and
HLA-DR.sup.+ (MHC class II) but CD14.sup.-, CD3.sup.- and
CD19.sup.- confirms identity of the DC population.
6.1.4. T Cell Proliferation Assays
[0081] One million prostate cancer patients' PBMC were plated in
microtiter plates in T cell media (TCM) consisting of RPMI 1640,
HEPES, 2-mercaptoethanol, L-glutamine and penicillin-streptomycin,
supplemented with 10% human AB serum (Sigma, St. Louis, Mo.) and 1
U/ml recombinant human Il-2. Ten thousand mitomycin-C inactivated
autologous DC and antigen was added to the well prior to
culture.
[0082] The antigens used in these assays were: (1) tetanus toxoid
at 500 ng/ml (TT; Sigma, St. Louis, Mo.); (2) the lysate of LNCaP
cells from an equivalent of 10.sup.5 LNCaP cells/ml; or (3)
purified PSMA. Lysate was prepared as described previously Topalian
et al., 1994, Melanoma-Specific CD4+ T Lymphocytes Recognize Human
Melanoma Antigens Processed and Presented by Epstein-Barr
Virus-Transformed B Cells, Int. J. Cancer 58:69-79. Briefly,
10.sup.7 LNCaP in 1 ml phosphate buffer saline (PBS) was subjected
to cycles of repeated freezing in liquid nitrogen and quick thawing
in a 37.degree. C. water bath. Purified PSMA was prepared as
follows:
Preparation of the Protein-A 7E11-05 Column.
[0083] Protein-A agarose beads were washed with 10.times. volume 3
M NaCl pH=9.0. The pH of the 2 ml of 7E11-05 monoclonal antibody
solution was adjusted to pH 9.0 and the NaCl concentration adjusted
to 3 M. Antibody solution and beads were mixed 1 hr at room
temperature. After the incubation the beads were washed with
10.times. volume 3 M NaCl, 50 mM Sodium Borate. The beads were
resuspended in 10.times. volumes 3 M NaCl, 0.2 M Sodium Borate,
pH=9.0. Dimethylpimeladate was added to a concentration to 20 mM.
The mixture was allowed to mix 30 minutes at room temperature. The
reaction was stopped by washing the beads with 0.2 M methanolamine
pH 8.0. Then 0.2 M ethanolamine pH 8.0 was added at 10.times.
volume and the mixture was allowed to incubate with mixing for 2
hours. The final wash with 2.times. volume PBS 0.01%
merthiolate.
Immunoprecipitation of PSMA From Semen.
[0084] Approximately 75 ml of human semen was collected from paid
donors under the WHO guidelines for fertility testing. The semen
was spun down at 10,000 RPMS for thirty minutes. The supernatant
was then removed. The pellet was washed two times with PBS pH=7.4
then subjected to 1 ml of lysis buffer [1% Triton X-100, 50 mM
HEPES 10% glycerol, 15 mM MgCl.sub.2, 1 mM phenylmethylsulfonyl
fluoride, and 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid
for one hour. The lysate was spun down at 10,000 RPMS for thirty
minutes and the supernatant was collected.
[0085] The 7E11-05 bound Protein-A beads were washed with 15 ml
binding buffer (20 mM HEPES pH=7.5). After washing, the beads were
added to 1 ml of the seminal pellet lysate and 2 ml of binding
buffer. The mixture was allowed to incubate over night.
[0086] The next day the beads were washed with 15 ml binding
buffer, followed by 10 ml of wash buffer (10 mM Sodium Phosphate).
The elution buffer (100 mM Glycine pH=1.8) was added at 2 ml
volumes and the fractions containing PSMA were collected.
[0087] The cell suspension was then added to a T cell proliferation
assays. T cell cultures were incubated in a humidified 37.degree.
C. incubator supplemented with 5% CO.sub.2 for 5 days prior to
addition of 1 .mu.Ci .sup.3H-Thymidine/well. After a 24 h
incubation, cells were harvested in a semi-automatic cell harvester
(Skatron, Stevina, Va.) and radioactivity of the collected cells
was determined. T cell proliferation was assessed by measurement of
average .sup.3HTdR incorporation.
6.2. Isolation and Characterization of Dendritic Cells from
Prostate Cancer Patients
[0088] DC were obtained from PBMC of prostate cancer patients as
described in Section 6.1.3. above and cultured in DCPM as
described. After 4-7 days in culture, clusters of dividing cells
started to form and became less adherent to the tissue culture
flask. These cells increased in size and showed a typical dendritic
morphology (results not shown). In addition to these slightly
adherent cells, tightly adherent macrophages were also present. The
average number of cells with dendritic morphology obtained after a
7 day culture was 2-7.times.10.sup.6 from 50 ml peripheral blood,
representing 4-14% of the starting number of PBMC cultured (see
Table 2).
[0089] In order to confirm the DC identity of the cultured cells,
the cells were harvested by pipetting (leaving tightly bound
macrophages behind) and were subjected to flow cytometric analyses
for surface expression of different protein markers for cells of
hematopoietic origin. Results are illustrated in FIG. 1.
[0090] As illustrated in FIG. 1, the cultured cells do not express
lineage specific markers for T cells (CD3), B cells (CD19), or
macrophages (CD14). CD1a, a marker for Langerhans cells (e.g.,
dendritic cells isolated from the skin), was expressed at a high
level early in the culture, but the level decreased when the cells
were maintained in culture for more than 14 days. CD11c
(beta-2-integrin) and HLA-DR were expressed at high levels while
B7/BB1 was expressed at moderate levels by these cells, confirming
further the identify of the cultured cells as DC's.
[0091] These cultures ceased to expand after 2 passages, although
the DC's maintained their characteristic morphology and surface
antigen expression for up to one month when fed weekly with fresh
dendritic cell propagation medium (DCPM).
6.3. Stimulation of Antigen Specific T Cells by Dendritic Cells
[0092] In order to assess the capacity of the cultured DC's to
present antigen to and stimulate autologous T cells from the same
patients, T cell proliferation assays were conducted as described
above in Section 6.1.4. in triplicate. Tetanus toxoid (TT) was
chosen as a representative antigen in these experiments to
determine whether patients' memory T cells could be activated in
vitro. Results are presented in FIG. 2.
[0093] FIG. 2 shows that autologous T cells cultured with the
patients' DC's and TT proliferated at levels significantly higher
than background levels (in the absence DC) and at levels
significantly higher than T cells cultured with DC without TT,
i.e., showing an autologous mixed lymphocyte reaction.
[0094] Thus, the results demonstrate that the presentation of TT by
DC's is useful for T cell proliferation. More particularly, the
results demonstrate that DC's obtained from prostate cancer
patients are useful to activate antigen specific T cell
proliferation.
6.4. Stimulation of Prostate Specific T Cells by Dendritic
Cells
[0095] The ability of DC's to present antigen specific for prostate
cancer and stimulate autologous T cells of a prostate cancer
patient was determined. In one study, a crude cellular lysate of
LNCaP cells, a metastatic prostate cancer cell line, was used as a
representative prostate cancer antigen in a T cell proliferation
assay generally as described above in Section 6.1.4. Results are
illustrated in FIG. 3.
[0096] FIG. 3 shows that significant increases in .sup.3HTdR
incorporation were observed in 2 of 4 cases when both DC's and
LNCaP lysates were included in the T cell cultures. Thus, in 2/4
cases presentation of prostate specific antigen stimulated
autologous T cell proliferation in vitro. It is our belief that the
2 negative cases with LNCaP lysate as antigen reflect the
limitations of using a crude cellular lysate with variable
concentration of prostate antigen. Additional experiments using
purified prostate-specific membrane antigen (PSMA) support this
view (results not shown).
[0097] In another experiment, T cells proliferated as a result of
DC presentation of LNCaP lysate were expanded in culture, for 2
weeks, and subjected to fluorescence flow cytometric analysis to
determine the representation of the two T cell subtypes, i.e.,
cytolytic T lymphocytes (CTL) and helper T cells (T.sub.H)
elicited. In particular, T cells proliferated as a result of DC
presentation of LNCaP lysate were expanded, for two weeks, by
culture in T cell propagation medium in the presence of mitomycin
C-inactivated autologous DC and LNCaP lysate (equivalent to
10.sup.4 LNCaP cell/ml). A 14 day old culture of T cells was
harvested and analyzed for the expression of cytolytic T cell
specific antigen; CD8, by fluorescence flow cytometry as described
above. Results are illustrated in FIG. 4.
[0098] As demonstrated in FIG. 4, the CTL's (CD8+) represented
about 40-50% of the T cells elicited against LNCaP by the DC
presentation of LNCaP lysate. Thus, presentation of prostate
antigen by autologous DC's is useful to elicit both cytolic (CTL's)
and helper (T.sub.H) T cells specific for prostate cancer.
[0099] In yet another experiment, T cells (both CD4.sup.+ and
CD8.sup.+) activated as described above by DC's presenting LNCaP
lysate as antigen demonstrated cytolytic activity using, as target
cells, autologous DC's presenting LNCaP antigen.
[0100] Briefly, DC's were isolated from a prostate cancer patient,
as described in Section 6.2 and were exposed to LNCaP lysate as
described above. Autologous T cells were obtained as PBMC's and
cultured in the presence of the LNCaP presenting DC's. T cells
proliferated as a result of prostate antigen presentation by the
DC's were expanded for a total of 21 days as described above.
[0101] The ability of the activated T cells to specifically lysis
target cells was assessed using the CytoTox 96.TM. assay (Promega,
Madison; WI). Briefly, effector cells (the 21 day T cells including
both CD4+ and CD8+ cells) were incubated with 2.times.10.sup.4
target cells in T cell propagation medium at effector: target cell
ratios of 20:1, 10:1 and 3:1 for 5 hours at 37.degree. C. in a
CO.sub.2 incubator. Target cells included: autologous DC's alone or
autologous DC's exposed to LNCaP lysate [equivalent of 10.sup.5
cells/ml (DC+LNCaP Lysate)] overnight. Released lactate
dehydrogenase, a stable cytosolic enzyme that is released upon cell
lysis was measured from culture supernatants with a coupled
enzymatic assay which was monitored by reading absorbance at 490
mm. All experiments were performed in triplicate. Results are
illustrated in FIG. 5.
[0102] As shown in FIG. 5, T cells proliferated as a result of
prostate antigen presentation by DC's according to the present
invention showed enhanced specific lytic activity against cells
presenting the same prostate antigen. Thus, presentation of
prostate antigen by DC's is useful to elicit T cells having
specific cytolytic activity against cells expressing prostate
antigen.
7. Example
Use of Cryopreserved Dendritic Cells to Stimulate Prostate Specific
T cells
7.1. Cryopreserved Dendritic Cells From a Prostate Cancer
Patient
[0103] Dendritic cells were isolated from PBMC of a prostate cancer
patient as described above in Section 6 and cultured, as described
above, in Section 6, for 7 days in the presence of 500 units/ml
GM-CSF and IL-4.
[0104] On day 7, the isolated DC's were harvested and cryopreserved
using 90% fetal calf serum and 10% dimethylsulfoxide. The
cryopreserved DC's were stored frozen for a period of time, thawed
in a 37.degree. C. water bath and transferred to a 15 ml
polypropylene tube and centrifuged at 1200 rpm for 5 min. The
thawed DC's were then resuspended in medium containing 10%
heat-inactivated human serum and counted.
[0105] One hundred thousand previously frozen T cells obtained from
peripheral blood of the same prostate cancer patient (PBMC) were
cultured in the presence or absence of 10.sup.4 DC's together with
5 .mu.l/well purified PSMA (see Section 6.1.4 above) in a total
volume of 200 .mu.l medium containing 10% heat-inactivated human
serum and 1 unit/ml IL-2. One .mu.Ci/well .sup.3H-Thymidine was
added on day 6 and cultures were harvested 18 hours later.
.sup.3H-TdR incorporation was counted in a liquid scintillation
counter. Each experiment was done in 4 replicates. Results are
presented in FIG. 6. The average cpm and standard deviation are
shown in the graph.
[0106] FIG. 6 demonstrates that a highly significant increase in
.sup.3HTdR incorporation was observed when both previously
cryopreserved DC's and prostate specific antigen, i.e., PSMA, were
included in the T cell cultures. Thus, presentation of prostate
specific antigen by previously cryopreserved DC's stimulated
autologous T cell proliferation in vitro. The effect was
significantly greater than that observed with antigen alone or with
DC's alone but no exogenous prostate antigen.
7.2. Cryopreserved Dendritic Cells of Normal Patients
[0107] PBMC are obtained from a normal or healthy individual known
not to be suffering from prostate cancer. The PBMC are cross-typed
and the relevant HLA antigens expressed noted. Relevant HLA
antigens include such as HLA-A, B, C and DR. DC are isolated from
the PBMC's as described in Section 6. The DC's are then
cryopreserved as described in Section 7.1 above.
[0108] The cryopreserved DC are used according to the present
invention to stimulate T cells in vitro, from a similarly matched
HLA-typed individual or from a patient suffering from prostate
cancer for use in the cancer patient.
7.3. Alternate Protocol for Cryopreservation And Use of Dendritic
Cells
[0109] Dendritic cells can be isolated from a prostate cancer
patient or from a healthy donor, cryopreserved and used according
to the methods of the invention as follows.
[0110] If desired, viable DC count, e.g., by trypan blue exclusion
(Kucker, 1977, Biochemical Methods in Cell Culture and Virology,
Dwoder, Hutchinsen & Ross, Stroudsburg, Pa., pp. 18-19) and
manual cell counting can be performed.
[0111] The DC from the prostate cancer patient are cryopreserved
using the following protocol: [0112] 1. Gently resuspend cells to a
concentration of 4.times.10.sup.6 viable cells/ml, using a mixture
of cold (4.degree. C.) 50% autologous plasma/RPMI-1640 or 50%
heat-inactivated FCS/RPMI, and place the suspension on ice. [0113]
2. In a cryovial containing 1 ml of a chilled sterile
cryoprotective medium of 20% DMSORPMI-1640, carefully layer a 1 ml
portion of the above cell suspension on top of the cryoprotective
medium. [0114] 3. Approximately 10 minutes prior to freezing,
slowly invert the 1:1 mixture to promote mixing, then place it on
ice to allow equilibrium between the cells and the cryoprotective
medium. NOTE: The "layered" tube should not remain unfrozen for
very long, so freezing should preferably be done within 20-30
minutes after exposure of cells to DMSO/RPMI solution. [0115] 4.
Place the vials in a freezing rack, which in turn is placed in a
4.degree. C. methanol bath, just deep enough to cover the cell
suspension. This is then placed in the bottom (to ensure proper
temperature) of a -80.degree. C. freezer for at least 2 hours and
less than 24 hours. [0116] 5. After cells reach the frozen state,
carefully and quickly transfer them to a long term liquid nitrogen
containment vessel. A cryogenic storage vessel which can be used is
the LR 1000 refrigerator (Union Carbide Corp., Indianapolis, Ind.)
which accommodates up to 40,000 cryules.
[0117] Following any desired length of time post-cryopreservation,
the protocol below can be used to thaw the DC cells for use to
stimulate autologous T cells specific to prostate cancer according
to the present invention. [0118] 1. Remove vial of frozen cells
from liquid nitrogen. Immediately the cell suspension by gently
agitating the vial in a 37.degree. C. water bath until just a small
amount of ice remains. [0119] 2. Aseptically, begin to add
drop-wise, a chilled mixture of 50% autologous serum/RPMI-1640
medium or 50% FCS/RPMI-1640 medium with a slight mixing between
each drop, until the suspension volume is doubled. [0120] 3.
Transfer this suspension to a larger centrifuge tube (12-15 ml) and
continue to add, drop-wise, 505 serum/RPMI mixture with mixing
between every other drop until the volume reaches 6-7 ml. Diluent
may now be added, drop-wise, with mixing at every 0.5 ml increment
until the volume reaches 9-10 ml. (NOTE: The reason for stepwise
addition of diluent is to prevent osmotic shock to the cells as
DMSO is diluted in the cell suspension). [0121] 4. Pellet cells by
centrifugation at 4.degree. C. 200.times.g, for 10 minutes.
Aspirate the supernatant. [0122] 5. Slowly add drop-wise 1 ml of
chilled 20% autologous serum/RPMI-1640 mixture to the pellet.
"Resuspend" the pellet by gently "flicking" the tube with a finger.
After the pellet is resuspended (clumps may remain), resuspend it
further by gently aspirating up and down with a 1 ml pipet. [0123]
6. Add an additional 4 ml chilled 20% autologous serum/RPMI,
dropwise, with mixing between every drop; then add 0.5 ml as volume
increases, as previously described. [0124] 7. Pellet cells by
centrifugation at 4.degree. C. 200.times.g, for 10 minutes.
Aspirate the supernatant. [0125] 8. Resuspend with 2-5 ml of
chilled 20% serum/RPMI mixture. [0126] 9. Perform cell counts
(e.g., by use of a hemocytometer) and viability testing (e.g., by
trypan blue exclusion).
[0127] Loss of cells due to clumping during the stepwise removal of
DMSO can be diminished by including Dnase (20 U per
2.times.10.sup.6 cells) or low molecular weight dextran and citrate
(to reduce the Ph to 6.5).
8. Example
Use of Extended Life Span Dendritic Cells to Stimulate Prostate
Specific T Cells
[0128] Extended life span dendritic cells were prepared according
to the present invention as follows:
[0129] DC's were isolated from human peripheral blood and cultured
for 4-6 days as described above in Section 6.1.3. After 4-6 days in
culture, in the presence of GM-CSF and IL-2, the DC's were infected
with Epstein Barr Virus (EBV) generally as described by Walls &
Crawford, in Lymphocytes: A Practical Approach, GCB Klaus, ed., IRL
Press, Oxford, England, pp. 149-162. Briefly, DC's were harvested
(.about.3-5.times.10.sup.6 cells) and resuspended in a 5 ml B95-8
(an EBV-producing marmoset cell line, American Type Culture
Collection, Rockville, Md.) culture supernatant. The cell
suspension was transferred to a 25 cm.sup.2 flask (Nunc, Inc.,
Naperville, Ill.) and incubated in a 37.degree. C. CO.sub.2
incubator for 24 hours. Five ml of fresh DCPM were added to the
culture. Clusters of growing cells were observed 2-3 weeks
post-infection. Extended life span DC cells were subcultured
weekly, at 3-5 weeks post-infection.
[0130] The ability of the extended life span DC's to present
antigen to stimulate autologous T cells of a prostate cancer
patient was determined. In this study, either tetanus toxoid (TT)
at 500 ng/ml or a lysate of LNCaP cells at an equivalent of
10.sup.4 cells/ml was used as representative antigen.
[0131] The autologous T cells used in the proliferation assay were
obtained from peripheral blood and had been previously
cryopreserved. One hundred thousand thawed T cells (designated
"PBMC") were cultured in complete medium containing 10%
heat-inactivated human serum and 1 unit/ml IL-2 as follows: in the
presence of LNCaP antigen alone (+LNCaP); in the presence of TT
antigen alone (+TT); in the presence of EBV-transformed DC's (+EBV
cells); in the presence of EBV-transformed dendritic cells plus
LNCaP antigen (+EBV-cells+LNCaP); or in the presence of
EBV-transformed dendritic cells plus TT (+EBV-cells+TT). T cells in
the presence of medium alone (PBMC+medium only) served as a
background sample.
[0132] One .mu.Ci/well .sup.3H-Thymidine (.sup.3H-TdR) was added to
all wells on day 6 and cultures were harvested 18 hours later.
.sup.3H-TdR incorporation was assessed using a liquid scintillation
counter. Each experiment was conducted in triplicate. .sup.3H-TdR
incorporation, average cpm for each culture, was determined after
subtracting average background cpm. Results are presented in FIG.
7.
[0133] FIG. 7 demonstrates that extended life span human dendritic
cells, exposed either to TT antigen or PSMA antigen, significantly
increased .sup.3HTdR incorporation in previously cryopreserved T
cell cultures. Thus, it is clear that extended life span DC's are
useful to present prostate specific antigen to stimulate autologous
T cell proliferation in vitro.
9. Example
Use of a PSMA Peptide to Stimulate T Cells
[0134] The ability of a peptide having the amino acid sequence,
LLHETDSAV (SEQ. ID. NO. 1) (designated PSM-P1), which corresponds
to amino acid residues 4-12 of the PSMA antigen as deduced from
cDNA (of the PSMA gene) to stimulate T cells was assessed in order
to determine whether presentation of such antigenic peptide would
be useful using the methods of the present invention. The following
data show that this antigenic peptide is suitable for use in the
methods of the invention.
[0135] A peptide having amino acid sequence LLHETDSAV (designated
PSM-P1 peptide) was synthesized and purified by Genemed
Biotechnologies (San Francisco, Calif.).
[0136] One hundred thousand previously frozen T cells (designated
"PBMC") obtained from a prostate cancer patient were cultured in
the presence or absence of 20
[0137] PSMA peptide in complete medium containing 10%
heat-inactivated human serum and 1 unit/ml IL2. One .mu.Ci/well
.sup.3H-Thymidine was added on day 6 and cultures were harvested 18
hours later. .sup.3H-TdR incorporation was counted in a liquid
scintillation counter. Each experiment was done in 4 replicates.
The average cpm and standard deviation are shown in FIG. 8.
[0138] As illustrated in FIG. 8, the antigenic peptide
corresponding to amino acid residues 4-12 of PSMA peptide elicited
a greatly enhanced T cell proliferation compared to T cells in the
absence of this peptide.
[0139] The T cells proliferated as a result of the PSMA peptide
were expanded in culture for 5 days, in complete medium containing
2 .mu.M PSMA peptide and 1 unit/ml Il2 for 5 days prior to harvest.
One million cells were incubated for 30 minutes on ice with
anti-CD4, anti-CD8 antibodies or medium alone, followed by another
30 minute incubation with fluorescein isothiocyanate
labelled-2.degree. antibody. Fluorescence-bound was analyzed in a
FACScan (Becton Dickinson, San Jose, Calif.) flow cytometer as
described above. The percent CD4+ and CD8+ cells were calculated.
Results are illustrated in FIG. 9.
[0140] As shown in FIG. 9, the CTL's represented about 41% of the T
cells stimulated by the PSMA peptide antigen. Thus, this peptide
antigen should be useful to be presented by DC's to T cells to
elicit prostate specific responses, both cytolytic (CTL's) and
helper (T.sub.H) cells against prostate cancer according to the
methods of the invention.
[0141] In another series of experiments, the ability of a peptide,
having the amino acid sequence, ALFDIESKV (SEQ. ID. NO. 2)
(designated PSM-P2), to stimulate T cells was assessed. The
following data show that this antigenic peptide is suitable for use
in the methods of the invention.
[0142] Peptide PSM-P2 was synthesized and purified.
[0143] One hundred thousand T cells, i.e. PBMC obtained from
HLA-A2(+) or HLA-A2(-) prostate cancer patients were cultured in
the presence or absence (i.e. "medium control") of 20 .mu.g PSM-P2
peptide for five (5) days in a humidified, 37.degree. C., 5%
CO.sub.2 incubator. On day 6, 1 .mu.Ci .sup.3H-Thymidine was added
to each well. After another 24 h incubation, cells were harvested
and the .sup.3H-TdR incorporated was counted in a liquid
scintillation counter. Each experiment was done in triplicate.
Results are presented in FIG. 10 as average cpm and standard error
of the mean.
[0144] As demonstrated in FIG. 10, the peptide corresponding to
amino acid residues 711-719 of PSMA elicited a greatly enhanced
cell proliferation of HLA-A2(T cells compared to HLA-A2(+) T cells
in the absence of this peptide. A less dramatic enhanced T cell
proliferation was observed when HLA-A2 (-) T cells were exposed to
this peptide.
10. Example
Use of Dendritic Cells to Produce an Immune Response in Human
Prostate Cancer Patients
[0145] A phase 1 clinical trial has been established using five
treatment groups of late stage prostate cancer patients. The groups
were treated as follows: Group I, received peptide PSM-P1 having
amino acid sequence LLHETDSAV (SEQ. ID. NO 1) only at 0.2, 2.0 and
20.0 .mu.g; Group II, peptide PSM-P2 having amino acid sequence
ALFDIESKV (SEQ. ID. NO. 2) only at 0.2, 2.0 and 20.0 .mu.g; Group
III, 1.times.10.sup.6 (#3-4) or 5.times.10.sup.6 (#1, 2) autologous
dendritic cells only; Group IV, up to 2.times.10.sup.7 autologous
dendritic cells loaded with PSM-P1 (SEQ. ID. NO. 1); and Group V,
up to 2.times.10.sup.7 autologous DC's loaded with PSM-P2 (SEQ. ID.
NO. 2). All treatments were delivered by infusion. Patients were
monitored 7 days post-infusion and their immune function was
analyzed by assessing T cell proliferation pre- and post-infusion
using .sup.3H-TdR incorporation as described above in Section 9.
Results, after the first infusion of Groups I-V are presented in
FIG. 11 (A-E).
[0146] As shown in FIG. 11A, patients receiving peptide PSM-P1
alone did not demonstrate any increase in T cell proliferation,
regardless of their HLA-type. As shown in FIG. 11B, only one
patient (out of 8 patients) receiving peptide PSM-P2 alone
demonstrated an increased T cell proliferation. The sole positive
patient was an HLA-A2(+) patient.
[0147] As demonstrated in FIG. 11C, patients receiving dendritic
cells only did not demonstrate any increase in T cell
proliferation. As indicated in FIG. 11C, three of the four patients
were HLA-2(A)(+) and one was HLA-2(A)(-).
[0148] In complete contrast to the results presented in FIG. 11
(A-C), results in FIGS. 11(D-E) demonstrate that administration of
DC's exposed to and presenting either peptide PSM-P1 or PSM-P2
induced an enhanced T cell proliferation. More particularly,
patients receiving dendritic cells containing PSM-P1 peptide showed
an increased T cell proliferation in 3 of 5 HLA-A(2)(+) as
illustrated in FIG. 11(D). In addition, the sole HLA-A(2)(-)
patient did not demonstrate an increased T cell proliferation.
These results demonstrate firstly, that administration of dendritic
cells in Combination with the PSM-P1 peptide effectively produced
an immune response in late stage prostate cancer patients.
Secondly, it appears that the immune response is an HLA-A(2)
restricted effect when this peptide was used. The two patients
receiving dendritic cells containing PSM-P2 peptide also showed an
increased T cell proliferation as illustrated in FIG. 11(E). Both
of these patients were HLA-A2(+).
[0149] Based on the results obtained, we have concluded that
dendritic cells exposed either to PSM-P1 or to PSM-P2 peptide are
useful to produce an immune response in prostate cancer patients
and that the methods and compositions of this invention are
advantageously useful for cancer immunotherapy.
[0150] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
[0151] All publications cited herein are incorporated by reference
in their entirety.
Sequence CWU 1
1
* * * * *