U.S. patent application number 09/981239 was filed with the patent office on 2003-06-26 for antigen presenting cells, method for their preparation and their use for cancer vaccines.
Invention is credited to De Santis, Rita.
Application Number | 20030119187 09/981239 |
Document ID | / |
Family ID | 11133712 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119187 |
Kind Code |
A1 |
De Santis, Rita |
June 26, 2003 |
Antigen presenting cells, method for their preparation and their
use for cancer vaccines
Abstract
The present invention discloses a method of generation of
antigen presenting cells, comprising: a. collecting said cells from
a subject; b. activating said collected cells; c. culturing and
optionally expanding ex vivo said activated cells; d. treating said
cultured and optionally expanded cells with DNA hypomethylating
agents so that said cells concomitantly express multiple tumor
associated antigens. The cells obtainable according to the method
of the present invention, as well as the cellular components
thereof whether alone or in combination with said cells, are useful
for prevention and treatment of malignancies of different histotype
that constitutively express one or more of the multiple tumor
associated antigens that are expressed in said cells. Conveniently,
said cells and/or cellular components are in the form of a vaccine.
Said vaccines are advantageous over the prior art in that as they
concomitantly express multiple/all methylation-regulated tumor
associated antigens.
Inventors: |
De Santis, Rita; (Pomezia,
IT) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Rd.
Arlington
VA
22201-4714
US
|
Family ID: |
11133712 |
Appl. No.: |
09/981239 |
Filed: |
October 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09981239 |
Oct 18, 2001 |
|
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PCT/IT01/00419 |
Jul 30, 2001 |
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Current U.S.
Class: |
435/455 ;
424/93.21; 435/372 |
Current CPC
Class: |
A61K 39/001184 20180801;
A61K 2039/5152 20130101; A61K 2039/5154 20130101; C12N 2502/99
20130101; A61P 35/00 20180101; C12N 5/0694 20130101; C12N 2501/06
20130101; C12N 2510/04 20130101; C12N 2506/11 20130101; C12N
2501/52 20130101; C12N 2501/59 20130101; C12N 2501/23 20130101 |
Class at
Publication: |
435/455 ;
435/372; 424/93.21 |
International
Class: |
A61K 048/00; C12N
005/08; C12N 015/85 |
Claims
1. A method for the generation of antigen presenting cells
comprising: a) collecting said cells from a subject, b) activating
said collected cells; c) culturing and optionally expanding ex vivo
said activated cells; d) treating said cultured and optionally
expanded cells with DNA hypomethylating agents so that said cells
concomitantly express multiple tumor associated antigens.
2. A method according to claim 1, wherein said subject is a
mammal.
3. A method according to claim 2, wherein said subject is a
human.
4. A method according to claim 2, wherein said subject is a cancer
patient.
5. A method according to any of claims 1-4, wherein said cells are
immune cells.
6. A method according to any of claims 1-4, wherein said cells are
non-immune cells.
7. A method according to any of claims 1-6, wherein said cells
express shared immunodominant cancer antigens.
8. A method according to any of claims 1-6, wherein said cells
express shared not immunodominant cancer antigens.
9. A method according to any of claims 1-5 and any of claims 7-8,
wherein said cells are Epstein-Barr virus-immortalized
B-lymphoblastoid cell lines.
10. A method according to any of claims 1-5 and any of claims 7-8,
wherein said cells are Pokeweed mitogen (PWM)-activated
B-lymphocytes.
11. A method according to any of claims 1-5 and any of claims 7-8,
wherein said cells are CD40 activated B-lymphocytes.
12. A method according to any of claims 1-5 and any of claims 7-8,
wherein said cells are Phytohemagglutinin (PHA)+recombinant human
interleukin-2 (rhIL-2)-activated PBMC.
13. A method according to any of claims 1-5 and any of claims 7-8,
wherein said cells are Phytohemagglutinin (PHA)+recombinant human
interleukin-2 (rhIL-2)+pokeweed mitogen (PWM)-activated PBMC.
14. A method according to any of claims 1-4 and any of claims 6-8,
wherein said cells are dendritic cells, monocytes, macrophages.
15. A method according to any of claims 1-4 and any of claims 6-8,
wherein said cells are CD34+ cells, fibroblasts, stem cells,
fibroblasts and cheratinocytes.
16. A method according to any of claims 1-15, wherein histone
deacetylase inhibitors are used in step d).
17. A method according to any of claims 1-16, wherein said DNA
hypomethylating agent is selected from 5-aza-cytidine or
5-aza-2'-deoxycytidine.
18. Cells obtainable by the method according to any one of claims
1-17.
19. Use of cells of claim 18, and/or their cellular components for
prevention and treatment of malignancies of different histotype
that constitutively express one or more of cancer antigens.
20. Use according to claim 19, wherein said shared cancer antigens
are immunodominant cancer antigens.
21. Use according to claim 18, wherein said shared cancer antigens
are not immunodominant.
22. Use according to claim 18, wherein said cancer antigens are
Cancer Testis Antigens.
23. Use according to any of claims 19-22, wherein said cells are
stored as reservoir of pooled antigens.
24. Pooled antigens as referred in claim 23 for use as cancer
vaccine.
25. Cancer vaccine comprising cells of claim 18.
26. Vaccine according to claim 25, said vaccine being
autologous.
27. Vaccine according to claim 25, said vaccine being
allogeneic.
28. Vaccine according to claim 27, wherein the cells are used as
according to claim 23.
29. Vaccine according to claim 27 or 28, wherein cellular
components according to claim 19 are used.
30. Use of cells of claim 18 and/or their cellular components in a
method for generating effector immune cells, said effector immune
cells being used for the preparation of a product useful in
adoptive immunotherapy.
31. An article of manufacture comprising a vaccine according to any
of claims 25-29 and a pharmaceutical composition suitable for
systemic administration of a hypomethylating agent.
Description
[0001] The present invention relates to the medical field, in
particular to products, substances and compositions for use in
methods for the treatment of human or animal subjects, more in
particular for the diagnosis, treatment, and prevention of cancer.
The present invention relates to cancer vaccines and methods for
their preparation.
BACKGROUND OF THE INVENTION
[0002] Several tumor-associated antigens (TAA) constitutively
expressed by transformed cells of different histotype have been
recently identified (Renkvist N. et al. Cancer Immunol. Immunother.
50:3-15, 2001).
[0003] A number of these TAA can provide multiple immunodominant
antigenic peptides recognized by CD8+cytotoxic T lymphocytes (CTL)
in the context of specific HLA class I allospecificities (Renkvist
N. et al. Cancer Immunol. Immunother. 50:3-15, 2001); furthermore
selected TAA, such as for example MAGE (Jager E. et al., J. Exp.
Med., 187: 265-270, 1998), NY-ESO-1 (Jager E. et al., J. Exp. Med.,
187: 265-270, 1998), SSX (Tureci O, et al. Cancer Res;
56(20):4766-72 1996), tyrosinase (Topalian S. L. et al., J. Exp.
Med., 183: 1965-1971, 1996.), Melan-A/MART-1 (Zarour H. M. et al.,
Proc. Natl. Acad. Sci. USA, 97: 400-405, 2000) concomitantly
include epitopes recognized by CD4+T lymphocytes in the context of
specific HLA class II allospecificities, thus being able to induce
a TAA-directed humoral immune response (Wang R. F., Trends
Immunol., 22: 269-276, 2001).
[0004] Different classes of TAA that may play a major role as
therapeutic targets have been identified:
[0005] i) cancer-testis antigens (CTA), expressed in tumors of
various histology but not in normal tissues, other than testis and
placenta such as for example MAGE, GAGE, SSX SART-1, BAGE,
NY-ESO-1, XAGE-1, TRAG-3 and SAGE, some of which represent multiple
families (Traversari C., Minerva Biotech., 11: 243-253, 1999);
[0006] ii) differentiation-specific antigens, expressed in normal
and neoplastic melanocytes, such as for example tyrosinase,
Melan-A/MART-1, gp100/Pmel17, TRP-1/gp75, TRP-2 (Traversan C.,
Minerva Biotech., 11: 243-253, 1999);
[0007] iii) antigens over-expressed in malignant tissues of
different histology but also present in their benign counterpart,
for example PRAME (Ikeda H. et al., Immunity, 6: 199-208, 1997),
HER-2/neu (Traversari C., Minerva Biotech., 11: 243-253, 1999),
CEA, MUC-1(Monges G. M. et al., Am. J. Clin. Pathol., 112: 635-640,
1999), alpha-fetoprotein (Meng W. S. et al., Mol. Immunol., 37:
943-950, 2001);
[0008] iv) antigens derived from point mutations of genes encoding
ubiquitously expressed proteins, such as MUM-1, .beta.-catenin,
HLA-A2, CDK4, and caspase 8 (Traversari C., Minerva Biotech., 11:
243-253, 1999);
[0009] v) viral antigens (Traversari C., Minerva Biotech., 11:
243-253, 1999).
[0010] In addition to TAA, the cellular elements that are crucial
for their effective immunogenicity and efficient recognition by
host's T lymphocytes include HLA class I and HLA class II antigens,
and co-stimulatory/accessory molecules (e.g., CD40, CD54, CD58,
CD80, CD81) (Fleuren G. J. et al., Immunol. Rev., 145: 91-122,
1995).
[0011] Among known classes of TAA, CTA are particularly suitable
therapeutic targets for active specific immunotherapy of cancer
patients, because of their limited expression in normal tissues and
their known in vivo immunogenicity in living subjects, in
particular mammals, humans included (Jager E. et al., J. Exp. Med.,
187: 265-270, 1998; Rejnolds S. R. et al., Int. J Cancer, 72:
972-976, 1997). However, the heterogeneous expression of specific
CTA among neoplastic lesions of different patients limits their
biological eligibility to CTA-directed therapeutic vaccination. In
fact, malignant lesions of distinct cancer patients can frequently
express only selected CTA (Sahin U. et al., Clin. Cancer Res., 6:
3916-3922, 2000), additionally down-regulated (Leth{haeck over (z)}
B. et al., Melanoma Res., 7: S83-S88, 1997) and/or heterogeneous
(dos Santos N. R. et al., Cancer Res., 60: 1654-1662, 2000)
expression of specific CTA within individual neoplastic lesions has
also been reported (Jungbluth A. A. et al., Br. J. Cancer, 83:
493-497, 2000). These events, that can occur in vivo separately or
concomitantly, may also contribute to the constitutively poor
immunogenicity of malignant cells favouring disease progression
(Speiser D. E. et al., J. Exp. Med., 186: 645-653, 1997), and may
as well lead to in vivo immunoselection of neoplastic cells with
the emergence of CTA-negative clones, in the course of immunologic
treatment against specific CTA. Thus, immunotherapeutic approaches
that focus on the immunologic targeting of distinct immunogenic
epitopes of single CTA cannot be applied to large numbers of cancer
patients, due to the absence or the possibly down-regulated
expression of target CTA in their neoplastic lesions; furthermore,
the immunological targeting of single CTA in vivo may generate
CTA-loss tumor variants that efficiently escape
treatment-induced/amplified CTA-specific immune response. An
additional limit to therapeutic approaches that target single CTA
derive from their heterogeneous intralesional expression (
Schultz-Thater E. et al., Br. J. Cancer, 83: 204-208, 2000),
moreover, the presentation of distinct immunogenic epitopes of
single CTA by specific HLA class I or HLA class II
allospecificities allows treatment only of patients with certain
defined HLA phenotypes.
[0012] To partially obviate to these limitations, recent
therapeutic strategies are utilizing more than one immunogenic
epitope of single or multiple CTA, or the whole CTA protein as
vaccinating agent (Conference on Cancer Vaccines, Eds. Ferrantini
M. and Belardelli F., Rome-Italy, Nov. 15-16, 1999;
http://www.cancerresearch.org).
[0013] Accordingly, there is a strongly felt need for a cancer
vaccine which can overcome the drawbacks of the state of the art,
in particular poor immunogenicity, in vivo immunoselection, the
possibility to practice a cancer vaccine on a wide population of
cancer patients, not limited to the specific single targeted CTA,
or TAA, and in that the cancer vaccine not be "restricted" to
selected HLA class I and/or HLA class II antigens.
[0014] Recent in vitro evidences have demonstrated that the
expression of all CTA genes that have been investigated, among the
so far known, is induced or up-regulated in neoplastic cells of
different histology following their exposure to DNA hypomethylating
agents (dos Santos N. R. et al., nCancer Res., 60: 1654-1662, 2000;
Weber J. et al., Cancer Res., 54: 1766-1771, 1994). CTA induction
was found to be persistent being still detectable several weeks
after the end of treatment. These findings support the notion that
CTA belong to a class of TAA that is comprehensively regulated by
DNA methylation. Furthermore, treatment of neoplastic cells with
DNA hypomethylating agents induced a concomitant and persistent
up-regulation of their expression of HLA class I antigens and of
investigated HLA class I allospecificities, and also up-modulated
the expression of the co-stimulatory/accessory molecules CD54 and
CD58 (Coral S. et al., J. Immunother., 22: 16-24, 1999).
[0015] Notwithstanding their promising therapeutic profile, CTA,
however, show a number of drawbacks, such as that specific CTA so
far investigated show a heterogeneous expression within distinct
neoplastic lesions, with the co-existence of CTA-positive and
-negative malignant cells; that only selected CTA among the ones so
far identified may be expressed on distinct neoplastic lesions,
independently from their hystological origin; that threshold levels
of expression of specific CTA on neoplastic cells are required for
their recognition by CTA-specific CTL and that vaccination against
a specific CTA requires an appropriate HLA class I and, for
selected CTA also HLA class II phenotype of patients.
[0016] Due to their unique biologic features, selected CTA are
being utilized in different clinical trials that aim to induce or
potentiate a CTA-specific immune response in patients affected by
malignant diseases of different histology. Diverse strategies are
currently utilized for the in vivo administration of therapeutic
CTA in the clinic or for the generation of more powerful
vaccinating tools at pre-clinical level (dos Santos N. R. et al.,
Cancer Res., 60: 1654-1662, 2000; Weber J. et al., Cancer Res., 54:
1766-1771, 1994) as the person expert in the art is aware of.
Noteworthy, mainly due to a number of technical and practical
limitations, only a limited number of immunogenic epitopes of
specific CTA, or single whole CTA protein are currently utilized in
the clinic for the therapeutic purposes. Following is a list
including the main strategies already utilized, or hypothesised so
far, to administer CTA to cancer patients; it should also be
emphasised that identical strategies are utilized to administer to
patients TAA that belong to the other classes of so far known TAA,
and that different adjuvants and/or carriers are sometimes utilized
to potentiate the immunogenicity of therapeutic agents.
[0017] Synthetic peptides representing immunogenic epitope(s) of
single or multiple CTA recognized by CD8+T cells (Conference on
Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy,
Nov. 15-16, 1999; http://www.cancerresearch.org).
[0018] Liposome-encapsulated synthetic peptides representing
immunogenic epitope(s) of single or multiple CTA (Steller M. A. et
al., Clin. Cancer Res., 4: 2103-2109, 1998).
[0019] Whole synthetic protein of a single CTA (Conference on
Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy,
Nov. 15-16, 1999; http://www.cancerresearch. org).
[0020] Recombinant viral vectors expressing epitopes of single or
multiple CTA recognized by CD8+T cells (Jenne L. et al., Trends
Immunol., 22:102-107, 2001).
[0021] Naked DNA shooting (Park J. H. et al., Mol. Cells, 9:
384-391, 1999).
[0022] Autologous PBMC/macrophages loaded ex vivo with synthetic
peptides representing epitopes of single or multiple CTA recognised
by CD8+T cells (Conference on Cancer Vaccines, Eds. Ferrantini M.
and Belardelli F., Rome-Italy, Nov. 15-16, 1999;
http://www.cancerresearch.org).
[0023] Autologous dendritic cells loaded ex vivo with synthetic
peptides representing epitopes of single or multiple CTA recognised
by CD8+ T cells or loaded with whole synthetic protein of a single
CTA, or loaded with whole tumour cell preparations (Conference on
Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy,
Nov. 15-16, 1999; http://www.cancerresearch.org; Jenne L. et al.,
Trends Immunol., 22:102-107, 2001).
[0024] Autologous dendritic cells transfected or transduced ex vivo
with DNA/RNA to express full-length CTA or fused with whole tumor
cells (Jenne L. et al., Trends Immunol., 22:102-107, 2001);
[0025] Autologous T lymphocytes transfected or transduced ex vivo
with DNA/RNA to express full-length CTA.
[0026] As far as autologous cancer vaccines, which the present
invention refers to as the main object, a number of patent
references may be cited. WO 99/42128 discloses methods for
determining the HLA transcription or expression profile of a solid
tumor, for selection of appropriate treatments and/or for
monitoring progress of the tumor. The purpose of this reference is
to inhibit some isoforms of HLA-G in order to increase the native
antitumor response. The method comprises extracting cells from a
tumor sample, lysing them and reacting the lysate with antibodies
directed against HLA Class I antigens.
[0027] DE 29913522 provides an apparatus for preparing a cancer
vaccine by submitting tumor cells extracted from a patient to
pressures of 200-9000 bar, in order to kill or damage the cells
while leaving their surface intact then reinjecting the cells to
the patient.
[0028] WO 00/02581 discloses a telomerase protein or peptide,
capable of inducing a T cell response against an oncogene or mutant
tumor suppressor protein or peptide. Said peptides are used for a
cancer vaccine.
[0029] WO 00/18933 discloses DNA constructs causing expression of
functionally inactive, altered antigens which are unaltered with
respect to the efficiency of transcription and translation of DNA,
RNA or the generation of antigenic peptides. The patient affected
by cancer is treated by the administration of the RNA or plasmid
DNA encoding an altered human cancer associated antigen, in
particular PSMA antigen. In a different embodiment, autologous
dendritic cells that have been exposed in vitro to the RNA or the
plasmid DNA are used as vaccine.
[0030] WO 00/20581 discloses a cancer vaccine comprising a new
isolated MAGE-A3 human leukocyte antigen (HLA) class II-binding
peptide. The peptide can also be used to enrich selectively a
population of T lymphocytes with CD4+ T lymphocytes specific the
said peptide. Said enriched lymphocytes are also used as cancer
vaccine.
[0031] WO 00/25813 discloses universal Tumor-Associated Antigen
(TAA) binding to a major histocompatibility complex molecule. The
method of treatment comprises administering a nucleic acid molecule
encoding the TAA, which is processed by an antigen-presenting cell
which activates cytotoxic lymphocytes and kills cells expressing
TAA. Other than the specific hTERT peptide, the identification of
different TAAs is enabled by a complex computer-aided method
synthesis of the computer-designed peptide and biological assays
for confirmation of the usefulness of the peptide.
[0032] WO 00/26249 discloses fragments of human WT-1 protein or
human gata-1 protein. These peptide fragments are used for cancer
vaccine through activation of cytotoxic T lymphocytes (CTL).
[0033] U.S. Pat. No. 6,077,519 provides a cancer vaccine comprising
a composition of T cell epitopes recovered through acid elution of
epitopes from tumor tissue.
[0034] WO 00/46352 provides a cancer vaccine comprising human T
lymphocytes that express a functional CD86 molecule. T lymphocytes
are obtained by subjecting T cells to at least two sequential
stimuli, each involving at least one activator (an antibody anti
CD2, 3 or 28) and a cytokine (interleukine) that stimulates T cell
proliferation.
[0035] Coral S. et al. Journal of Imnmunotherapy 22(1):16-24, 1999,
teach that the immunogenic potential of melanoma cells and their
recognition by the host's cytotoxic cells depend on the presence
and on the level of expression of Human Leukocytic Antigen (HLA)
class I antigens, costimulatory molecules and melanoma-associated
antigens (MAA) on neoplastic cells. There may be a suggestion that
5-AZA-CdR for use in active and/or passive specific immunotherapy
for human melanoma through its systemic administration might
enhance melanoma cells recognition by cytotoxic cells.
[0036] Momparler, Anticancer Drugs Apr; 8(4):358-68, 1997, mentions
5-AZA-CdR as chemotherapic.
[0037] Shichijo S. et al Jpn. J. Cancer Res. 87, 751-756, July
1996, investigated whether the demethylating agent 5-AZA-CDR
induces MAGE 1, 2, 3 and 6 in normal and malignant lymphoid cells
in order to better understand the mechanisms of their expression in
the cells. The authors showed the induction of investigated CTA in
selected samples tested and discussed that demethylation is not a
sufficient stimulus to induce MAGE genes in all cases and that
their results should lead to a better understanding of mechanisms
of MAGE genes expression in cells. No perspective therapeutic
implications were suggested.
[0038] Abstract of the Invention
[0039] It has now been found a method of generation of antigen
presenting cells, comprising:
[0040] a) collecting said cells from a subject,
[0041] b) activating said collected cells;
[0042] c) culturing and optionally expanding ex vivo said activated
cells;
[0043] d) treating said cultured and optionally expanded cells with
DNA hypomethylating agents so that said cells concomitantly express
multiple tumor associated antigens.
[0044] The cells obtainable according to the method of the present
invention, as well as the cellular components thereof whether alone
or in combination with said cells, are useful for prevention and
treatment, in particular in a mammal, human beings included, of
malignancies of different histotype that constitutively express one
or more of the multiple tumor associated antigens that are
expressed in said cells.
[0045] In the foregoing of the present invention, said cells are
briefly named ADHAPI-Cells.
[0046] Most conveniently, the cells obtainable from the method
above mentioned are used in the form of a cancer vaccine.
[0047] In the foregoing, the present invention shall be disclosed
in detail also by means of examples and figures, wherein:
[0048] FIG. 1 shows the proliferation of autologous (aMLR)PBMC (R)
stimulated with ADHAPI-Cells/B-EBV or control B-EBV cells (S);
[0049] FIG. 2 shows the proliferation of autologous (aMLR)PBMC (R)
stimulated with ADHAPI-Cells/PWM-B or control PWM-B cells (S);
[0050] FIG. 3 shows the proliferation of autologous (aMLR)PBMC (R)
stimulated with ADHAPI-Cells/CD40L-B or control CD40L-B cells
(S);
[0051] FIG. 4 shows the proliferation of autologous (aMLR)PBMC (R)
stimulated with ADHAPI-Cells/PWM-PBMC or control PWM-PBMC cells
(S);
[0052] FIG. 5 shows the proliferation of autologous (aMLR)PBMC (R)
stimulated with ADHAPI-Cells/PHA-PBMC and control PHA-PBMC;
[0053] FIG. 6 shows the proliferation of autologous (aMLR)PBMC (R)
stimulated with ADHAPI-Cells/PHA-+PWM-PBMC or control PHA-+PWM-PBMC
(S);
DETAILED DISCLOSURE OF THE INVENTION
[0054] According to the present invention, there is virtually no
limit as to the type of cells that can be treated in order to
generate the antigen-presenting cells, provided that they are
suitably activated and treated with a hypomethylating agent.
[0055] According to the present invention, the cells are collected
from a subject, in particular a mammal, more in particular a human.
In a possible embodiment of the present invention, said human is a
cancer patient.
[0056] In a first preferred embodiment of the present invention,
antigen-presenting cells obtainable by the method above described
are immune cells.
[0057] In a second preferred embodiment of the present invention,
antigen-presenting cells obtainable by the method above described
are non-immune cells.
[0058] The cells obtainable according to present invention can
express shared immunodominant cancer antigens or can express shared
not immunodominant cancer antigens.
[0059] In certain specific embodiments of the present invention,
cells suitable for the method herein disclosed are:
[0060] Epstein-Barr virus-immortalized, DNA hypomethylating
agent-treated B-lymphoblastoid cell lines, generated from
peripheral blood mononuclear cells (PBMC) of cancer patients in
advanced stage of disease or healthy subjects
(ADHAPI-Cells/B-EBV).
[0061] Pokeweed mitogen (PWM)-activated, DNA hypomethylating
agent-treated B-lymphocytes, generated from B-lymphocytes purified
from PBMC of cancer patients in advanced stage of disease or
healthy subjects (ADHAPI-Cells/PWM-B).
[0062] CD40 activated, DNA hypomethylating agent-treated
B-lymphocytes, generated from B-lymphocytes purified from PBMC of
cancer patients in advanced stage of disease or healthy subjects
(ADHAPI-Cells/CD40-B).
[0063] Pokeweed mitogen (PWM)-activated, DNA hypomethylating
agent-treated PBMC, generated from purified PBMC of cancer patients
in advanced stage of disease or healthy subjects
(ADHAPI-Cells/PWM-PBMC)
[0064] Phytohemagglutinin (PHA)+recombinant human interleukin-2
(rhIL-2)-activated, DNA hypomethylating agent-treated PBMC,
generated from purified PBMC of cancer patients in advanced stage
of disease or healthy subjects (ADHAPI-Cells/PHA-rhIL2-PBMC)
[0065] Phytohemagglutinin (PHA)+recombinant human interleukin-2
(rhIL-2)+pokeweed mitogen (PWM)-activated, DNA hypomethylating
agent-treated PBMC, generated from purified PBMC of cancer patients
in advanced stage of disease or healthy subjects
(ADHAPI-Cells/PHA-rhIL2-PWM- -PBMC)
[0066] Dendritic cells, monocytes, macrophages.
[0067] CD34+cells, fibroblasts, stem cells, fibroblasts and
cheratinocytes.
[0068] The cells obtainable by the method according to the present
invention are suitable for use as agents for the prevention and
treatment of malignancies of different histotype that
constitutively express one or more of cancer antigens, whether
immunodominant or not immunodominant.
[0069] Another possible embodiment of the present invention is
applicable to those cases wherein it is not wished or necessary to
utilize the direct antigen presenting ability of vaccinating cells.
In this case, vaccinating cells or their cellular components
obtainable by the method of the present invention can be used as
"reservoir" of pooled cancer antigens to vaccinate patients.
[0070] In a preferred embodiment of the present invention, the
selected TAA are CTA.
[0071] This embodiment of the present invention offers to the
skilled person the following advantages:
[0072] CTA are immunogenic since they include epitopes recognized
by HLA class I-restricted CTA-specific CD8+ CTL.
[0073] CTA are immunogenic since they include epitopes recognized
by HLA class II-restricted CTA-specific CD4+ T lymphocytes.
[0074] Selected CTA simultaneously include epitopes presented by
HLA class I and by HLA class II antigens; thus, selected CTA can
concomitantly induce CD8+ CTL and CD4+ T lymphocytes reactions.
[0075] CTA are not expressed in benign tissues with the exception
of testis and placenta.
[0076] Different CTA can be concomitantly expressed in neoplastic
cells of solid and hemopoietic malignancies, providing multiple
therapeutic targets that are co-expressed on transformed cells.
[0077] Distinct CTA are homogeneously expressed among concomitant
and sequential metastatic lesions of given patients.
[0078] Distinct CTA can be expressed in malignant tissues of
different hystological origin providing common therapeutic targets
shared by human neoplasia regardless of their specific
hystotype.
[0079] Distinct CTA may encode for multiple immunogenic peptides
presented in the context of different HLA class I and HLA class II
allospecificities.
[0080] In a further embodiment of the present invention, histone
deacetylase inhibitors can sinergize with DNA hypomethylating
agents in inducing/up-regulating the expression of CTA, of HLA
antigens and of co-stimulatory/accessory molecules on neoplastic
cells of different histology. In fact, DNA methylation and histone
deacetylation act as synergistic layers for the epigenetic gene
silencing in cancer (Fuks F. et al., Nat. Genet., 24: 88-91, 2000),
and a strong reactivation of selected hypermethylated genes, with
tumor suppressor function, has been observed in colorectal
carcinoma cells after treatment with histone deacetylase
inhibitors, following an initial minimal DNA demethylation (Cameron
E. E. et al., Nat. Genet., 21: 103-107, 1999).
[0081] The activation step in the method according to the present
invention is carried out following the general common knowledge, in
any case reference can be made to Current Protocols in Immunology,
Coligan J. E. et al. Eds, Wiley.
[0082] The demethylation treatment in the method according to the
present invention is generally well-known and the literature
generally reports the procedure, for further information see also
Santini V. et al., Ann. Intern. Med., 134: 573-586, 2001.
[0083] Hypomethylating agents, also known in the art as
demethylating agents, useful for the purposes of the present
invention are well known in the art. DNA demethylating agents are
widely disclosed in the literature, see for example WO 01/29235,
U.S. Pat. No. 5,851,773. A preferred DNA demethylating agent is
5-aza-cytidine or, more preferred, 5-aza-2'-deoxycytidine
(5-AZA-CdR).
[0084] Antigen presenting cells according to the present invention
are suitable for the preparation of cancer vaccines. In a preferred
embodiment of the present invention, said vaccines are autologous
vaccines.
[0085] In another preferred embodiment of the present invention,
said vaccines are allogeneic vaccines. In this embodiment, the
cells obtainable according to the method above disclosed may be
used both as antigen presenting cells and as in the form of
"reservoir" of pooled cancer antigens, whether as cells or cellular
components thereof.
[0086] In a still further another embodiment of the present
invention, the cells and/or the cellular components can be used in
a method for generating effector immune cells, said effector immune
cells being used for the preparation of a product useful in the
well-known adoptive immunotherapy. In another embodiment of the
present invention, the vaccine herein disclosed can be used in
combination with a systemic pre-treatment of the cancer patient
with a hypomethylating agent, for example decitabine. This
embodiment may be performed with an article of manufacture, for
example a kit, comprising a vaccine according to the present
invention and a pharmaceutical composition suitable for systemic
administration of a hypomethylating agent, for example
decitabine.
[0087] Vaccines can be prepared according to techniques well-know
to the person skilled in this art, just resorting to the general
common knowledge. For example, the patent references mentioned in
the present description are a sufficient disclosure for the
preparation of cancer vaccines, see for example WO 00/25813 or WO
00/46352.
[0088] The skilled person will have no difficulty in establishing
the proper manner for using the vaccines according to the present
invention, in particular as to the administration protocol.
[0089] The following examples further illustrate the present
invention.
EXAMPLE 1
[0090] ADHAPI-Cells/B-EBV
[0091] PBMC purification
[0092] PBMC were purified by standard Ficoll-Hypaque density
gradient centrifugation from heparinized peripheral blood of cancer
patients in advanced stage of disease or healthy subjects.
[0093] Generation of Autologous B-lymphoblastoid Cell Lines by the
Immortalization of PBMC with Epstein-Barr Virus (EBV)
[0094] B-EBV+lymphoblastoid cell lines were generated by incubating
PBMC with supernatant from B95.8 marmoset cell line at 37.degree.
C. in a 5% CO.sub.2 humidified atmosphere, in RPMI 1640 medium
supplemented with 10% heat-inactivated foetal calf serum (or human
AB serum) and 2 mM L-glutamine.
[0095] Generation of ADHAPI-Cells/B-EBV and Control B-EBV Cells
[0096] B-EBV+lymphoblastoid cell lines (7.5.times.10.sup.5
cells/ml) were cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated foetal calf serum (or 10% heat-inactivated human
AB serum) and 2 mM L-glutamine at 37.degree. C. in a 5% CO.sub.2
humidified atmosphere, and pulsed four times with 1 .mu.M
5-aza-2'-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the
culture medium was replaced with fresh medium and cultures were
allowed to proceed for additional 48 h. Then cells were utilized
for experimental procedures and/or frozen under viable conditions.
Control cells (B-EBV cells) were cultured under similar
experimental conditions but without pulses of 5-AZA-CdR.
[0097] Final Recovery of ADHAPI-Cells/B-EBV and Control B-EBV
Cells
[0098] For the results, see Table I.
[0099] Autologous Mixed Lymphocyte Reaction (aMLR) and MLR
[0100] ADHAPI-Cells/B-EBV and control B-EBV cells (stimulators=S)
were collected, washed twice with Hanks' balanced salt solution
(HBSS) and x-ray treated (75 Gy). For aMLR and MLR scalar
concentrations (from 1.times.10.sup.6 cells/ml to 6.times.10.sup.4
cells/ml) of ADHAPI Cells/B-EBV or control B-EBV cells were added
to autologous or allogeneic PBMC (1.times.10.sup.6 cells/ml)
(responder=R) in Basal Iscove's medium supplemented with 10%
heat-inactivated human AB serum, 2 mM L-glutamine, 100 U/ml
penicillin, 100 .mu.g/ml streptomycin sulphate, and seeded in 96
well U-bottom plates to a final volume of 200 .mu.l/well. After a
24 h incubation at 37.degree. C. in a 5%CO.sub.2 humidified
atmosphere, 100 .mu.l of culture supernatant were collected and
immediately stored at -80.degree. C. until use for cytokine assay.
Then, 100 .mu.l of fresh medium were added to each well and
cultures were allowed to proceed for additional five days, when
cultures were pulsed O/N with .sup.3H-TdR (1 .mu.LCi/well); then
plates were harvested and .sup.3H-TdR incorporation by R cells was
measured by a .beta.-counter.
[0101] Proliferation of Autologous PBMC (R) Stimulated with
ADHAPI-Cells/B-EBV or Control B-EBV Cells (S) in aMLR
[0102] See FIG. 1.
[0103] Phenotypic Profile of ADHAPI-Cells/B-EBV and Control B-EBV
Cells
[0104] See Table II for results.
[0105] RT-PCR Analysis of CTA Expressed by ADHAPI-Cells/B-EBV and
Control B-EBV Cells
[0106] Experimental conditions and primers utilized to assess CTA
expression on investigated cells were as follows:
[0107] for MAGE-1, -2, -3, -4 Brasseur, F., et al. Int. J. Cancer
63: 375-380, 1995; for GAGE 1-6 Van den Eynde, B., et al. J. Exp.
Med. 182: 689-698, 1995; for NY-ESO-1 Stockert, E. et al. J. Exp.
Med. 187: 265-270, 1998; for SSX-2 ; Sahin, U., et al. Clin. Cancer
Res. 6: 3916-3922, 2000.
1 5-AZA-CdR - + MAGE-1 0/4.sup.a 4/4 MAGE-2 NT NT MAGE-3 0/4 4/4
MAGE-4 NT NT NY-ESO-1 0/4 4/4 GAGE-1-6 0/4 4/4 SSX-2 2/4 4/4
.sup.aposittive/tested; NT, not tested;
[0108] ELISA Evaluation of IFN-.gamma. Released by PBMC (R)
Stimulated in aMLR by ADHAPI-Cells/B-EBV or Control B-EBV Cells
(S)
[0109] See Table III for results.
EXAMPLE 2
[0110] ADHAPI-Cells/PWM-B
[0111] B-Lymphocyte Purification
[0112] PBMC were purified by standard Ficoll-Hypaque density
gradient centrifugation from heparinized peripheral blood of cancer
patients in advanced stage of disease or healthy subjects, and
purified B lymphocytes were obtained by conventional E resetting
technique utilizing neuraminidase-treated sheep red blood
cells.
[0113] Generation of PWM-activated B Cells
[0114] Purified B-Lymphocytes (1.5.times.10.sup.6 cells/ml) were
added with PWM (3 .mu.g/ml) and cultured for 48 h at 37.degree. C.
in a 5% CO.sub.2 humidified atmosphere in Basal Iscove's medium
supplemented with 10% heat-inactivated human AB serum, 2 mM
L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin
sulphate.
[0115] Generation of ADHAPI-Cells/PWM-B and Control PWM-B Cells
[0116] PWM-activated B-Lymphocytes were pulsed four times with 1
.mu.M 5-aza-2'-deoxycytidine (5-AZA-CdR) every 12 h; then, half of
the culture medium was replaced with fresh medium and cultures were
allowed to proceed for additional 48 h. Then cells were utilized
for experimental procedures and/or frozen under viable conditions.
Control cells (PWM-B) were cultured under similar experimental
conditions but without pulses of 5-AZA-CdR.
[0117] Final Recovery of ADHAPI-Cells/PWM-B and Control PWM-B
Cells
[0118] See Table I for results
[0119] Autologous Mixed Lymphocyte Reaction (aMLR) and MLR
[0120] ADHAPI-Cells/PWM-B and control PWM-B cells (stimulators=S)
were collected, washed three times with Hanks' balanced salt
solution supplemented with 0.5% .alpha.-methylmannopyranoside, and
x-ray treated (30 Gy). For aMLR and MLR scalar concentrations (from
1.times.10.sup.6 cells/ml to 6.times.10.sup.4 cells/ml) of
ADHAPI-Cells/PWM-B or control PWM-B cells were added to autologous
or allogeneic PBMC (1.times.10.sup.6 cells/ml) (responder=R) in
Basal Iscove's medium supplemented with 10% heat-inactivated human
AB serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin sulphate, and seeded in 96 well U-bottom plates to a
final volume of 200 .mu.l/well. After a 6 day incubation at
37.degree. C. in a 5% CO.sub.2 humidified atmosphere, 100 .mu.l of
culture supernatant were collected from each well and immediately
stored at -80.degree. C. until use for cytokine assay Then, 100
.mu.l of fresh medium were added to each well and cultures were
pulsed O/N with .sup.3H-TdR (1 .mu.Ci/well); then, plates were
harvested and .sup.3H-TdR incorporation by R cells was measured by
a .beta.-counter.
[0121] Phenotypic Profile of ADHAPI-Cells/PWM-B and Control PWM-B
Cells.
[0122] See Table II for results
[0123] RT-PCR Analysis of CTA Expressed by ADHAPI-Cells/PWM-B and
Control PWM-B Cells
2 5-AZA-CdR - + MAGE-1 0/4.sup.a 4/4 MAGE-2 0/4 4/4 MAGE-3 0/4 4/4
MAGE-4 0/4 4/4 NY-ESO-1 0/4 4/4 GAGE- 1-6 0/4 4/4 SSX-2 1/4 4/4
.sup.apositive/tested; NT, not tested.
[0124] Proliferation of Autologous PBMC (R) Stimulated with
ADHAPI-Cells/PWM-B or Control PWM-B Cells (S) in aMLR
[0125] See FIG. 2 for results.
[0126] ELISA Evaluation of IFN-y Released by Allogeneic (MLR) and
Autologous (aMLR) PBMC (R) Stimulated with ADHAPI-Cells/PWM-B or
Control PWM-B Cells (S)
[0127] See Table III for results.
EXAMPLE 3
[0128] ADHAPI-Cells/CD40L-B
[0129] PBMC Purification
[0130] PBMC were purified by standard Ficoll-Hypaque density
gradient centrifugation from heparinized or acid citrate dextrose
(ACD)-anticoagulated peripheral blood of cancer patients in
advanced stage of disease or healthy subjects.
[0131] Generation of NIH3T3-CD40L-activated PBMC
[0132] PBMC (2.times.10.sup.6 cells/ml) were co-cultured with
semiconfluent, x-ray treated (75 Gy) NIH3T3-CD40L at 37.degree. C.
in a 5% CO.sub.2 humidified atmosphere, in Basal Iscove's medium
supplemented with 10% heat-inactivated human AB serum, 2 mM
L-glutamine, 2 ng/ml recombinant human (rh) interleukin 4 (rhIL-4),
50 .mu.g/ml human transferrin, 5 .mu.g/ml rh insulin,
5.5.times.10.sup.-7 M cyclosporin A (CsA), 100 U/ml penicillin, and
100 .mu.g/ml streptomycin sulphate (complete medium). After six
days of incubation, PBMC were collected, washed twice with HBSS,
resuspended at 1.times.10.sup.6 cells/ml in complete medium and
co-cultured for additional 3 days at 37.degree. C. in a 5% CO.sub.2
humidified atmosphere with NIH3T3-CD40L freshly prepared as
described above. This procedure was repeated every 2-3 days to a
maximum culture time of 16-18 days.
[0133] Generation of ADHAPI-Cells/CD40L-B and Control CD40L-B
Cells
[0134] After 16-18 days of culture, activated PBMC were harvested
and restimulated with NIH3T3-CD40L as described above; after an O/N
incubation at 37.degree. C. in a 5% CO.sub.2 humidified atmosphere,
cultures were pulsed four times with 1 .mu.M 5-aza-2'-deoxycytidine
(5-AZA-CdR) every 12 h; then, cells were harvested and restimulated
with NIH3T3-CD40L as described above and cultures were allowed to
proceed for additional 48 h. Then cells were utilized for
experimental procedures and/or frozen under viable conditions.
Control cells (CD40L-B cells) were cultured under similar
experimental conditions but without pulses of 5-AZA-CdR.
[0135] Final Recovery of ADHAPI-Cells/CD40L-B and Control CD40L-B
Cells
[0136] See Table I for results.
[0137] Autologous Mixed Lymphocyte Reaction (aMLR) and MLR
[0138] ADHAPI-Cells/CD40L-B and control CD40L-B cells
(stimulators=S) were collected, washed three times with Hanks'
balanced salt solution and x-ray treated (50 Gy). For aMLR and MLR
scalar concentrations (from 1.times.10.sup.6 cells/ml to
6.times.10.sup.4 cells/ml) of ADHAPI-Cells/CD40L-B or control
CD40L-B cells were added to autologous or allogeneic PBMC
(1.times.10.sup.6 cells/ml) (responder=R) in Basal Iscove's medium
supplemented with 10% heat-inactivated human AB serum, 2 mM
L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin
sulphate, and seeded in 96 well U-bottom plates to a final volume
of 200 .mu.l/well. After a 24 h incubation at 37.degree. C. in a
CO.sub.2 humidified atmosphere, 100 .mu.l of culture supernatant
were collected and immediately stored at -80.degree. C. until use
for cytokine assay. Then, 100 .mu.l of fresh medium were added to
each well and cultures were allowed to proceed for additional 5
days when cultures were pulsed O/N with .sup.3H-TdR (1
.mu.Ci/well); then plates were harvested and .sup.3H-TdR
incorporation by R cells was measured by a .beta.-counter.
[0139] Phenotypic Profile of ADHAPI-Cells/CD40L-B and Control
CD40L-B Cells
[0140] See Table II for results.
[0141] RT-PCR Analysis of CTA Expressed by ADHAPI-Cells/CD40L-B and
Control CD40L-B Cells
3 5-AZA-CdR - + MAGE-1 0/10.sup.a 10/10 MAGE-2 0/10 9/10 MAGE-3
0/11 10/11 MAGE-4 0/11 11/11 NY-ESO-1 0/14 14/14 GAGE- 1-6 0/14
14/14 SSX-2 0/14 13/14 .sup.apositive/tested.
[0142] Proliferation of Autologous (aMLR) PBMC (R) Stimulated with
ADHAPI-Cells/CD40L-B or Control CD40L-B Cells (S) in aMLR
[0143] See FIG. 3 for results.
[0144] ELISA Evaluation of IFN-y Released by PBMC (R) Stimulated in
aMLR by ADHAPI-Cells/CD40L-B or Control CD40L-B Cells (S)
[0145] See Table III for results.
EXAMPLE 4
[0146] ADHAPI-Cells/PWM-PBMC
[0147] PBMC Purification
[0148] PBMC were purified by standard Ficoll-Hypaque density
gradient centrifugation from heparinized peripheral blood of cancer
patients in advanced stage of disease or healthy subjects.
[0149] Generation of PWM-activated PBMC
[0150] PBMC (1.5.times.10.sup.6 cells/ml) were added with PWM (3
.mu.g/ml) and cultured for 48 h at 37.degree. C. in a 5% CO.sub.2
humidified atmosphere in Basal Iscove's medium supplemented with
10% heat-inactivated human AB serum, 2 mM L-glutamine, 100 U/ml
penicillin, 100 .mu.g/ml streptomycin sulphate.
[0151] Generation of ADHAPI-Cells/PWM-PBMC and Control PWM-PBMC
Cells
[0152] PWM-activated PBMC were pulsed four times with 1 .mu.M
5-aza-2'-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the
culture medium was replaced with fresh medium and cultures were
allowed to proceed for additional 48 h. Then cells were utilized
for experimental procedures and/or frozen under viable conditions.
Control cells (PWM-PBMC) were cultured under similar experimental
conditions but without pulses of 5-AZA-CdR.
[0153] Final Recovery of ADHAPI-Cells/PWM-PBMC and Control PWM-PBMC
Cells
[0154] See Table I for results.
[0155] Autologous Mixed Lymphocyte Reaction (aMLR) and MLR
[0156] ADHAPI-Cells/PWM-PBMC and control PWM-PBMC cells
(stimulators=S) were collected, washed three times with Hanks'
balanced salt solution supplemented with 0.5%
.alpha.-methylmannopyranoside, and x-ray treated (30 Gy). For aMLR
and MLR scalar concentrations (from 1.times.10.sup.6 cells/ml to
6.times.10.sup.4 cells/ml) of ADHAPI-Cells/PWM-PBMC or control
PWM-PBMC cells were added to autologous or allogeneic PBMC
(1.times.10.sup.6 cells/ml) (responder=R) in Basal Iscove's medium
supplemented with 10% heat-inactivated human AB serum, 2 mM
L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin
sulphate, and seeded in 96 well U-bottom plates to a final volume
of 200 .mu.l/well. After a 6 day incubation at 37.degree. C. in a
5% CO.sub.2 humidified atmosphere, 100 .mu.l of culture supernatant
were collected from each well and immediately stored at -80.degree.
C. until use for cytokine 100 .mu.l of fresh medium were added to
each well were pulsed O/N with .sup.3H-TdR (1 .mu.Ci/well); then
harvested and .sup.3H-TdR incorporation by R cells was measured by
a .beta.-counter.
[0157] Phenotvpic Profile of ADHAPI-Cells/PWM-PBMC and Control
PWM-PBMC Cells
[0158] See Table II for results.
[0159] RT-PCR Analysis of CTA Expressed by ADHAPI-Cells/PWM-PBMC
and Control PWM-PBMC Cells
4 5-AZA-CdR - + MAGE-1 0/4.sup.a 4/4 MAGE-2 0/4 3/4 MAGE-3 0/4 4/4
MAGE-4 1/4 3/4 NY-ESO-1 0/4 4/4 GAGE-1-6 0/4 3/4 SSX-2 0/4 3/4
.sup.apositive/tested; NT, not tested.
[0160] Proliferation of Autologous (aMLR) PBMC (R) Stimulated with
ADHAPI-Cells/PWM-PBMC or Control PWM-PBMC Cells (S) in aMLR
[0161] See FIG. 4 for results.
[0162] ELISA Evaluation of IFN-y Released by Autologous PBMC (R)
Stimulated in aMLR by ADHAPI-Cells/PWM-PBMC or Control PWM-PBMC
Cells (S)
[0163] See Table III for results.
EXAMPLE 5
[0164] ADHAPI-Cells/PHA-PBMC
[0165] PBMC Purification
[0166] PBMC were purified by standard Ficoll-Hypaque density
gradient centrifugation from heparinized peripheral blood of cancer
patients in advanced stage of disease or healthy subjects.
[0167] Generation of PHA-activated PBMC
[0168] PBMC (1.5.times.10.sup.6 cells/ml) were added with PHA-M (10
.mu.g/ml) and 100 UI/ml rhIL-2, and cultured for 48 h at 37.degree.
C. in a 5% CO.sub.2 humidified atmosphere in RPMI 1640 medium
supplemented with 10% heat-inactivated foetal calf serum (or in
Basal Iscove's medium supplemented with 10% heat-inactivated human
AB serum), 2 mM L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin sulphate (complete medium).
[0169] Generation of ADHAPI-Cells/PHA-PBMC and Control PHA-PBMC
[0170] PHA-activated PBMC were pulsed four times with 1 .mu.M
5-aza-2'-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the
culture medium was replaced with fresh complete medium without
PHA-M and cultures were allowed to proceed for additional 48 h.
Then cells were utilized for experimental procedures and/or frozen
under viable conditions. Control cells (PHA-PBMC) were cultured
under similar experimental conditions but without pulses of
5-AZA-CdR.
[0171] Final Recovery of ADHAPI-Cells/PHA-PBMC and Control
PHA-PBMC
[0172] See Table I for results.
[0173] Autologous Mixed Lymphocyte Reaction (aMLR) and MLR
ADHAPI-Cells/PHA-PBMC and control PHA-PBMC (stimulators=S) were
collected, washed three times with Hanks' balanced salt solution
supplemented with 0.5% .alpha.-methylmannopyranoside, and x-ray
treated (50 Gy). For aMLR and MLR scalar concentrations (from
1.times.10.sup.6cells/ml to 6.times.10.sup.4cells/ml) of
ADHAPI-Cells/PHA-PBMC or control PHA-PBMC were added to autologous
or allogeneic PBMC (1.times.10.sup.6 cells/ml) (responder=R) in
Basal Iscove's medium supplemented with 10% heat-inactivated human
AB serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin sulphate and seeded in 96 well U-bottom plates to a
final volume of 200 .mu.l/well. After a 24 h incubation at
37.degree. C. in a 5% CO.sub.2 humidified atmosphere, 100 .mu.l of
culture supernatant were collected from each well and immediately
stored at -80.degree. C. until use for cytokine assay. Then, 100
.mu.l of fresh medium were added to each well and cultures were
allowed to proceed for additional 5 days when cultures were pulsed
O/N with .sup.3H-TdR (1 .mu.Ci/well); then, plates were harvested
and .sup.3H-TdR incorporation by R cells was measured by a
.beta.-counter.
[0174] Phenotvpic Profile of ADHAPI-Cells/PHA-PBMC and Control
PHA-PBMC.
[0175] See Table II for results.
[0176] RT-PCR Analysis of CTA Expressed by ADHAPI-Cells/PHA-PBMC
and Control PHA-PBMC
5 5-AZA-CdR - + MAGE-1 0/12.sup.a 12/12 MAGE-2 0/3 3/3 MAGE-3 0/12
12/12 MAGE-4 0/4 4/4 NY-ESO-1 0/6 6/6 GAGE-1-6 0/4 4/4 SSX-2 0/6
6/6 .sup.apositive/tested; NT, not tested.
[0177] Proliferation of Autologous PBMC (R) Stimulated with
ADHAPI-Cells/PHA-PBMC and Control PHA-PBMC in aMLR
[0178] See FIG. 5 for results.
[0179] ELISA Evaluation of IFN-.gamma. (Released by Allogeneic
(MLR) and Autologous (aMLR) PBMC (R) Stimulated with
ADHAPI-Cells/PHA-PBMC or Control PHA-PBMC(S)
[0180] See Table III for results.
EXAMPLE 6
[0181] ADHAPI-Cells/PHA+PWM-PBMC
[0182] PBMC Purification
[0183] PBMC were purified by standard Ficoll-Hypaque density
gradient centrifugation from heparinized or ACD-anticoagulated
peripheral blood of cancer patients in advanced stage of disease or
healthy subjects.
[0184] Generation of PHA+PWM-activated PBMC
[0185] PBMC (1.5.times.10.sup.6 cells/ml) were added with PHA-M (10
.mu.g/ml), PWM (3 .mu.g/ml), 100 UI/ml rhIL-2 and cultured for 48 h
at 37.degree. C. in a 5% CO.sub.2 humidified atmosphere in Basal
Iscove's medium supplemented with 10% heat-inactivated human AB
serum (or with 10% heat-inactivated autologous serum), 2 mM
L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin
sulphate (complete medium).
[0186] Generation of ADHAPI-Cells/PHA+PWM-PBMC and Control
[0187] PHA+PWM-PBMC
[0188] PHA+PWM-activated PBMC were pulsed four times with 1 .mu.M
5-aza-2'-deoxycytidine (5-AZA-CdR) every 12 h; then, half of the
culture medium was replaced with fresh complete medium without PHA
or PWM and cultures were allowed to proceed for additional 48 h.
Then cells were utilized for experimental procedures and/or frozen
under viable conditions. Control cells (PHA+PWM-PBMC) were cultured
under similar experimental conditions but without pulses of
5-AZA-CdR.
[0189] Final Recovery of ADHAPI-Cells/PHA+PWM-PBMC and Control
PHA+PWM-PBMC
[0190] See Table I for results.
[0191] Autologous Mixed Lymphocyte Reaction (aMLR) and MLR
[0192] ADHAPI-Cells/PHA+PWM-PBMC and control PHA+PWM-PBMC
(stimulators=S) were collected, washed three times with Hanks'
balanced salt solution supplemented with 0.5%
.alpha.-methylmannopyranoside, and x-ray treated (50 Gy). For aMLR
and MLR scalar concentrations (from 1.times.10.sup.6 cells/ml to
6.times.10.sup.4cells/ml) of ADHAPI-Cells/PHA-rhIL2-+PWM-PBMC or
control PHA+PWM-PBMC were added to autologous or allogeneic PBMC
(1.times.10.sup.6 cells/ml) (responder=R) in Basal Iscove's medium
supplemented with 10% heat-inactivated human AB serum, 2 mM
L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin
sulphate, and seeded in 96 well U-bottom plates to a final volume
of 200 .mu.l/well. After a 6 day incubation at 37.degree. C. in a
5% CO.sub.2 humidified atmosphere, 100 .mu.l of culture supernatant
were collected from each well and immediately stored at -80.degree.
C. until use for cytokine assay. Then, 100 .mu.l of fresh medium
were added to each well and cultures were pulsed O/N with
.sup.3H-TdR (1 .mu.Ci/well); then, plates were harvested and
.sup.3H-TdR incorporation by R cells was measured by a
.beta.-counter.
[0193] Phenotvpic Profile of ADHAPI-Cells/PHA+PWM-PBMC and
Control
[0194] PHA+PWM-PBMC
[0195] See Table II for results.
[0196] RT-PCR Analysis of CTA Expressed by
ADHAPI-Cells/PHA+PWM-PBMC and Control PHA+PWM-PBMC
6 5-AZA-CdR - + MAGE-1 0/7.sup.a 7/7 MAGE-2 0/7 7/7 MAGE-3 0/7 7/7
MAGE-4 0/7 7/7 NY-ESO-1 0/7 7/7 GAGE-1-6 0/7 7/7 SSX-2 0/7 7/7
.sup.apositive/tested.
[0197] Proliferation of Autologous (aMLR) PBMC (R) Stimulated with
ADHAPI-Cells/PHA+PWM-PBMC or Control PHA+PWM-PBMC in aMLR
[0198] See FIG. 6 for results.
[0199] ELISA Evaluation of IFN-.gamma.Released by Allogeneic (MLR)
and Autologous (aMLR) PBMC (R) Stimulated with
ADHAPI-Cells/PHA+PWM-PBMC or Control PHA+PWM-PBMC (S).
[0200] See Table III for results.
[0201] In Vivo Tumorigenicity of ADHAPI-Cells
[0202] Single subcutaneous xenografts of viable ADHAPI
Cells/PHA-rhIL2-PWM-PBMC (12.times.10.sup.6) and their control
cells (14.times.10.sup.6), ADHAPI-Cells /CD40L-B (8.times.10.sup.6)
and their control cells (8.times.10.sup.6) or x-ray-treated (30 Gy)
ADHAPI-Cells/PHA-rhIL2 PWM-PBMC (12.times.10.sup.6) and their
control cells (14.times.10.sup.6), x-ray treated (50 Gy)
ADHAPI-Cells/CD40L-B (15.times.10.sup.6) and their control cells
(18.times.10.sup.6), neither induced tumor formation at injection
or distant (clinically explorable) sites, nor affected general
health and weight of BALB/c nu/nu mice 180 days after ADHAPI-Cells
administration. Repeated subcutaneous xenografts of viable
ADHAPI-Cells/B-EBV (5.times.10.sup.6/1.sup.st injection;
1.times.10.sup.7/2.sup.nd and subsequent injections) and control
B-EBV cells (5.times.10.sup.6/1.sup.st injection;
1.times.10.sup.7/2.sup.nd and subsequent injections) or
x-ray-treated ADHAPI-Cells/B-EBV (75 Gy) (5.times.10.sup.6/1.sup.st
injection; 1.times.10.sup.7/2.sup.nd and subsequent injections) and
x-ray-treated (75 Gy) control B-EBV cells
(5.times.10.sup.6/1.sup.st injection; 1.times.10.sup.7/2.sup.nd and
subsequent injections), at day 0, 33, 63 and 96, neither induced
tumor formation at injection or distant (clinically explorable)
sites, nor affected general health and weight of BALB/c nu/nu mice
180 days after the first administration. General health and weight
of ADHAPI-Cells-treated animals was comparable to control animals,
untreated or grafted with B-EBV cells.
[0203] Advantages of ADHAPI-Cells as Polyvalent Cellular CTA
Vaccines
[0204] As compared to the main strategies already utilized, or so
far hypothesised, to most effectively administer known CTA to
cancer patients, ADHAPI-Cells represent a totally new and
innovative approach, and comprise a number of prominent/remarkable
advantages. Among these:
[0205] ADHAPI-Cells vs not Genetically-modified Cellular CTA
Vaccines
[0206] ADHAPI-Cells are new and unique APC vaccines as they
concomitantly express multiple/all methylation-regulated CTA; being
endogenously synthesised, CTA can directly and simultaneously
access both HLA class I and HLA class II antigen processing
pathways within ADHAPI-Cells (Jenne L. et al., Trends Immunol.,
22:102-107, 2001).
[0207] Thus, due to their constitutive cell membrane expression of
both HLA class I and HLA class II antigens, ADHAPI-Cells can
concomitantly present immunogenic epitopes of endogenously
synthesised CTA both to CD8+ and to CD4+ T autologous lymphocytes;
therefore, ADHAPI-Cells can simultaneously induce/amplify a
CTA-directed CTL and humoral immune responses. Additionally,
ADHAPI-Cells may express and present to host's T cells
methylation-regulated CTA that have not been identified and
characterized yet (as well as not immunodominant epitopes of known
and still unknown CTA).
[0208] Opposite to ADHAPI-Cells, synthetic CTA peptide(s)-pulsed,
synthetic CTA whole protein-pulsed, or whole tumor cell
preparations-pulsed autologous APC vaccines (e.g., dendritic cells,
PBMC), as well as electrofusion-generated tumor cell dendritic cell
hybrids (Kugler A. et al., Nat. Med., 6: 332-336, 2000. Tureci O.
et al., Cancer Res., 56: 4766-4772, 1996. Eds), share major
limitations including: i) the unknown fate in vivo of the ex
vivo-loaded synthetic CTA peptide(s), of whole synthetic CTA
protein or of tumor-derived CTA, which may significantly affect the
longevity of antigen presentation to host's immune system; ii)
limited amounts of synthetic CTA peptide(s), of whole synthetic CTA
protein or of tumor-derived CTA that can be loaded ex vivo onto HLA
class I and/or HLA class II antigens of cellular vaccines, which
may significantly hamper the immunogenicity of administered CTA;
iii) the restriction by the patient's HLA phenotype, and the still
relatively limited number of known HLA class I antigens- and even
more HLA class II antigens restricted immunogenic epitopes of so
far identified CTA; iv) availability of adequate amounts of fresh
tumor tissue, that should also be sufficiently representative of
the diverse CTA expressed in neoplastic lesions (Jenne L. et al.,
Trends Immunol., 22:102-107, 2001).
[0209] The expression of endogenously synthesised CTA by
ADHAPI-Cells is long lasting; thus, at variance with ex vivo
synthetic CTA peptide(s)-pulsed or synthetic CTA whole
protein-pulsed or whole tumor cell preparations-pulsed autologous
APC vaccines, ADHAPI-Cells can provide a prolonged stimulation in
vivo of hosts immune response and with a lower number of
administrations to patients. This hypothesis is reinforced by the
foreseen possibility to administer ADHAPI-Cells as a viable, not
x-ray-treated, cellular vaccines due to their absence of long term
tumorigenicity in vivo. Furthermore, once ADHAPI-Cells would
undergo physiological death in vivo, they could still act as a
"reservoir" of endogenously synthesised CTA peptides and proteins,
that could further and efficiently boost the presentation of HLA
class I-restricted epitopes of CTA to CD8+ T cells by patient's
dendritic cells, through the immunologic mechanism of
cross-priming, as well as the presentation of HLA class
II-restricted epitopes of CTA to CD4+ T cells, through the
well-defined exogenous pathway of antigen processing.
[0210] ADHAPI-Cells retain their APC function; in fact, they
efficiently stimulate the proliferation and IFN-.gamma. release of
autologous and allogeneic PBMC; furthermore, ADHAPI-Cells are in
most instances more potent stimulators as compared to their
respective control cells. In this respect, it is relevant that in
addition to CTA, ADHAPI-Cells may concomitantly express higher
levels of HLA class I antigens and/or of different
co-stimulatory/accessory molecules as compared to their respective
control cells. These evidences clearly represent a great advantage
of ADHAPI-Cells as autologous cellular vaccines, compared to
autologous tumor cells that are poorly immunogenic, and do not
constitutively express several co-stimulatory/accessory molecules.
Furthermore, as compared to ex vivo-generated and expanded
autologous dendritic cells, ADHAPI-Cells vaccines are generated by
fully mature and immunocompetent APC; this aspect overcomes the
potential limitation represented by the maturation stage of
dendritic cells utilized for the generation of cellular vaccines,
which may influence their tolerogenic rather than immunogenic
potential.
[0211] As compared to other cellular vaccines, the ex vivo
generation of ADHAPI-Cells vaccines, that concomitantly express
multiple/all methylation-regulated CTA, is simple, in most cases
rapid, does not require cumbersome in vitro cellular manipulations,
does not involve genetic manipulations, does not require autologous
tumor tissue, and it is highly reproducible both from PBMC of
healthy individuals and cancer patients.
[0212] Furthermore, close to 100% of ADHAPI-Cells preparations
express all investigated CTA that are demethylation-inducible in
APC. Due to these characteristics, the generation of ADHAPI-Cells
vaccines is easier to standardize and to control for quality (for
example by flow cytometry for selected cell surface molecules and
RT-PCR for selected CTA) and potency (for example by quantitative
RT-PCR for selected CTA). Additionally, compared to other cellular
vaccines that to date must be freshly prepared each time they must
be administered to patients, thus generating obvious
inter-preparations variability (e. g., cellular viability,
phenotypic profile of vaccinating cells, amount of loaded synthetic
CTA peptide(s) or synthetic CTA whole protein or of whole tumor
cell preparations, efficiency of generation of tumor cell-dendritic
cell hybrids by electrofusion), ADHAPI-Cells vaccines, once
prepared and checked for viability, quality and potency, can be
aliquoted, appropriately frozen, and stored under viable conditions
until use for therapeutic purposes. Furthermore, since they do not
require the availability of autologous tumor tissue to pulse
autologous cellular vaccines or to generate tumor cell-dendritic
cell hybrids ex vivo, and since they can be rapidly prepared in
large number from repeated leukaphereses, ADHAPI-Cells vaccines
represent a practically unlimited source of therapeutic agent for
each patient.
[0213] In light of their concomitant expression of multiple/all
methylation-regulated CTA that are endogenously synthesised, and
that can directly and simultaneously access the HLA class I and HLA
class II antigen-processing pathway, owing to their possibility to
express and present to host's T cells methylation regulated CTA
that have not been identified and characterized yet (as well as not
immunodominant epitopes of known and still unknown CTA), and due to
the still limited number of known HLA class I antigens- and HLA
class II antigens-restricted immunogenic epitopes of so far
identified CTA that can thus be utilized for therapeutic
applications according to patient's HLA phenotype, an additional
advantage of ADHAPI-Cells is that they are most likely able to
concomitantly present known and still unknown immunogenic epitopes
of different CTA in the context of any and multiple HLA class I and
HLA class II allospecificities. Thus, as compared to synthetic CTA
peptide(s)-pulsed or synthetic CTA whole protein-pulsed cellular
vaccines, treatment with ADHAPI-Cells vaccines is not limited to
patients with defined HLA phenotypes; hence, all cancer patients
whose neoplastic lesions express one or more CTA can be candidate
to treatment with ADHAPI-Cells vaccines, regardless of their HLA
phenotype. In this respect, among the so far known CTA, one or more
of them is generally expressed in most investigated malignancies of
different histotype; therefore, vaccination with ADHAPI-Cells is
suitable in the large majority of cancer patients. A significant
information is that MAGE, GAGE or NY-ESO-1 are expressed in 96% of
human tumors (Cancer Immunol. Immunother. 50:3-15, 2001).
[0214] Compared to synthetic CTA peptide(s)-pulsed and synthetic
CTA whole protein-pulsed cellular vaccines, in which limited
amounts of protein(s) can be loaded ex vivo onto HLA class I and/or
HLA class II antigens of cellular vaccines, significantly hampering
the immunogenicity of administered CTA, and due to their
concomitant expression of multiple all methylation-regulated CTA,
ADHAPI-Cells vaccines can overcome the immunoselection of
CTA-negative tumor variants occurring in the course of treatment
against single or few CTA, and overcome the constitutively
heterogeneous and sometimes down-regulated expression of distinct
CTA occurring in specific neoplastic lesions.
[0215] ADHAPI-Cells vaccines are constituted by autologous
functional APC that concomitantly express multiple/all known
methylation-regulated CTA, and that most likely express still
unidentified CTA whose expression is regulated by DNA methylation;
furthermore, ADHAPI-Cells vaccines can be utilized in patients
affected by CTA-positive tumors of different histotype. These
functional and phenotypic features represent a clear advantage over
currently utilized allogeneic tumor cell vaccines (e.g., lysates of
whole pooled neoplastic cell lines or their non-purified extracts,
shed antigens from pooled neoplastic cell lines). In fact, these
tumor cell vaccines may not contain or may contain insufficient
amounts of known and of still unknown immunologically-relevant CTA,
contain irrelevant cellular components that may compete with CTA
for immunological responses, may have increased toxicity being
allogeneic, require efficient processing by patients' immune
system, and can be utilized exclusively in patients affected by
malignancies of the same histologic type.
[0216] ADHAPI-Cells vs Genetically Modified Cellular CTA
Vaccines
[0217] The generation of ADHAPI-Cells does not involve the ex vivo
genetic manipulations of autologous dendritic cells or of other
autologous APC, that are required to produce genetically-modified
cellular vaccines expressing selected CTA following transfection or
transduction. Furthermore, as compared to ADHAPI/Cells, a number of
limitations affect genetically-modified cellular vaccines; among
these are: i) the relative low efficiency of available transfection
methodologies; ii) the induction of cellular immune responses
against antigens of the viral vectors utilized for cellular
transduction, which leads to the destruction of
genetically-modified vaccinating cells; iii) the presence of
pre-existing or vaccination-induced neutralizing antibodies that
interfere with vaccine administration(s); iv) direct effects of
viral vectors on the viability, maturation and antigen-presentation
ability of transduced cells (Jenne L. et al., Trends Immunol.,
22:102-107, 2001).
7TABLE I Recovery of ADHAPI-Cells and control cells Cell Type
ADHAPI-Cells Control cells B-EBV.sup.a 114 .+-. 25 175 .+-. 51
PWM-B.sup.b 16 .+-. 5 38 .+-. 17 CD40L-B.sup.c 75 .+-. 27 96 .+-. 5
PWM-PBMC.sup.d 26 .+-. 11 45 .+-. 16 PHA-PBMC.sup.e 23 .+-. 10 63
.+-. 25 PHA+PWM-PBMC.sup.f 35 .+-. 28 63 .+-. 36 .sup.aData
represent the mean % .+-.SD of recovered cells as compared to the
number of cells (100%) utilized for their generation in 3 (a), 4
(b), 4 (c), 4 (d), 7 (e) and 5 (f) independent experiments.
[0218]
8TABLE II Phenotypic profile of ADHAPI-Cells compared to autologous
control cells* ADHAPI Cells PHA + CD40L- Pwm- PHA- PWM-PB Antigen
B.sup.a.dagger. B-EBV.sup.b PBMC.sup.c PWM-B.sup.d PBMC.sup.e
MC.sup.f HLA ns.sup.f ns ns ns ns 0.02.sup.g Class I HLA-A ns ns
nt.sup.h nt 0.004 nt Locus HLA-B ns ns nt nt 0.05 nt Locus HLA-A
0.01 ns ns ns 0.008 0.006 Alleles HLA-B ns ns nt nt ns nt Alleles
CD40 ns 0.01 ns 0.005 ns ns CD54 0.03 0.01 ns ns 0.003 ns HLA ns ns
ns 0.03 ns 0.05 Class II CD56 nt nt nt ns nt nt CD58 ns ns nt nt ns
ns CD59 nt nt ns ns nt 0.04 CD80 0.05 ns ns ns ns ns CD81 nt 0.002
nt nt ns nt CD86 0.008 nt ns ns nt ns *Data were obtained comparing
by Student's paired t-test the mean values of mean fluorescence
intensity obtained by flow cytometry in 6 (a), 6 (b), 4 (c), 4 (d),
6 (e), and 2 (f) independent experiments. Statistically significant
differences were invariably representative of an up-regulated
expression of investigated antigen on ADHAPI-Cells compared to
autologous control cells. .sup.fnot significant; .sup.gp value;
.sup.hnot tested; .dagger.ADHAPI-Cells/CD40L-B = 82-100% CD20+;
Control CD40L-B cells = 87-99% CD20+.
[0219]
9TABLE III Enzyme-linked immunosorbent assay (ELISA) evaluation of
IFN-.gamma. released by autologous (R) (aMLR) and allogeneic (MLR)
PBMC (R) stimulated by ADHAPI-Cells (S) or by control cells (S).*
AMLR Control ADHAPI- MLR Cell type cells Cells Control cells
ADHAPI-Cells B-EBV.sup.a 1770 .+-. 919 2360 .+-. nt.sup.h nt
850.5.sup.g PWM-B.sup.b 4330 .+-. 629 5530 .+-. 4040 .+-. 721 4950
.+-. 8040.06 4760.08 CD40L-B.sup.c 330 .+-. 197 429 .+-. nt nt
1530.1 PWM-PBMC.sup.d 1500 .+-. 135 1520 .+-. nt nt 1750.6
PHA-pBMC.sup.e 140 .+-. 70 956 .+-. 267 .+-. 119 1040 .+-. 4360.1
5450.07 PHA+PWM+ 790 .+-. 236 831 .+-. 819 .+-. 184 830 .+-.
PBMC.sup.f 2440.09 1690.7 *Data represent the mean values .+-. SD
of IFN-.gamma. (pg/ml) released in two (a), four (b), four (c),
four (d), three (e) and four (f) independent experiments. S/R
ratios were: 3:1 (a), 1:1 (b), 1:2 (e), 1:1 (d), 1:1 (e), 1:2 (f).
IFN-.gamma.release was assayed 24 h (a), six days (b), 24 h (c),
six days (d), 24 h (e) and 6 days (f) after the beginning of
culture; .sup.gp value vs control cells obtained by Student's
paired t-test; .sup.hnot tested.
* * * * *
References