U.S. patent application number 10/080013 was filed with the patent office on 2003-04-24 for cell therapy method for the treatment of tumors.
Invention is credited to Degraw, Juli, Heiskala, Marja, Jackson, Michael R., Leturqc, Didier J., Moriarty, Ann, Peterson, Per A..
Application Number | 20030077248 10/080013 |
Document ID | / |
Family ID | 23030546 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077248 |
Kind Code |
A1 |
Moriarty, Ann ; et
al. |
April 24, 2003 |
Cell therapy method for the treatment of tumors
Abstract
T cell responses are often diminished in humans with a
compromised immune system. We have developed a method to isolate,
stimulate and expand naive cytotoxic T lymphocyte precursors (CTLp)
to antigen-specific effectors, capable of lysing tumor cells in
vivo. This ex vivo protocol produces fully functional effectors.
Artificial antigen presenting cells (AAPCs; Drosophila
melanogaster) transfected with human HLA class I and defined
accessory molecules, are used to stimulate CD8.sup.+ T cells from
both normal donors and cancer patients. The class I molecules
expressed to a high density on the surface of the Drosophila cells
are empty, allowing for efficient loading of multiple peptides that
results in the generation of polyclonal responses recognizing tumor
cells endogenously expressing the specific peptides. The responses
generated are robust, antigen-specific and reproducible if the
peptide epitope is a defined immunogen. This artificial antigen
expression system can be adapted to treat most cancers in a
significant majority of the population.
Inventors: |
Moriarty, Ann; (Poway,
CA) ; Leturqc, Didier J.; (San Diego, CA) ;
Degraw, Juli; (San Diego, CA) ; Jackson, Michael
R.; (Del Mar, CA) ; Peterson, Per A.; (Basking
Ridge, NJ) ; Heiskala, Marja; (San Diego,
CA) |
Correspondence
Address: |
Philip S. Johnson, Esq.
Johnson & Johnson
One Johnson & Johnson Plaza
New Brunswick
NJ
08933-7003
US
|
Family ID: |
23030546 |
Appl. No.: |
10/080013 |
Filed: |
February 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60270252 |
Feb 20, 2001 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
424/277.1; 424/93.7; 435/372 |
Current CPC
Class: |
A61K 2039/55533
20130101; A61K 2039/5154 20130101; A61P 43/00 20180101; C12N 5/0636
20130101; A61K 2035/124 20130101; A61K 41/00 20130101; A61K
2039/5158 20130101; A61P 35/00 20180101; C12N 2501/515 20130101;
A61K 2039/5156 20130101; A61P 35/04 20180101; C12N 5/0601 20130101;
C12N 2510/00 20130101; A61K 2039/55538 20130101; C12N 2502/99
20130101; A61K 2039/55527 20130101; A61P 37/04 20180101; C12N
2501/23 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/85.2 ;
424/93.7; 424/277.1; 435/372 |
International
Class: |
A61K 045/00; A61K
039/00; C12N 005/08; A61K 038/20 |
Claims
What is claimed:
1. A method for treating a subject with cancer comprising: a.
preparing a non-naturally occurring antigen-presenting cell
line(nnAPC), wherein said nnAGC is capable of presenting up to
about fifteen different peptide molecules associated with said
cancer simultaneously wherein said peptide molecules are each about
six to twelve amino acids in length, b. harvesting CD8.sup.+ cells
from said subject or a suitable donor; c. stimulating said
CD8.sup.+ cells with said nnAPC cell line; d. adding said CD8.sup.+
cells to media that contains a cytokine selected from the group
consisting of IL-2, IL-7 or conditioned growth medium (CGM),
wherein said cytokines can be used individually or in combination;
e. mixing unsuspended peripheral blood monocytes, or CD-8 depleted
peripheral blood monocytes collected from said subject or a
suitable donor with about 1 to 50 .mu.g/ml of one of said peptides
that said nnAPC can simultaneously present; f. irradiating said
peripheral blood monocyte suspension with a sufficient dose of
.gamma.-radiation necessary to sterilize all components in the
suspension, except the desired peripheral blood monocytes; g.
isolating adherent peripheral blood monocytes; h. loading said
adherent peripheral blood monocytes with about 1 .mu.g/ml to 50
.mu.g/ml of said each peptide; i. combining said CD8.sup.+ cells
with said adherent peripheral blood monocytes at a ratio of about
ten CD8.sup.+ cells to one peripheral blood monocyte; and j.
inoculating said subject with CD8.sup.+ suspension.
2. The method of claim 1 wherein said nnAPC is capable of
presenting up to about ten peptide molecules.
3. The method of claim 1 wherein said peptide molecules are about
eight to ten amino acids in length
4. The method of claim 1 wherein said peptide molecules are in a
concentration range of about 10 nM to 100 .mu.M.
5. The method of claim 1 wherein said cytokine component is
IL-2.
6. The method of claim 1 wherein said cytokine component is IL-2
and IL-7 in combination
7. The method of claim 1 wherein the dose of .gamma.-radiation is
about 3,000 to 7,000 rads.
8. The method of claim 1 wherein the dose of .gamma.-radiation is
about 5,000 rads.
9. A method for treating a subject with cancer comprising, a.
preparing a non-naturally occurring antigen-presenting cell
line(nnAPC), wherein said nnAPC is capable of presenting up to
about fifteen different peptide molecules associated with said
cancer simultaneously; b. harvesting CD8.sup.+ cells from said
subject; c. stimulating said CD8.sup.+ cells with said nnAPC cell
line for about six to seven days; d. stimulating said CD8.sup.+
cells with IL-2 and IL-7 in media; e. mixing peripheral blood
monocytes collected from said subject with about 20 .mu.g/ml of
each peptide; f. irradiating said CD8-depleted peripheral blood
monocyte suspension with about 5,000 rads of .gamma.-radiation; g.
isolating adherent peripheral blood monocytes; h. loading said
adherent peripheral blood monocytes with about 1 ug/ml to 50
.mu.g/ml of said epitope; i. combining said CD8.sup.+ cells with
said adherent peripheral blood monocytes at a ratio of about ten
CD8+ cells to one peripheral blood monocyte; j. stimulating said
combined suspension of CD8.sup.+ cells and peripheral blood
monocytes for about six to seven days; k. stimulating said
suspension of CD8.sup.+ cells and peripheral blood monocytes with
IL-2 and IL-7 in media; l. assaying CD8.sup.+ suspension for
suitable CTL activity, purity, sterility and endotoxin content; and
m. inoculating said subject with CD8.sup.+ suspension.
10. The method of claim 9 wherein each peptide is about eight to
ten amino acids in length.
11. A method for treating a subject with melanoma comprising, a.
preparing a non-naturally occurring antigen-presenting cell
line(nnAPC), wherein said nnAPC is capable of presenting up to
about fifteen different peptide molecules associated with said
melanoma simultaneously where each peptide is eight to ten amino
acids in length; b. harvesting CD8.sup.+ cells from said subject;
c. stimulating said CD8.sup.+ cells with said nnAPC cell line for
about six to seven days; d. stimulating said CD8.sup.+ cells with
IL-2 and IL-7 in media; e. mixing peripheral blood monocytes
collected from said subject with about 20 .mu.g/ml of each peptide
said nnAPC can present; f. irradiating said CD8-depleted peripheral
blood monocyte suspension with about 5,000 rads of
.gamma.-radiation; g. isolating adherent peripheral blood
monocytes; h. loading said adherent peripheral blood monocytes with
about 1 ug/ml to 50 .mu.g/ml of said epitope; i. combining said
CD8.sup.+ cells with said adherent peripheral blood monocytes at a
ratio of about ten CD8+ cells to one peripheral blood monocyte; j.
stimulating said combined suspension of CD8.sup.+ cells and
peripheral blood monocytes for about six to seven days; k.
stimulating said suspension of CD8.sup.+ cells and peripheral blood
monocytes with IL-2 and IL-7 in media; l. assaying CD8.sup.+
suspension for suitable CTL activity, purity, sterility and
endotoxin content; and m. inoculating said subject with CD8.sup.+
suspension.
12. The method of claim 11 wherein said nnAPC present ten
peptides.
13. The method of claim 12 wherein said peptides are
Tyrosinase.sub.369-377, Tyrosinase.sub.207-216, gp100.sub.209-217,
gp100.sub.154-162, MART-1.sub.27-35, HER-2/neu.sub.789-797,
HER-2/neu.sub.369-377, C-lectin.sub.8-16, Pec60.sub.20-29, and
Pec60.sub.25-35.
14. A non-naturally occurring antigen-presenting cell (nnAPC)
derived from Drosophila melanogaster cells transfected with nucleic
acid that expresses human class I HLA molecules, binding molecules,
and co-stimulatory molecules, wherein said nnAPC is capable of
presenting up to fifteen different peptide molecules that are
encoded by said nucleic acid simultaneously.
15. The non-naturally occurring antigen-presenting cell nnAPC of
claim 14 wherein said nnAPC is capable of presenting up to ten
different peptide molecules simultaneously.
16. The non-naturally occurring antigen-presenting cell nnAPC of
claim 15 wherein said nnAPC is presents the following ten peptide
molecules simultaneously, Tyrosinase.sub.369-377,
Tyrosinase.sub.207-216, gp100.sub.209-217, gp100.sub.154-162,
MART-1.sub.27-35, HER-2/neu.sub.789-797, HER-2/neu.sub.369-377,
C-lectin.sub.8-16, Pec60.sub.20-29, and Pec60.sub.25-33.
17. A method for manufacturing non-naturally occurring
antigen-presenting cell (nnAPC) capable of presenting up fifteen
peptide molecules simultaneously, said method comprising of the
step, a. preparing a insect cell line from Drosophila melanogaster
eggs; b. growing said insect cells a media that is suitable for
growing insect cells, preferably Schneider.TM.'s Drosophila Medium;
c. making a pRmHa-3 plasmid from a pRmHa-1 expression vector, where
said pRmHa-3 plasmid includes a metallothionein promoter, metal
response consensus sequences and an alcohol dehydrogenase gene
bearing a polyadenylation signal isolated from Drosophila
melanogaster; d. inserting into said pRmHa-3 plasmid complementary
DNA for human class I HLA A2.1, B7.1, B7.2, ICAM-1, .beta.-2
microglobulin and LFA-3, wherein A2.1 can be substituted with any
human class I DNA sequence; e. transfecting said insect cells with
a phshneo plasmid and said pRmHa-3 plasmid containing complementary
DNA; f. creating nnAPC by contacting said insect cells with
CuSO.sub.4 to induce expression of the transfected genes in said
insect cells.
18. The method of claim 17 wherein said nnAPC is capable of
presenting up to ten peptides.
19. The method of claim 17 wherein said insect cell line is
prepared by growing cells for twelve days, selecting desired cells
with peptides that are capable of identifying said desired cells,
and expanding said desired cells with OKT3 and IL-2.
20. The method of claim 19 wherein said peptides capable of
identifying said desired cells are tetramers.
21. The method of claim 1 wherein said peptide molecules of step
(a) are selected from the peptides having an amino acid sequence
set forth in SEQ ID NO: 1 through 42.
22. The method of claim 9 wherein said peptide molecules of step
(a) are selected from the peptides having an amino acid sequence
set forth in SEQ ID NO: 1 through 42.
23. The method of claim 17 wherein said peptide molecules of step
(a) are selected from the peptides having an amino acid sequence
set forth in SEQ ID NO: 1 through 42.
Description
BACKGROUND OF THE INVENTION
[0001] Cancer continues to be a major health problem, despite
significant progress made in the area of treatment. The standard
treatment regimes of chemotherapy, radiation therapy, surgical
intervention and combinations of the three, often fail to produce a
long lasting cure. In many cases, the cancer patient having
undergone the treatment often relapses back to the disease
condition after some period of time, further exacerbating the
problem, is the severity of these treatment regimes to the
patient.
[0002] Another factor complicating development of a cancer
treatment is that cancers have been found to be caused not by a
single biological agent or factor, but rather by a combination of
agents and factors. Unlike most medical treatments where a single
causative agent or event is the focus of the treatment, cancer
therapy requires addressing a plurality of biological factors.
[0003] In recent years, research has been directed to developing
cancer therapies that utilize the patient's own immune system. One
such approach is adoptive immunotherapy. Adoptive immunotherapy
calls for using the patient's own cells to generate cytotoxic T
lymphocytes (CTLs) to treat a tumor or cancerous cells. However,
this technique remains largely unproven as a viable clinical
treatment regime for human patients. Aside from the problem of
identifying the proper epitopes with which to immunize the CTL's,
the current technology does not provide for a method of presenting
a sufficient number of different epitopes to APCs in order to
adequately target multiple antigens to effectively treat the
cancer. The present invention fulfills unmet needs, as well as
providing other benefits.
SUMMARY OF THE INVENTION
[0004] The present invention provides a non-naturally occurring
antigen-presenting cell (nnAPC) capable of presenting up to ten or
more different peptides simultaneously, methods of manufacturing
nnACP, methods of using said nnACP for the treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1: This figure is a graphic depiction of the
interaction between CD8.sup.+ cells, also known as cytotoxic T
lymphocytes with antigen-presenting cells or target cells, in this
case tumor cells.
[0006] FIG. 2, Panels A and B:
[0007] This figure is a two panel graphical depiction of mechanisms
of lymphocyte-mediated cytosis.
[0008] FIG. 3: This figure shows the result of an experiment where
several different peptides were tested in a competition assay to
identify peptide binders that could be used to load multiple
peptides onto Drosophila cells expressing human empty class I
molecules.
[0009] FIG. 4, Panels A, B and C:
[0010] This figure shows the result of an experiment where three
melanoma peptides were tested for the ability to raise CTLs when
added as single epitopes on Drosophila cells. In a single donor,
CTL activity was elicited to each of the peptides when added alone
to three different Drosophila preparations. The specificity of the
response was compared with control HBc peptide, a high affinity
binder.
[0011] FIG. 5, Panels A, B and C:
[0012] This figure shows the results of a series of experiments
where up to four different peptides were added to single Drosophila
cells. CTL activity in each of the represented peptides was seen
after a three-week stimulation protocol and is graphically depicted
in this figure.
[0013] FIG. 6, Panels A, B and C:
[0014] This figure shows CTL activity after three different primary
in vitro stimulation protocols.
[0015] FIG. 7, Panels A and B:
[0016] This figure compares the ability of Drosophila cells versus
dendritic cells to elicit CTL responses to a single peptide epitope
following standard stimulation protocols.
[0017] FIG. 8: This figure shows that the dendritic cells
displaying either mature or immature phenotype was not as efficient
as Drosophila cells in eliciting specific CTL responses when
defined peptides were used to pulse the cells.
[0018] FIG. 9, Panels A, B and C:
[0019] This figure shows CTL activity generated by a single donor
to three different in vitro stimulation protocols presenting four
peptides.
[0020] FIG. 10: This figure show CTL activity generated to ten (10)
peptides loaded, in combination, to Drosophila cells.
[0021] FIG. 11: This figure shows the peptide binding capacity of
the HER-2 peptides (826, 835, 861 and 863) on the Drosophila cells
transfected with the human HLA-A2.1 class I molecule.
[0022] FIG. 12: This figure demonstrates the anti-peptide and
anti-tumor response for MART-1 specific effector cells. T2 cells
were loaded with MART-1 peptide or a negative control (HBc).
Malme3M is a melanoma line, Malme3 is a non-tumor cell line.
[0023] FIG. 13, Panels A and B:
[0024] This figure shows the tetrameric staining of the HER-2
specific CD8 effector cells from two different donors.
[0025] FIG. 14: This figure reveals the anti-peptide response for
the HER-2 effector cells evaluated on peptide-loaded T2 cells.
[0026] FIG. 15, Panels, A, B, C, D:
[0027] This figure demonstrates the enhanced killing of an ovarian
tumor cell line (HTB-77) when transfected with HLA-A2.1.
[0028] FIG. 16: This figure shows the enhanced killing of a breast
cancer cell line (HTB-133) when transfected with HLA-A2.1
[0029] FIG. 17: This figure shows that IFN.gamma. pre-treatment is
required to demonstrate lysis of the tumor cell line
HTB-77/A2.1.
[0030] FIG. 18, Panels A and B:
[0031] This graph demonstrates that the surface expression of
HLA-A2 and HER-2 is unaffected by the IFN.gamma. induction in the
two cell lines (HTB-77 and HTB-77/A2.1).
[0032] FIG. 19: This graph shows which protein mRNA levels are
elevated in the HTB-77/A2.1 cells after an induction with
IFN.gamma..
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention provides a method for treating a
subject with cancer comprising:
[0034] a. preparing a non-naturally occurring antigen-presenting
cell line (nnAPC), wherein said nnAPC is capable of presenting up
to about fifteen different peptide molecules that is associated
with cancer, preferably about ten different peptide molecules,
simultaneously where each peptide is about six to twelve amino
acids in length, preferably about eight to ten amino acids in
length and in a concentration range of about 10 nM to 100
.mu.M;
[0035] b. harvesting CD8.sup.+ cells from said subject or a
suitable donor;
[0036] c. stimulating said CD8.sup.+ cells with said nnAPC cell
line;
[0037] d. adding said CD8+ cells to media that contains a cytokine,
such as, IL-2, IL-7 or conditioned growth medium (CGM), preferably,
IL-2, or IL-2 and IL-7 in combination;
[0038] e. mixing unsuspended peripheral blood monocytes, or
alternatively, CD-8 depleted peripheral blood monocytes collected
from said subject or a suitable donor with about 10 to 50 .mu.g/ml
of a peptide;
[0039] f. irradiating said peripheral blood monocyte suspension
with a sufficient dose of .gamma.-radiation necessary to prevent
proliferation of these cells in the suspension, such as a dose in
the range of about 3,000 to 7,000 rads, preferably about 5,000
rads, alternatively, the peripheral blood lymphocyte suspension may
be treated with cytostatic agents including, but not limited to,
mitomycin C;
[0040] g. isolating adherent peripheral blood monocytes;
[0041] h. loading said adherent peripheral blood monocytes with
about 10 ng/ml to 10 .mu.g/ml of said each peptide;
[0042] i. combining said CD8.sup.+ cells with said adherent
peripheral blood monocytes at a ratio of about ten CD8.sup.+ cells
to one peripheral blood monocyte;
[0043] j. optionally stimulating said combined suspension of
CD8.sup.+ cells and peripheral blood monocytes for about six to
seven days;
[0044] k. optionally stimulating said suspension of CD8.sup.+ cells
and peripheral blood monocytes with IL-2 and IL-7 in media;
[0045] l. optionally assaying CD8.sup.+ suspension for suitable CTL
activity, and optionally assaying for CTL purity, sterility and
endotoxin content; and
[0046] m. inoculating said subject with CD8.sup.+ suspension.
[0047] Another embodiment of the present invention provides a
method for treating a subject with cancer comprising:
[0048] a. preparing a non-naturally occurring antigen-presenting
cell line (nnAPC), wherein said nnAPC is capable of presenting up
to about fifteen different peptide molecules that is associated
with cancer, preferably about ten peptides, simultaneously where
each peptide is eight to ten amino acids in length;
[0049] b. harvesting CD8.sup.+ cells from said subject;
[0050] c. stimulating said CD8.sup.+ cells with said nnAPC cell
line for about six to seven days;
[0051] d. stimulating said CD8.sup.+ cells with IL-2 and IL-7 in
media;
[0052] e. mixing peripheral blood monocytes collected from said
subject with about 20 .mu.g/ml of each peptide;
[0053] f. irradiating said CD8-depleted peripheral blood monocyte
suspension with about 5,000 rads of .gamma.-radiation;
[0054] g. isolating adherent peripheral blood monocytes;
[0055] h. loading said adherent peripheral blood monocytes with
about 10 .mu.g/ml of said epitope;
[0056] i. combining said CD8.sup.+ cells with said adherent
peripheral blood monocytes at a ratio of about ten CD8.sup.+ cells
to one peripheral blood monocyte;
[0057] j. stimulating said combined suspension of CD8.sup.+ cells
and peripheral blood monocytes for about six to seven days;
[0058] k. stimulating said suspension of CD8.sup.+ cells and
peripheral blood monocytes with IL-2 and IL-7 in media;
[0059] l. assaying CD8.sup.+ suspension for suitable CTL activity,
purity, sterility and endotoxin content; and
[0060] m. inoculating said subject with CD8.sup.+ suspension.
[0061] Another embodiment of the present invention provides a
method for treating a subject with melanoma comprising:
[0062] a. preparing a non-naturally occurring antigen-presenting
cell line(nnAPC), wherein said nnAPC is capable of presenting up to
about fifteen different peptide molecules that is associated with
melanoma, preferably about ten peptides, simultaneously where each
peptide is eight to ten amino acids in length;
[0063] b. harvesting CD8.sup.+ cells from said subject;
[0064] c. stimulating said CD8.sup.+ cells with said nnAPC cell
line for about six to seven days;
[0065] d. stimulating said CD8.sup.+ cells with IL-2 and IL-7 in
media;
[0066] e. mixing peripheral blood monocytes collected from said
subject with about 20 .mu.g/ml of each peptide said nnAPC can
present;
[0067] f. irradiating said CD8-depleted peripheral blood monocyte
suspension with about 5,000 rads of .gamma.-radiation;
[0068] g. isolating adherent peripheral blood monocytes;
[0069] h. loading said adherent peripheral blood monocytes with
about 10 .mu.g/ml of said epitope;
[0070] i. combining said CD8.sup.+ cells with said adherent
peripheral blood monocytes at a ratio of about ten CD8.sup.+ cells
to one peripheral blood monocyte;
[0071] j. stimulating said combined suspension of CD8.sup.+ cells
and peripheral blood monocytes for about six to seven days;
[0072] k. stimulating said suspension of CD8.sup.+ cells and
peripheral blood monocytes with IL-2 and IL-7 in media;
[0073] l. assaying CD8.sup.+ suspension for suitable CTL activity,
purity, sterility and endotoxin content; and
[0074] m. inoculating said subject with CD8.sup.+ suspension.
[0075] Another embodiment of the present invention is a method of
treating melanoma wherein the nnAPC presents the following
peptides, Tyrosinase.sub.369-377, Tyrosinase.sub.207-216,
gp100.sub.209-217, gp100.sub.154-162, MART-1.sub.27-35,
HER-2/neu.sub.789-797, HER-2/neu.sub.369-377, C-lectin.sub.8-16,
Pec60.sub.20-29, Pec60.sub.25-33.
[0076] Another embodiment of the present invention is a method of
treating a disease or disease condition that results in an
insufficient or inadequate immune response that is normally
associated with Class I HLA molecules, wherein the treatment
eliminates infected or transformed cells has been demonstrated to
be achieved by CTLs.
[0077] Another embodiment of the present invention is a method of
treating a disease or disease condition that results in an
insufficient or inadequate immune response that is normally
associated with Class I HLA molecules, wherein infected or
transformed cells that have been shown to be susceptible to
elimination by CTL are treated by the method comprising:
[0078] a. preparing a non-naturally occurring antigen-presenting
cell line (nnAPC), wherein said nnAPC is capable of presenting up
to about fifteen different peptide molecules that is associated
with said disease or disease condition, preferably about ten
different peptide molecules, simultaneously where each peptide is
about six to twelve amino acids in length, preferably about eight
to ten amino acids in length and in a concentration range of about
10 nM to 100 .mu.M;
[0079] b. harvesting CD8.sup.+ cells from said subject or a
suitable donor;
[0080] c. stimulating said CD8.sup.+ cells with said nnAPC cell
line;
[0081] d. adding said CD8.sup.+ cells to media that contains a
cytokine, such as, IL-2, IL-7 or CGM, preferably, IL-2, or IL-2 and
IL-7 in combination;
[0082] e. mixing unsuspended peripheral blood monocytes, or
alternatively, CD-8 depleted peripheral blood monocytes collected
from said subject or a suitable donor with about 10 to 50 .mu.g/ml
of a peptide;
[0083] f. irradiating said peripheral blood monocyte suspension
with a sufficient dose of .gamma.-radiation necessary to sterilize
all components in the suspension, except the desired peripheral
blood monocytes, such as a dose in the range of about 3,000 to
7,000 rads, preferably about 5,000 rads;
[0084] g. isolating adherent peripheral blood monocytes;
[0085] h. loading said adherent peripheral blood monocytes with
about 10 .mu.g/ml to 10 .mu.g/ml of said each peptide;
[0086] i. combining said CD8.sup.+ cells with said adherent
peripheral blood monocytes at a ratio of about ten CD8.sup.+ cells
to one peripheral blood monocyte;
[0087] j. optionally stimulating said combined suspension of
CD8.sup.+ cells and peripheral blood monocytes for about six to
seven days;
[0088] k. optionally stimulating said suspension of CD8.sup.+ cells
and peripheral blood monocytes with IL-2 and IL-7 in media;
[0089] l. optionally assaying CD8.sup.+ suspension for suitable CTL
activity, and optionally assaying for CTL purity, sterility and
endotoxin content; and
[0090] m. inoculating said subject with CD8.sup.+ suspension.
[0091] The present invention provides a non-naturally occurring
antigen-presenting cell (nnAPC) derived from Drosophila
melanogaster cells transfected with DNA encoding human class I HLA,
binding, and co-stimulatory molecules for expression, wherein the
nnAPC is capable of presenting up to fifteen different peptide
molecules, preferably ten peptide molecules.
[0092] Another embodiment of the present invention provides a nnAPC
that presents peptides that are associated with various desired
functions that enhance the treatment of the subject. For example,
in addition to peptides associated with the disease or disease
condition being treated, the nnAPC can present peptides associated
with accessory molecules such as, lymphocyte function antigens
(LFA-1, LFA-2 and LFA-3), intercellular adhesion molecule 1
(ICAM-1), T-cell co-stimulatory factors (CD2, CD28, B7) enhance
cell-cell adhesion or transduce additional cell activation
signals.
[0093] Another embodiment of the present invention provides a nnAPC
that presents peptides that are associated with several types of
cancers. For example, the peptides associated or derived from a
breast cancer related polypeptide, such as, HER-2/neu, may be
presented with peptides associated or derived from a melanoma
related polypeptide, such as, MART-1, or MAGE.
[0094] Another embodiment of the present invention provides a
method for manufacturing non-naturally occurring antigen-presenting
cell (nnAPC) capable of presenting up to ten different peptide
molecules simultaneously, said method comprising of the step:
[0095] a. preparing an insect cell line from Drosophila
melanogaster eggs; alternatively preparing an insect cell line for
expressing human MHC Class I molecules and co-stimulatory adhesion
molecules;
[0096] b. growing said insect cells a media that is suitable for
growing insect cells, preferably Schneider.TM.'s Drosophila
Medium;
[0097] c. making a pRmHa-3 plasmid from a pRmHa-1 expression
vector, where said pRmHa-3 plasmid includes a metallothionein
promoter, metal response consensus sequences and an alcohol
dehydrogenase gene bearing a polyadenylation signal isolated from
Drosophila melanogaster;
[0098] d. inserting into said pRmHa-3 plasmid complementary DNA for
human class I HLA A2.1, B7.1, B7.2, ICAM-1, .beta.-2 microglobulin
and LFA-3, wherein A2.1 can be substituted with any human class I
DNA sequence;
[0099] e. transfecting said insect cells with a phshneo plasmid and
said pRmHa-3 plasmid containing complementary DNA; and,
[0100] f. creating nnAPC by contacting said insect cells with
CuSO.sub.4 to induce expression of the transfected genes in said
insect cells.
[0101] The insect cells of the present invention are grown in a
media suitable for growing insect cells, hereinafter referenced to
as "insect growth media". Insect growth media are commercially
available from a number of vendors, such as, Schneider.TM.'s
Drosophila Medium, Grace's Insect Media, and TC-100 Insect Media.
Alternatively, insect growth media can be prepared by one of
ordinary skill in the art. Typically the media will include
components necessary to promote and sustain the growth of insects
cells, such as, inorganic salts (for example, calcium chloride,
magnesium sulfate, potassium chloride, potassium phosphate, sodium
bicarbonate, sodium chloride, and sodium phosphate), amino acids
various carbohydrate and chemical species (Imogene Schneider, Exp.
Zool. (1964) 156(1): pg. 91). Alternatively, the media can also
include vitamins, minerals, and other components that aid in the
growth of insect cells.
[0102] Following is a list of abbreviations and definitions used in
the present specification.
1 Abbreviations APC Antigen-presenting cells CD8+ CD8+ T cells CTL
Cytotoxic T lymphocyte E Effector Fas Also known as CD95, epitope
on T cells ICAM Intercellular adhesion molecule IL Interleukin LAK
Lymphokine-activated killer cells LFA Lymphocyte function antigens
MHC Major histocompatibility complex nnAPC non-naturally occurring
antigen-presenting cell NP Nuclear protein PBMC Peripheral blood
mononuclear cell PBS Phosphate-buffered saline PCR Polymerase chain
reaction RPMI Roswell Park Memorial Institute RWJPRI The R. W.
Johnson Pharmaceutical Research Institute T Target TCR T cell
antigen receptor TIL Tumor-infiltrating lymphocytes
[0103] Following is a list of abbreviations used in the present
specification for various peptide epitopes. The individual amino
acid residues are identified according to a single letter code that
is readily known and used by those of ordinary skill in the
art.
2 Abbreviations Amino Acid 3-Letter 1-Letter alanine ala A valine
val V leucine leu L isoleucine ile I proline pro P phenylalanine
phe F trytophan tyr W methionine met M glycine gly G serine ser S
threonine thr T cysteine cys C tyrosine tyr Y asparagine asn N
glutamine gln Q aspartic acid asp D glutamic acid glu E lysine lys
K arginine arg R histidine his H
[0104] Peptide Epitope Abbreviations
[0105] As used herein the term "tyrosinase 369-377" or
"tyrosinase.sub.369-377" refers to the amino acid sequence
YMNGTMSQV (SEQ ID NO: 1). Also included within this definition is
the peptide of the sequence YMDGTMSQV (SEQ ID NO: 2), which results
from a post-translational event that modifies the amino acid
residue "N" of sequence YMNGTMSQV (SEQ ID NO: 1) to "D" resulting
in the amino acid sequence of YMDGTMSQV (SEQ ID NO: 2) (Skipper et
al., J. Exp. Med. (1996) 183:527-534).
[0106] As used herein the term "tyrosinase 207-216" or
"tyrosinase.sub.207-216" refers to the amino acid sequence
FLPWHRLFLL (SEQ ID NO: 3).
[0107] As used herein the term "gp100 209-217" or
"gp100.sub.209-217" refers to the amino acid sequence ITDQVPFSV
(SEQ ID NO: 4).
[0108] As used herein the term "gp100 154-162" or
"gp100.sub.154-162" refers to the amino acid sequence KTWGQYWQV
(SEQ ID NO: 5).
[0109] As used herein the term "MART-1 27-35" or "MART-1.sub.27-35"
refers to the amino acid sequence AAGIGILTV (SEQ ID NO: 6).
[0110] As used herein the term "HER-2/neu 789-797" or
"HER-2/neu.sub.789-797" refers to the amino acid sequence CLTSTVQLV
(SEQ ID NO: 7).
[0111] As used herein the term "HER-2/neu 369-377" or
"HER-2/neu.sub.369-377" refers to the amino acid sequence KIFGSLAFL
(SEQ ID NO: 8).
[0112] As used herein the term "C-lectin 8-16" or
"C-lectin.sub.8-16" refers to the amino acid sequence KMASRSMRL
(SEQ ID NO: 9).
[0113] As used herein the term "Pec60 20-29" or "Pec60.sub.20-29"
refers to the amino acid sequence ALALAALLVV (SEQ ID NO: 10).
[0114] As used herein the term "Pec60 25-33" or "Pec60 .sub.25-33"
refers to the amino acid sequence ALLVVDREV (SEQ ID NO: 11).
[0115] As used herein, the term "CD8 peptide 59-70" or "CD8
peptide.sub.59-70" refers to the amino acid sequence of
AAEGLDTQRFSG (SEQ ID NO: 12).
[0116] Terms and Definitions
[0117] As used herein, the term "adoptive immunotherapy" refers the
administration of donor or autologous T lymphocytes for the
treatment of a disease or disease condition, wherein the disease or
disease condition results in an insufficient or inadequate immune
response that is normally associated with Class I HLA molecules.
Adoptive immunotherapy is an appropriate treatment for any disease
or disease condition where the elimination of infected or
transformed cells has been demonstrated to be achieved by CTLs. For
example, disease or disease conditions include but are not limited
to cancer and/or tumors, such as, melanoma, prostate, breast,
colo-rectal, stomach, throat and neck, pancreatic, cervical,
ovarian, bone, leukemia and lung cancer; viral infections, such as,
hepatitis B, hepatitis C, human immunodeficiency virus; bacterial
infections, such as tuberculosis, leprosy and listeriosis, and
paracytic infections such as malaria.
[0118] As used herein, the term "B7.1" refers to a co-stimulatory
molecule associated with antigen-presenting cells.
[0119] As used herein, the term "BCNU" refers to carmustine, also
known as, 1,3-bis (2chloroethyl)-1-nitrosourea.
[0120] As used herein, the term "BSE" refers to bovine spongiform
encephalitis.
[0121] As used herein, the term "CD" refers to clusters of
differentiation, T lymphocytes (originally), B lymphocytes,
monocytes, macrophages, and granulocytes grouped by antigen
epitopes and function.
[0122] As used herein, the term "DTIC" refers to dacarbazine,
5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide.
[0123] As used herein, the term "ex vivo" or "ex vivo therapy"
refers to a therapy where biological materials, typically cells,
are obtained from a patient or a suitable alternate source, such
as, a suitable donor, and are modified, such that the modified
cells can be used to treat a pathological condition which will be
improved by the long-term or constant delivery of the therapeutic
benefit produced by the modified cells. Treatment includes the
re-introduction of the modified biological materials, obtained from
either the patient or from the alternate source, into the patient.
A benefit of ex vivo therapy is the ability to provide the patient
the benefit of the treatment, without exposing the patient to
undesired collateral effects from the treatment. For example,
cytokines are often administered to patients with cancer or viral
infections to stimulate expansion of the patient's CTLs. However,
cytokines often cause the onset of flu like symptoms in the
patients. In an ex vivo procedure, cytokines are used to stimulate
expansion of the CTLs outside of the patient's body, and the
patient is spared the exposure and the consequent side effects of
the cytokines. Alternatively under suitable situations, or
conditions, where appropriate and where the subject can derive
benefit, the subject can be treated concurrently with low level
dosages of .gamma. interferon, .gamma. interferon and/or IL-2. The
expected effect of the interferons is to possibly sensitize the
tumor cells to lysis by antigen specific CTL, and the effect of the
IL-2 is to possibly enhance antigen specific CTL persistence.
[0124] As used herein, the term "HEPES" refers to
N-2-hydroxyethylpiperazi- ne-N'2-ethanesulfonic acid buffer.
[0125] As used herein, the term "HLA-A2.1" refers to a HLA Class I
molecule found in approximately 45% of Caucasians.
[0126] As used herein, the term "MART-1" or "(melanoma antigen
recognized by T-Cells-1" refers to a melanoma-associated antigen.
The amino acid and nucleic acid sequences, as well as various
characteristics of this antigen are disclosed in U.S. Pat. No.
5,994,523, issued Nov. 30, 1999 entitled "Melanoma Antigens and
Their Use in Diagnostic and Therapeutic Methods"; U.S. Pat. No.
5,874,560, issued Feb. 23, 1999 entitled "Melanoma Antigens and
Their Use in Diagnostic and Therapeutic Methods"; and U.S. Pat. No.
5,844,075, issued Dec. 1, 1998 entitled "Melanoma Antigens and
Their Use in Diagnostic and Therapeutic Methods." In particular,
U.S. Pat. No. 5,994,523 discloses full length nucleic acid and
amino acid sequences of MART-1 in FIG. 1 as SEQ ID NO: 1, and SEQ
ID NO: 2, respectively. The aforementioned FIG. 1 is herein
incorporated by reference.
[0127] As used herein, the term "MAGE" refers to a
melanoma-associated antigen. The amino acid and nucleic acid
sequences, as well as various characteristics of this antigen are
disclosed in U.S. Pat. No. 6,140,050, issued Oct. 31, 2000 entitled
"Methods for Determining Breast Cancer and Melanoma by Assaying for
a Plurality of Antigens Associated Therewith"; U.S. Pat. No.
5,759,783, issued Jun. 2, 1998 entitled "Method of Screening for
Cancer by Detecting Messenger RNA for a MAGE-XP Gene"; and U.S.
Pat. No. 5,662,907, issued Sep. 2, 1997 entitled "Induction of
Anti-Tumor Cytotoxic T Lymphocytes in Humans Using Synthetic
Peptide Epitopes."
[0128] As used herein, the term "MPC-10" refers to a magnetic
particle concentrator.
[0129] As used herein, the term "NK cells" refers to natural killer
cells.
[0130] As used herein, the term "OKT3"refers to ORTHOCLONE OKT3,
muromonab-CD3, anti-CD3 monoclonal antibody.
[0131] As used herein, the term "TAP-1,2" refers to Transporter
Associated with Antigen Processing-1,2.
[0132] As used herein, the term "Th cells" refers to Helper T
cells, CD4.sup.+.
[0133] As used herein, the term, "tyrosinase" refers to a protein
associated with melanoma (Brichard et al., J. Exp. Med. (1993)
178:489-495; Robbins et al., Cancer Res. (1994) 54: 3124-3126).
U.S. Pat. No. 5,843,648, issued Dec. 1, 1998 entitled "P15 and
Tyrosinase Melanoma Antigens and Their Use in Diagnostic and
Therapeutic Methods" discloses antigenic peptides and associated
polynucleic acids related to tyrosinase in FIG. 7, Panels A to D,
the aforementioned figure incorporated herein by reference. U.S.
Pat. No. 5,487,974, issued Jan. 30, 1996 entitled "Method for
Detecting Complexes Containing Human Leukocyte Antigen A2 (HLA-A2)
Molecules and a Tyrosinase Derived Peptide on Abnormal Cells"
discloses an additional peptide that is associated with tyrosinase
and melanoma in Example 9, at Table 3, the aforementioned
incorporated herein by reference.
[0134] As used herein, the term "gp100" refers to a melanoma
antigen recognized by tumor infiltrating lymphocytes (TIL). The TIL
which recognize gp100 is associated with in vivo tumor rejection
(Bakker et al., J. Exp. Med. (1994) 179:1005-1009; Kawakami et al.,
J. Immunol. (1995) 154:3961-3968). Antigenic peptides related to
gp100 are disclosed in U.S. Pat. No. 5,994,523, issued Nov. 30,
1999 entitled "Melanoma Antigens and Their Use in Diagnostic and
Therapeutic Methods"; U.S. Pat. No. 5,874,560, issued Feb. 23, 1999
entitled "Melanoma Antigens and Their Use in Diagnostic and
Therapeutic Methods"; and U.S. Pat. No. 5,844,075, issued Dec. 1,
1998 entitled "Melanoma Antigens and Their Use in Diagnostic and
Therapeutic Methods." In particular, U.S. Pat. No. 5,994,523
discloses nucleic acid and amino acid sequences related to GP100 in
FIGS. 4 and 5, respectively. Also disclosed are antigenic peptides
derived from the amino acid sequences, including those identified
as SEQ ID NOs: 27, 33, 34, 35, 36, 37, 38, 39, 40, and 41. All of
the aforementioned FIGS. 4 and 5, and the peptides identified by
SEQ ID NOs are herein incorporated by referenced.
[0135] As used herein, the term "melanoma" refers to, but is not
limited to, melanomas, metastatic melanomas, melanomas derived from
either melanocytes or melanocytes related nevus cells,
melanosarcomas, melanocarcinomas, melanoepitheliomas, melanoma in
situ superficial spreading melanoma, nodular melanoma, lentigo
maligna melanoma, acral lentiginous melanoma, invasive melanoma or
familial atypical mole and melanoma (FAM-M) syndrome. Such
melanomas in mammals may be caused by, chromosomal abnormalities,
degenerative growth and developmental disorders, mitogenic agents,
ultraviolet radiation (UV), viral infections, inappropriate tissue
expression of a gene, alterations in expression of a gene, and
presentation on a cell, or carcinogenic agents. The aforementioned
melanomas can be diagnosed, assessed or treated by methods
described in the present application.
[0136] As used herein, the term "C-lectin" refers to a peptide of
the sequence that has been found to be associated with ovarian
cancer.
[0137] As used herein, the term "major histocompatibility complex"
or "MHC" is a generic designation meant to encompass the
histo-compatibility antigen systems described in different species
including the human leucocyte antigens (HLA).
[0138] As used herein, the terms "epitope," "peptide epitope,"
"antigenic peptide" and "immunogenic peptide" refers to a peptide
derived from an antigen capable of causing a cellular immune
response in a mammal. Such peptides may also be reactive with
antibodies from an animal immunized with the peptides. Such
peptides may be about five to twenty amino acid in length
preferably about eight to fifteen amino acids in length, and most
preferably about nine to ten amino acids in length.
[0139] As used herein, the term "Pec60" refers to a peptide of the
sequence that has been found to be associated with ovarian and
breast cancer.
[0140] As used herein, the term "analog" includes any polypeptide
having an amino acid residue sequence substantially identical to
the sequences of the present invention, specifically shown herein
in which one or more residues have been conservatively substituted
with a functionally similar residue and which displays the
functional aspects of the present invention as described herein.
Examples of conservative substitutions include the substitution of
one non-polar (hydrophobic) residue such as isoleucine, valine,
leucine or methionine for another, the substitution of one polar
(hydrophilic) residue for another such as between arginine and
lysine, between glutamine and asparagine, between glycine and
serine, the substitution of one basic residue such as lysine,
arginine or histidine for another, or the substitution of one
acidic residue, such as aspartic acid or glutamic acid or
another.
[0141] As used herein, the term "conservative substitution" also
includes the use of a chemically derivatized residue in place of a
non-derivatized residue.
[0142] As used herein, the term "chemical derivative" refers to a
subject polypeptide having one or more residues chemically
derivatized by reaction of a functional side group. Examples of
such derivatized molecules include for example, those molecules in
which free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups may be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups may be derivatized to form O-acyl or O-alkyl derivatives.
The imidazole nitrogen of histidine may be derivatized to form
N-imbenzylhistidine. Also included as chemical derivatives are
those proteins or peptides which contain one or more
naturally-occurring amino acid derivatives of the twenty standard
amino acids. For examples: 4-hydroxyproline may be substituted for
proline; 5-hydroxylysine may be substituted for lysine;
3-methylhistidine may be substituted for histidine; homoserine may
be substituted for serine; and ornithine may be substituted for
lysine. Proteins or polypeptides of the present invention also
include any polypeptide having one or more additions and/or
deletions or residues relative to the sequence of a polypeptide
whose sequence is encoded is the corresponding nucleic sequence of
the present invention, so long as the requisite activity is
maintained.
[0143] As used herein, the term "HER-2/neu" refers to an oncogene,
which express or over-express, one or more membrane-associated,
receptor-like oncogene proteins. Among the cancers which have been
found to be associated with expression or over-expression of
HER-2/neu are certain breast, stomach, ovarian colon and salivary
gland cancers. The HER-2/neu oncogene is a member of the tyrosine
protein kinase family of oncogenes and shares a high degree of
homology with the epidermal growth factor receptor (EGFR).
HER-2/neu has been shown to play a role in cell growth and/or
differentiation. HER-2/neu appears to induce malignancies through
quantitative mechanisms that result from increased or deregulated
expression of an essentially normal gene product. U.S. Pat. No.
6,075,122, issued Jun. 13, 2000 entitled "Immune Reactivity to
HER-2/neu Protein for Diagnosis and Treatment of Malignancies in
Which the HER-2/neu Oncogene is Associated" discloses peptides that
elicit CD8.sup.+ T cell responses at column 12, line 31 to column
13, line 7, identified according to SEQ ID numbers are herein
incorporated by reference.
[0144] HER-2/neu (p185) is the protein product of the HER-2/neu
oncogene. The HER-2/neu gene is amplified and the HER-2/neu protein
is over-expressed in a variety of cancers including breast,
ovarian, colon, lung and prostate cancer. HER-2/neu is related to
malignant transformation. It is found in 50% to 60% of ductal in
situ carcinoma and 20% to 40% of all breast cancers, as well as a
substantial fraction of adenocarcinomas arising in the ovaries,
prostate, colon and lung. HER-2/neu is intimately associated not
only with the malignant phenotype, but also with the aggressiveness
of the malignancy, being found in one-fourth of all invasive breast
cancers. HER-2/neu over-expression is correlated with a poor
prognosis in both breast and ovarian cancer. HER-2/neu is a
transmembrane protein with a relative molecular mass of 185 kd that
is approximately 1255 amino acids (aa) in length. It has an
extracellular binding domain (ECD) of approximately 645 aa, with
40% homology to epidermal growth factor receptor (EGFR), a highly
hydrophobic transmembrane anchor domain (TMD), and a
carboxyterminal cytoplasmic domain (CD) of approximately 580 amino
acids with 80% homology to EGFR.
[0145] Ongoing research involving oncogenes has identified at least
forty oncogenes operative in malignant cells and responsible for,
or associated with, transformation. Oncogenes have been classified
into different groups based on the putative function or location of
their gene products (such as the protein expressed by the
oncogene). Oncogenes are believed to be essential for certain
aspects of normal cellular physiology.
[0146] Cancer continues to be a major health problem, despite
significant progress made in the area of treatment. The standard
treatment regimes of chemotherapy, radiation therapy, surgical
intervention and combinations of the three, often fail to produce a
long lasting cure. In many cases, the cancer patient having
undergone the treatment often relapses back to the disease
condition after some period of time, further exacerbating the
problem, is the severity of these treatment regimes to the patient.
In the instance of melanoma, a cure for metastatic melanoma has not
been achieved using conventional chemotherapy. Response rates of
35% to 50% have been reported with the Dartmouth regimen of
combination chemotherapy (DTIC, cis-platin, BCNU and tamoxifen),
but the duration of survival has remained at six to ten months.
High rates of remission have been reported for aggressive "high
dose intensity" chemotherapy and repletion of hematopoeisis with
autologous bone marrow transplants. Nevertheless, the median
duration of survival was short, approximately four months.
[0147] Rosenberg and colleagues have attempted to use infusion of
activated lymphocytes as a treatment for various cancers.
Initially, lymphokine-activated killer cells (LAK) and later
tumor-infiltrating lymphocytes (TIL) activated ex vivo with IL-2
were used, but evidence for efficacy is equivocal. In fact,
controlled clinical trials have failed to show an advantage to the
use of ex vivo-activated cells over direct administration of IL-2
to patients. Thus, the benefits of LAK and TIL therapy are
marginal, and the side effects are typically so severe that many
trials have been discontinued prematurely.
[0148] Studies in mouse tumor models have demonstrated that
adoptive immunotherapy, in vivo immunization of T cells specific
for a tumor antigens(s), is very efficacious with minimal toxicity.
A major obstacle to applying this strategy to the treatment of
human tumors is the identification of immunogenic antigens that
render the tumor cells susceptible to cytotoxic T lymphocyte
(CTL)-mediated destruction. The isolation of tumor-reactive T cells
from melanoma patients has led to the identification of some of the
tumor antigens (epitopes) against which CTLs are directed. These
include tyrosinase (Brichard et al., J. Exp. Med. (1993)
178:489-495; Robbins et al., Cancer Res. (1994) 54:3124-3126), MART
1/Melan A (Kawakami et al., J. Exp. Med. (1994) 180:347-352), gp
100 (Bakker et al., J. Exp. Med. (1994) 179:1005-1009; and Kawakami
et al., J. Immunol. (1995) 154:3961-3968) and MAGE (Gaugler et al.,
J. Exp. Med. (1994) 179:921-930). Of these, tyrosinase and MART-1
are nearly universally expressed on melanomas and thus are the
logical choice for adoptive immunotherapy.
[0149] In recent years, significant improvements in survival on the
order of several years have been noted in a small percentage of
melanoma patients undergoing immunological therapy. This includes
active specific immunotherapy with "cancer vaccines" as well as the
use of non-specific boosters of the immune system such as
cytokines, like IL-2, .alpha.-interferon and .gamma.-interferon.
However, the benefit of cytokines is lessened by side effects that
often accompany their use, such as, nausea, and fever.
[0150] Cytolytic T cells (CD8.sup.+) are the main line of defense
against viral infections. CD8.sup.+ lymphocytes specifically
recognize and kill host cells that are infected by a virus.
Theoretically, it should be possible to harness the immune system
to combat other types of diseases including cancer. However, few in
vitro/ex vivo procedures have been available for specifically
activating CTLs. The identification of key melanoma antigens noted
above and a method for specific in vitro activation CTLs described
below now allow testing of the concept of adoptive immunotherapy of
metastatic melanoma.
[0151] All naive T cells require two signals for activation to
elicit an immune response. For CD8.sup.+ lymphocytes (CTLs), the
first signal, which imparts specificity, consists of presentation
to the CD8.sup.+ cell of an immunogenic peptide fragment (epitope)
of the antigen bound to the Class I MHC (HLA) complex present on
the surface of antigen-presenting cells (APCs). This complex is
recognized specifically by a T cell antigen receptor (TCR), which
communicates the signal intracellularly.
[0152] Binding to the T cell receptor is necessary but not
sufficient to induce T cell activation, and usually will not lead
to cell proliferation or cytokine secretion. Complete activation
requires a second co-stimulatory signal(s), these signals serve to
further enhance the activation cascade. Among the co-stimulatory
molecules on antigen-presenting cells, B7 and cell adhesion
molecules (integrins) such as ICAM-1 assist in this process by
binding to CD28 and LFA-1, respectively, on the T cell. When a
CD8.sup.+ cell interacts with an antigen-presenting cell bearing an
immunogenic peptide (epitope) bound by a Class I MHC molecule in
the presence of appropriate co-stimulatory molecule interactions,
the CD8.sup.+ cell becomes a fully activated cytolytic T cell.
[0153] Lymphocyte-mediated cell killing involves a sequence of
biological events beginning with the binding of the CD8.sup.+ CTL
to an antigen-bearing target (tumor) cell by means of the
recognition process described above for T cell activation.
[0154] The interaction between CD8.sup.+ cells and
antigen-presenting cells or target (tumor) cells as described above
is depicted in FIG. 1. The interaction begins with the binding of
antigen in association with an MHC Class I molecule on the APC or
target cell to the T cell antigen receptor (TCR). Accessory
molecules such as lymphocyte function antigens (LFA-1, LFA-2 and
LFA-3), intercellular adhesion molecule 1 (ICAM-1), T cell
co-stimulatory factors (CD2, CD28, B7) enhance cell-cell adhesion
or transduce additional cell activation signals.
[0155] After cell-cell interaction, the CTL kills the target cell
through the action of soluble cytolytic mediators (perforin and
granzymes stored in cytoplasmic granules in the T cell) and a CTL
surface molecule (Fas ligand). After the cytolytic attack, target
cells die by necrosis (membrane perforation and organelle
destruction) or apotosis (chromatin condensation, DNA fragmentation
and membrane blebbing).
[0156] The mechanisms of lymphocyte-mediated cytolysis is
graphically depicted in FIG. 2. In Panel A of FIG. 2, after binding
to the target cell, cytoplasmic granules in the CTL are rapidly
reoriented toward the target cell for release of granules
containing perforin and granzymes into the intercellular space.
These proteolytic enzymes form pores in the plasma membrane of the
target cell eventually leading to cell necrosis. In Panel B, after
binding to the target cell, the level of Fas ligand expression on
the CTL increases. The interaction of Fas ligand and the Fas
receptor on the target cell leads to apoptosis. Proteases such as
CPP32 and others related to IL-1b-converting enzyme (ICE) have been
implicated in the induction of apoptosis. It is possible to use
naturally-occurring antigen-presenting cells, for example,
dendritic cells, macrophages, autologous tumor cells for in vitro
CD8.sup.+ activation. However, the efficiency of activation
following this approach is low. This is because the Class I
molecules of native APCs contain many other types of peptide
epitopes besides tumor epitopes. Most of the peptides are derived
from normal innocuous cell proteins, resulting in a dilution of the
number of active native APCs that would actually be effective
against a tumor (Allison et al., Curr. Op. Immunol. (1995)
7:682-686).
[0157] A more direct and efficient approach to this problem is to
specifically activate CD8.sup.+ cells only with those epitopes
relevant to combating a specific disease, (such as, cancer) or
tumor specific antigens (such as, melanoma-specific antigens). To
this end, an artificial antigen presenting cell is created by
expressing MHC Class I molecules in Drosophila melanogaster (fruit
fly) cells. Since Drosophila does not have an immune system, the
TAP-1,2 peptide transporters involved in loading peptide epitopes
onto class I molecules are absent. As a result, the class I
molecules appear on the Drosophila cell surface as empty vessels.
By incubating these transfected Drosophila cells with exogenous
peptides that bind to the class I molecules, such as, cancer or
tumor specific epitopes, including but limited to, melanoma
specific epitopes, it is possible to occupy every class I molecule
with the same peptide. High density expression of class I molecules
containing a single peptide in these Drosophila APCs permit
generation of cytotoxic CD8.sup.+ T cells in vitro which are
completely specific for the antigen peptide. Methods and procedures
for preparing Drosophila cells are taught in U.S. Pat. No.
5,529,921, issued Jun. 25, 1996 entitled "In Vitro Activation of
Cytotoxic T-Cells Using Insect Cells Expressing Human Class I MHC
and .beta.2-Microglobulin", and U.S. Pat. No. 5,314,813, issued May
24, 1994 entitled "Drosophila Cell Lines Expressing Genes Encoding
MHC Class I Antigens And .beta.2-Microglobulin and Capable of
Assembling Empty Complexes and Methods of Making Said Cell Lines".
In particular, U.S. Pat. No. 5,529,921 discloses at column 26, line
56 to column 28, line 22 various methods of separating out and/or
enriching cultures of precursor cells.
[0158] Additionally, this feature eliminates the need for in vivo
stimulation of the immune system with high doses of various
cytokines. Thereby resulting in a treatment that fore goes the side
effects caused by cytokines. Alternatively under suitable
situations, or conditions, where appropriate and where the subject
can derive benefit, the subject can be treated concurrently with
low level dosages of .alpha. interferon, .gamma.-interferon, and/or
IL-2.
[0159] Eliminating the need for in vivo stimulation with cytokines
provides an improvement to the quality of patient care. Treatment
regimes that include the administration of cytokines to patients
often result in the patient developing flu-like symptoms, such as
nausea, vomiting, and fever. These side reactions are generally not
life threatening, although a particularly severe reaction occurring
in a patient who is already in a weaken condition could result in a
life endangering situation. Another consideration is the adverse
impact such side reactions have on patient acceptance and
compliance of an otherwise beneficial treatment regime. Removing
the need for in vivo stimulation with cytokines results in a
treatment regime that improves the comfort of the patient, and
provides the clinician with an effective method of treatment that
his or her patient is more likely to comply with.
[0160] The utility of this method for adoptive immunotherapy of
tumors has been demonstrated in mice using transfected Drosophila
cells as APCs and CD8.sup.+ cells from the 2C line of T cell
receptor (TCR) transgenic mice. In this system, purified CD8.sup.+
2C cells are highly responsive to in vitro peptides presented by
MHC Class I (Ld)-transfected Drosophila cells also bearing the
co-stimulatory molecules B7-1 and ICAM-1. Transfected Drosophila
cells as a probe for defining the minimal requirements for
stimulating unprimed CD8+ T cells (Cai et al., P. N. A. S. USA
(1996) 93:14736-14741). Alternatively, when un-separated mouse
spleen cells are used as responders in place of purified 2C cells,
the need for co-stimulatory molecules does not apply. In this
instance, the CD8.sup.+ cells in the spleen population receive
"bystander" co-stimulation from activated B cells. Utilizing this
finding, it has been possible to show that MHC Class I
(L.sup.d)-transfected Drosophila cells are able to induce normal
DBA/2 mouse spleen cells to respond to syngeneic P815 mastocytoma
tumor-specific peptides in vitro in the absence of added
lymphokines. Injection of these CTLs into DBA/2 mice bearing P815
mastocytoma led to rapid tumor regression (Sun et al., Immunity
(1996) 4:555-564).
[0161] Procedurally, normal DBA/2 mouse spleen cells were cultured
in vitro with MHC Class I (L.sup.d)-transfected Drosophila cells
loaded with P1A.35-43 peptide, a tumor-specific epitope from the
DBA/2-derived P815 mastocytoma cell line. Lymphocytes harvested
from the cultures after five days displayed strong cytotoxic T
lymphocyte (CTL) activity toward P815 tumor cells in vitro, but
failed to lyse P1024, a mutant cell line of P815 that does not
express P1A.35-43, as shown in FIG. 3, Panel A. When these CTLs
were injected into DBA/2 mice previously inoculated with P815 cells
three days earlier, the tumors grew unimpeded during the first
week, but were subsequently eliminated within the next week, as
shown in FIG. 3, Panel B. Specificity was demonstrated by the
absence of any effect on P815 growth when CTLs were immunized in
vitro against an irrelevant antigen, such as, viral nucleoprotein
peptide, as shown in FIG. 3, Panel B. In summary, major
histocompatibility complex Class I (Ld)-transfected Drosophila
cells induced normal DBA/2 mouse spleen cells to respond to
syngeneic P815 mastocytoma tumor-specific peptides in vitro in the
absence of added lymphokines. Injection of these CTLs into DBA/2
mice bearing P815 mastocytoma led to rapid tumor regression (Wolfel
et al., J. Exp. Med. (1993) 178:489-495).
[0162] Human Studies in Vitro
[0163] Human CTLs from healthy subjects were immunized in vitro
against tyrosinase. Following primary stimulation only with
Drosophila cells, specific lysis of tyrosinase-bearing JY cells was
evident at all CTL effector to JY target ratios tested.
Tyrosinase-specific CTLs from healthy subjects were induced using
the full stimulation/re-stimulation protocol and tested for their
ability to kill the Malme 3M melanoma cell line. With one or two
possible exceptions, specific CTL activity against Malme 3M was
induced in all donors to a varying extent. For the most part,
reactivity toward control Malme 3 tumor cells was minimal. Cells
from melanoma patients were also immunized in vitro against the
tyrosinase epitope to generate CTLs of similar activity and
specificity to those derived from healthy volunteers.
[0164] The use of any natural, or artificial, antigen presenting
cell (APC) system to generate cytotoxic T lymphocytes in vitro is
limited by the antigen specificities these systems are capable of
generating.
[0165] The following APC systems have been utilized to generate
antigen-specific CTL's to single epitopes: 1) human dendritic cells
(DC) pulsed with defined peptides; 2) peripheral blood mononuclear
cells (PBMCs) which have been driven to lymphoblasts and pulsed
with peptides; 3) lymphoblastoid cell lines (LCL) where the natural
peptides are acid-stripped and loaded with the peptides of
interest; 4) Drosophila cells engineered to express empty class I
molecules; and Mouse 3T3 cells transfected with human class I and
co-stimulatory molecules (J. B. Latouche and M. Sadelain, Nature
Biotech (2000) 18:405-409).
[0166] Dendritic cells (DCs) are considered the primary antigen
presenting cell system in humans because of their wide application
in presenting primary antigen cells. Self or foreign proteins are
processed within a DC. The resultant peptide epitopes are presented
by HLA molecules, and are transported to the surface of the DC.
However, it was found that DCs would not consistently generate in
vitro, CTLs directed against four different peptides. This would
have provided CTLs having activity corresponding to each of the
four peptides. In addition, it was also found that the phenotype of
the DC at the time of peptide pulsing, mature or immature, did not
effect the outcome.
[0167] Alternatively, Drosophila cell stimulation usually resulted
in CTLs directed against up to ten different types of peptides.
This provides CTLs that are active to each of the ten peptides.
[0168] The ability of Drosophila cells and DC to elicit CTL
responses were evaluated, initially to a single peptide epitope,
following the standard stimulation protocols for each. In order to
compare DCs and transfected Drosophila cells. Immature DCs were
generated by culturing for one week autologous monocytes in the
presence of IL-4 and GM-CSF. Mature DCs were obtained from immature
DCs by addition of TNF a to the culture medium twenty-four hours
prior to harvesting. DCs (immature and mature) were harvested,
pulsed with peptides and mixed with purified CD8 cells following
the procedure used for the stimulation of CD8 cells and
peptide-pulsed Drosophila cells. Drosophila cells were found to be
generally better stimulators than DC when evaluated for tyrosinase
peptide epitope 689, as shown in FIG. 7. Further, DCs displaying
either the immature or mature phenotype (FIG. 8) were not as
efficient as Drosophila cells in eliciting specific CTL responses
when defined peptides were used to pulse the APCs. This is
particularly surprising, because of the dominant role played by DCs
in the immune system. A comparison study with one donor was
performed, as shown in FIG. 9. Specific killing was generated
against four different peptides when using fly cells as stimulators
whereas immature DCs resulted in marginal specific killing and
mature DCs resulted in specific killing against only one of the
four peptides used for stimulation.
[0169] Preparation of Cytotoxic Lymphocytes
[0170] CD8.sup.+ cells isolated from leukapheresis samples by
positive selection with anti-CD8 antibody are stimulated against
four different melanoma associated peptides presented by Drosophila
cells expressing Human Class I molecules (HLA-A2.1), B7.1, ICAM-1,
LFA-3 and B7.2. CD8.sup.+ cells are re-stimulated for two rounds
with autologous monocytes loaded with the peptide epitope in the
presence of IL-2 and IL-7. CTLs are non-specifically expanded with
OKT3 and IL-2. CTL activity is measured against Malme 3M cells and
purity of CD8.sup.+ T cells is assessed by flow cytometry.
[0171] The manufacturing processes and protocols are done according
to Good Laboratory Practices and Good Manufacturing Practices.
"Good Laboratory Practices" and "Good Manufacturing Practices" are
standards of laboratory and manufacturing practices which are set
by United States Food and Drug Administration, and are readily
known to those of skill in the art. The CTLs are monitored for
identity, viability, CTL activity, sterility, and endotoxin
content.
[0172] A listing of peptide epitopes suitable for use in the
methods of the present invention to treat breast and ovarian
cancers are shown in the following Table 1. It is readily apparent
to those of ordinary skill in the art that a wide variety of
peptide epitopes in addition to those listed in the following Table
1 will also be suitable for use in the methods of the present
invention to treat breast and ovarian cancers, provided that such
peptides are T cell epitopes.
3TABLE 1 Identified HLA-A2.1 Restricted Epitopes for Tumor
Associated Antigens as Targets for Breast and Ovarian Cancers
Target Sequence HLA Peptide (residues) Name PRI # AKA (SEQ ID NO:)
Binding Prediction Her-2/neu 789-797 826 E90 CLTSTVQLV 160 (SEQ ID
NO:7) 48-56 827 D113 HLYQGCQVV (SEQ ID NO:13) 369-377 835 E75
KIFGSLAFL 481 (SEQ ID NO:8) 654-662 837 GP2 IISAVVGIL (SEQ ID
NO:14) 650-658 838 GP1 PLTSIISAV (SEQ ID NO:15) 773-782 861
VMAGVGSPYV (SEQ ID NO:16) 851-859 862 E89 VLVKSPNHN 118 (SEQ ID
NO:17) 971-979 863 C85 ELVSEFSRM (SEQ ID NO:18) AES Amino enhancer
of the split Notch G128-135 893 G76 GPLTPLPV (SEQ ID NO:19) MUC-1
Mucin 950-958 908 1.1 STAPVHNV (SEQ ID NO:20) CEA Carcinoembryonic
Ag 571-579 879 CAP-1 YLSGANLNL (SEQ ID NO:21) FBP Folate binding
protein 191-199 914 E39 EIWTHSYKV (SEQ ID NO:22) C-Lectin MESM,
RELP 8-16 C8 KMASRSMRL CTL Activity (SEQ ID NO:9) 77-86 C77
SILSLKEAST CTL Activity (SEQ ID NO:23) NY-ESO-1 157-165C 894
SLLMWITQC native (SEQ ID NO:24) 157-165V 906 SLLMWITQV modified
(SEQ ID NO:25) 155-163 913 QLSLLMWIT (SEQ ID NO:26) Pec60 20 P20
ALALAALLVV CTL Activity (SEQ ID NO:10) 25 P25 ALLVVDREV CTL
Activity (SEQ ID NO:11) CA-125 157-165 900 YLETFREQV 38 (SEQ ID
NO:27) 255-263 902 VLLKLRRPV 88 (SEQ ID NO:28) 337-345 901
GLQSPKSPL 21 (SEQ ID NO:29) 546-554 903 ELYIPSVDL 5 (SEQ ID NO:30)
898-906 899 KALFAGPPV 13 (SEQ ID NO:31) 414-422 910 FMWGNLTLA 315
(SEQ ID NO:32) MAGE-3 271-279 909 FLWGPRALV (SEQ ID NO:33)
Telomerase hTRT 540-548 907 ILAKFLHWL (SEQ ID NO:34) 865-873 911
RLVDDFLLV (SEQ ID NO:35) G250 245-262 912 HLSTAFARV (SEQ ID
NO:36)
[0173] The following examples are provided for the purpose of
illustrating the present invention, but do not limit the present
invention to the content of the examples.
EXAMPLE 1
Manufacture of Drosophila Antigen-Presenting Cells
[0174] The Schneider S2 cell line was prepared from Drosophila
melanogaster (Oregon-R) eggs according to published procedures and
has been deposited with the American Type Culture Collection (CRL
10974). S2 cells are grown in commercial Schneider's Drosophila
medium supplemented with 10% fetal bovine serum.
[0175] The pRmHa-3 plasmid vector for expressing MHC Class I and
co-stimulatory proteins in S2 cells was derived from the pRmHa-1
expression vector constructed as described in the literature. It
contains a metallothionein promoter, metal response consensus
sequences and an alcohol dehydrogenase gene bearing a
polyadenylation signal isolated from Drosophila melanogaster.
[0176] Complementary DNAs for Transfection were Prepared as
Follows:
[0177] HLA-A2.1 and .beta.-2 microglobulin: Reverse
transcription-PCR from K562 cells using primers derived from the
published sequence
4 B7.1: Reverse transcription-PCR from K562 cells using primers
derived from the published sequence ICAM-1: Reverse
transcription-PCR from K562 cells using primers derived from the
published sequence B7.2: Reverse transcription-PCR from HL-60 cells
(ATCC CCL-240) using primers derived from the published sequence
LFA-3: Reverse transcription-PCR from HL-60 cells (ATCC CCL-240)
using primers derived from the published sequence
[0178] Complementary DNAs were individually inserted into the
pRmHa-3 vector. S2 cells were transfected with a mixture of
HLA-A2.1, B7.1 and ICAM-1 plasmid DNAs and the phshneo plasmid
using the calcium phosphate precipitation method. Stably
transfected cells were selected by culturing in Schneider's medium
containing geneticin. Twenty-four hours before use, expression of
the transfected genes was induced by addition of CuSO.sub.4. The
level of expression was assessed by flow cytometry using
anti-HLA-A2.1, anti-B7.1 and anti-ICAM-1 antibodies. HLA expression
by greater than 30% of the cells is necessary for efficient in
vitro activation of CD8.sup.+ lymphocytes.
[0179] Isolation of Human CD8.sup.+ Cells
[0180] CD8.sup.+ cells are isolated from leukapheresis samples by
positive selection using the Dynabeads.TM. isolation procedure
(Dynal). An anti-human CD8 mouse monoclonal antibody (50 .mu.g/ml
in human gamma globulin [Gammagard.RTM.]) is added to washed cells
in Dulbecco's PBS supplemented with 1% human serum albumin
(Baxter-Hyland) and 0.2% Na citrate. After incubation at 4.degree.
C. for forty-five minutes with gentle mixing, the cells are washed
and re-suspended in the same buffer containing Dynal magnetic beads
(Dynabeads.TM.) coated with sheep anti-mouse IgG at a bead to cell
ratio of 1:1. The cells and beads are placed into a sterile tube
and gently mixed at 4.degree. C. for forty-five minutes. At the end
of this time, the antibody-bound cells are removed magnetically
using the MPC-1.RTM. separator according to the manufacturer's
instructions (Dynal). Dissociation of the CD8 cell-bead complex is
achieved by incubation at 37.degree. C. for forty-five minutes in
the presence of CD8 peptide.sub.59-70 (AAEGLDTQRFSG; SEQ ID NO:
12). Free beads are removed magnetically and the CD8 cells are
counted and analyzed by flow cytometry to evaluate purity. Recovery
of CD8.sup.+ cells is typically greater than 80%. Table 1
summarizes the cell composition of fourteen separate CD8.sup.+
preparations from normal human PBMC preparations by positive
selection with anti-CD8 antibody.
5TABLE 2 Purification of CD8+ Cells by Positive Selection Analyzed
by Flow Cytometry PBMC POST SELECTION CELL TYPE Mean % (Range) Mean
% (Range) CD8 T cells 15% (7-24) 82% (56-95) CD4 T cells 36%
(14-52) 2% (0.1-10) CD14 Monocytes 15% (7-26) 0.8% (0.2-2) CD15
Neutrophils 12% (8-21) 0.6% (0.1-3) CD19 B cells 2% (0.4-7) 3%
(0.5-9) CD56 NK cells 6% (2-17) 6% (0.1-20)
[0181] In Vitro Immunization of Purified Human CD8.sup.+ Cells
[0182] Primary Stimulation
[0183] Transfected Drosophila S2 cells are incubated in Schneider's
medium (10.sup.6 cells/ml) supplemented with 10% fetal calf serum
and CuSO.sub.4 at 27.degree. C. for twenty-four hours. Cells are
harvested, washed and re-suspended in Insect X-press medium
(BioWhittaker) containing 100 .mu.g/ml human
tyrosinase.sub.369-377. Following incubation at 27.degree. C. for
three hours, the S2 cells are mixed with CD8.sup.+ cells at a ratio
of 1:10 in RPMI medium (Gibco) supplemented with 10% autologous
serum. The cell mixture is incubated for four days at 37.degree. C.
during which the Drosophila cells die off. On Day five, IL-2 (20
U/ml) and IL-7 (30 U/ml) are added to selectively expand the
tyrosinase-specific CTL population.
[0184] Re-Stimulation
[0185] Frozen, autologous, CD8-depleted PBMCs, obtained at the time
of leukapheresis, are thawed, washed and re-suspended at 10.sup.6
cells/ml in RPMI medium containing 10% autologous serum (as a
source of .beta.2 microglobulin) and 20 .mu.g/ml
tyrosinase.sub.369-377. Following .gamma.-irradiation (5,000 rads),
the cells are incubated at 37.degree. C. for two hours.
Non-adherent cells are removed by washing with Dulbecco's PBS.
Adherent monocytes are loaded with the tyrosinase epitope by
incubation for 90 minutes in Hepes-buffered RPMI medium containing
10% autologous serum and 10 .mu.g/ml tyrosinase.sub.369-377. The
supernatant is removed and the Drosophila-activated CD8.sup.+ cell
suspension (3.times.10.sup.6 cells/ml in RPMI medium with 10%
autologous serum) is added at a ratio of 10 CD8.sup.+ cells to 1
adherent monocyte. After three to four days of culture at
37.degree. C., IL-2 (20 U/ml) and IL-7 (30 U/ml) are added with a
medium change to selectively expand the tyrosinase-specific CTL
population.
[0186] Non-specific Expansion
[0187] Effector cells are non-specifically expanded by culturing
them in RPMI medium supplemented with autologous serum, anti-CD3
monoclonal antibody (OKT.RTM.3), IL-2 and .gamma. irradiated
autologous PBMCs.
[0188] Assays for Activity and Purity
[0189] CTL Assay
[0190] Malme 3M cells are used as target cells in a .sup.51Cr
release assay. 5.times.10.sup.6 Malme 3M cells in RPMI medium
containing 4% fetal calf serum, 1% HEPES buffer and 0.25%
gentamycin are labeled at 37.degree. C. for one hour with 0.1 mCi
.sup.51Cr. Cells are washed four times and diluted to 10.sup.5
cells/ml in RPMI with 10% fetal bovine serum (HyClone). In a
96-well microtiter plate, 100 .mu.l effector CTLs and 100 .mu.l
peptide-loaded, .sup.51Cr-labeled Malme 3M target cells are
combined at ratios of 100:1, 20:1 and 4:1 (effector: target). K562
cells are added at a ratio of 20:1 (K562:Malme 3M) to reduce
natural killer cell background lysis. Non-specific lysis is
assessed using the non-tumor HLA-A2.1 fibroblast cell line, Malme
3. Controls to measure spontaneous release and maximum release of
.sup.51Cr are included in duplicate. After incubation at 37.degree.
C. for six hours, the plates are centrifuged and the supernatants
counted to measure .sup.51Cr release.
[0191] Percent specific lysis is calculated using the following
equation:
cpm sample-cpm spontaneous release/cpm maximum release-cpm
spontaneous release.times.100
[0192] Flow Cytometry.
[0193] CD8.sup.+ cells, before and after in vitro activation, were
analyzed for a number of cell surface markers using fluorescent
monoclonal antibodies and FACS analysis. Results from a typical
activation protocol using cells from a healthy donor is shown in
Table 2.
6TABLE 3 Flow Cytometry Analysis of In Vitro Activated CD8+ Cells
PRE-ACTIVATION POST-ACTIVATION MARKER/CELL TYPE Mean % Mean % CD8 T
cell 98 99 TCR.alpha..beta. T cell receptor 98 92 CD 44 lymph node
horming 91 99 receptor CD45RO memory T cell 58 88 CD45RA 41 31
CID62L HEV homing 24 38 receptor CD56 NK cell 1 11 CD25 activated T
cell 0.1 29
[0194] In addition to activity and purity, CTL preparations will be
assayed for sterility and endotoxin content.
7 REAGENTS REAGENT SUPPLIER GRADE NOTES rh IL-2 Chiron USP sterile
solution rh IL-7 Genzyme Research lyophilized, sterile solution
human tyrosinase.sub.369-377 Research Dynabeads .RTM. M-450 Dynal
GMP sheep anti-mouse IgG magnetic beads human serum albumin Baxter
USP sterile, non-pyrogenic hepatitis virus-free, 25% solution fetal
bovine serum Gemini Research sterile, BSE-, endotoxin-,
mycoplasma-free Gammagard .RTM. Baxter USP sterile, human immune
globulin solution for injection anti-CD8 antibody Research mouse
anti-human CD8 monoclonal antibody CD8 peptide.sub.59-70 Research
release of CD8+ cells from magnetic beads W6/32 ATCC Research mouse
anti-human HLA-A, B, C monoclonal antibody
[0195]
8 CELL LINES CELL LINE SUPPLIER NOTES Drosophila S2 ATCC CRL 10974
M3 UCSD Non-HLA-A2.1 human melanoma Malme 3 ATCC Normal skin
fibroblast from a melanoma patient Malme 3M ATCC Metastatic
melanoma from lung (same patient as Malme 3) M14 UCSD HLA-A2.1
human melanoma K562 ATCC human erythroleukemic cell line; target
for NK cells JY cells ATCC EBV-transformed, human B cell line
expressing HLA- A2.1 and B7 P815 and P1024 ATCC DBA/2 mouse
mastocytoma cell lines Jurkat A2.1 ATCC acute T cell leukemia
transfected with human HLA- A2.1 ATCC: American Type Culture
Collection
EXAMPLE 2
Trial of Cytotoxic T Cell Infusions Against Melanoma
[0196] Purpose of Trial
[0197] This example teaches the effectiveness of cytotoxic T Cell
infusions in the treatment of melanoma as assessed according to the
following factors:
[0198] 1. safety and toleration of re-infused autologous CTLs after
in vitro immunization;
[0199] 2. kinetics of infused CTLs in the systemic circulation
factoring in limiting dilution analysis;
[0200] 3. whole body disposition of CTLs by radioscintography;
[0201] 4. cell composition of biopsied nodules by immunohistology
(CTLs, TH, NK, B cells); and
[0202] 5. regression of measurable lesions and duration of response
over two months.
[0203] Patient Populations
[0204] Eligibility for treatment required patients to have
histologically-documented, unresectable malignant melanoma that was
measurable or evaluable, and the HLA-A2 haplotype. Pretreatment
evaluation included radiologic evaluation of the brain by MRI or CT
scan, CT scanning of the chest and abdomen, and physical
examination, especially of the skin and lymph nodes. The total
number of patients treated was fifteen (nine male and six female).
The ages ranged from 33 to 75 years with an average of 58 years.
The average duration of metastatic disease was 1.5 years. A
pretreatment skin test to determine whether a state of anergy
existed was performed on 14/15 patients with 5/14 testing negative
for all seven of the common antigens evaluated. Patients were
screened for the HLA-A2 haplotype by FACS analysis with an HLA-A2
specific monoclonal antibody (BB7.2). Subtyping was performed by
PCR analysis. All, but one of the patients, were HLA-A*0201, the
exception (patient 08) was HLA-A*0205.
[0205] Treatment with Ex Vivo Generated Autologous CTLs
[0206] Fifteen patients were treated under this clinical protocol.
All patients received, at least, a single infusion of autologous
CTLs. The number of cycles and the dose of cells administered to
each patient are summarized in Table 1. The number of cells
generated in vitro was dependent on patient-related factors such as
the numbers of PBMCs isolated from the aphaeresis procedure and the
number of CD8.sup.+ T cells present in each PBMC preparation. Since
all of the cells generated in vitro were re-infused into the donor,
doses administered to each patient were necessarily varied. In an
attempt to normalize the doses between patients, a calculated
"potency" score was recorded for each dose. The value was obtained
by multiplying the total number of cells by the lytic activity
obtained with peptide-loaded target cells. Doses of T cells infused
ranged from a minimum of 4.times.10.sup.7 (patient 08) to a maximum
of 3.2.times.10.sup.9 (patient 13). Patients were entered into a
second, third or fourth cycle of treatment based on their clinical
status at the end of each cycle. The number of PBMCs obtained from
the aphaeresis samples tended to be lower in patients undergoing
additional cycles, especially if the start of the subsequent cycle
was close to the end of the previous one. This is attributed to
persistent lymphopenia due to the IFN.alpha.-2b administered during
the previous cycle. The total number of naive CD8.sup.+ T cells
isolated was dependent on its percentage in each of the PBMC
preparations. The percent of CD8.sup.+ T cells varied between 8% to
31% among the patients. The obtained expansion factor also
contributed to the final cell numbers and ranged from 0.1-6.0 fold.
The procedure for generating CTLs ex vivo is taught in the
Specification and Example 1, above.
[0207] Up-Regulation of Class I and Melanoma-Associated Antigens in
Response to IFN.alpha.-2b
[0208] In an attempt to enhance the ability of the antigen-specific
CTLs to lyse melanoma cells in vivo, low dose IFN.alpha.-2b was
administered for five consecutive days prior to the CTL infusion,
and thrice weekly for an additional four weeks. One way to measure
an in vivo response to the cytokine is to evaluate biopsies
obtained at serial time points by immunohistochemical analysis for
positive staining with specific antibodies. Serial biopsies were
obtained in one patient with multiple skin lesions (patient 04) for
evaluation of both class I and antigen expression. The biopsies
indicated Class I and MART-1 expression were weakly positive prior
to any treatment (biopsy A). Following five days of subcutaneous
injections of 10 MU/m.sup.2, a dramatic increase in these two
markers was noted (biopsy B). For tyrosinase and gp100,
immunohistochemical staining was negative to weakly positive,
respectively in the pretreatment samples (biopsy A). After the
initial five-day IFN.gamma. dose, and thirteen additional
treatments, expression of these later antigens was increased in the
stained tissue samples (biopsy C).
[0209] Antigenic Specificity of Ex Vivo-Generated CTLs
[0210] CTLs generated from all patients were evaluated on the day
of release against peptide-loaded T2 targets, an HLA-A2 melanoma
cell line (Malme3M) and an autologous melanoma line, if biopsy
material was available to establish a line. Each prepared dose of
cells was evaluated for its cytolytic activity. Peptide-loaded T2
cells, presenting either each peptide alone, or all four peptides
simultaneously, were used to determine the specificity of the CTL
response generated for each patient. The ability to lyse
endogenously-expressed, melanoma-associated antigen-bearing cells
was assessed with an HLA-A2 matched line or an autologous tumor
line. In addition to cytolytic activity, antigen-specificity was
evaluated with an established method for detecting intracellular
gamma interferon production, made in response to a specific peptide
stimulus. The CTLs generated at the end of the ex vivo protocol
were evaluated by this method. The percent of cells specific for
each of the peptides was recorded individually. The total number of
specific cells in each bulk CD8 culture from patient 13 was
calculated by adding each of the peptide specificities detected in
that population of T cells. An increase in the total number of
specific cells could be detected with each successive treatment
cycle.
[0211] Detection of CD8 and CD4 Cells Infiltrating Tumor Biopsies
Post-CTL Therapy
[0212] Biopsy samples from all patients prior to, during and after
treatment would have been ideal. However, the experimental
conditions allowed for biopsy samples from only a limited number of
patients. Tumor tissue was obtained from five of the fifteen
patients enrolled in the study. In two patients (patients 08 and
13) biopsy samples were available at five and six weeks post T cell
therapy, respectively. Examination of the tissue samples revealed
the presence of both infiltrating CD8 and CD4 cells. One of the
tumor samples was taken from a skin lesion in the occipital region
of the scalp, which increased in size by the time of the follow up
examination, four weeks after a second infusion of T cells. The
biopsy revealed necrosis of the tissue that was heavily infiltrated
with lymphocytes. The other biopsy was from the head of a femur
bone, removed during hip replacement surgery. The skin lesion from
patient 08 was strongly positive (4+) for both a general class I,
and a specific HLA-A2 marker. Tyrosinase and gp100 were weakly
positive (1+ and 2+, respectively), while MART-1 was negative in
this same sample. Regions of the biopsy from patient 13 were also
necrotic, with more heterogeneous staining; distinct populations of
tumor cells lacking expression of the HLA-A2.1 molecule, and one or
more of the MAAs. However, intact tissue regions revealed strong
class I (4+), and all of the melanoma-associated antigens. The
lymphocytic infiltrations in this later sample appeared to surround
the tumor nodules rather than to deeply infiltrate them. However,
the highest percentage of cells directly associated with the tumor
were CD8 cells. The lack of pretreatment biopsy samples from both
of these patients prevented a confirmation of similar types of
infiltrating cells in tissue samples prior to treatment.
[0213] CT Scans Post-T Cell Therapy Confirm an Objective
Response
[0214] CT scans were part of the pretreatment screening criteria
and the post treatment follow-up examination. Patient 10 received a
single infusion of 8.times.10.sup.8 CTLs (Jul. 7, 1999) five weeks
after the pretreatment scan (Jun. 23, 1999). When a CT scan of the
chest was repeated one month after the infusion (Aug. 27, 1999), a
dramatic decrease in the size of a lung lesion was noted.
Similarly, patient 14 underwent a chest CT scan as part of the
enrollment process (Sep. 10, 1999), three and one-half weeks before
a first infusion with 6.6.times.10.sup.8 cells (Oct. 5, 1999). A
follow-up CT scan (Jan. 7, 1999), one month after a second infusion
with 11.5.times.10.sup.8 cells, revealed dramatic shrinkage in
three separate lesions. Patient 13 also had an objective response
as measured in pre and post CT scans. Paratracheal adenopathy went
from 7.8 cm.sup.2 (pre-study) to 4.4 cm.sup.2 after cycle I, and
disappeared following cycle II.
[0215] Presence of an Anergic State Did Not Preclude Ability to
Generate CTLs or Prevent a Clinical Response
[0216] Most of the patients treated under this protocol had
received previous medical intervention. A pretreatment skin test
was performed to determine if an anergic response to a panel of
seven common antigens correlated with either an inability to
generate CTLs ex vivo, or prevent a documented clinical response.
The ability to generate CTLs ex vivo did not correlate with the
patient's pretreatment skin test results. It should be noted that
patients 03 and 04 (both mixed responders) had repeat skin tests
prior to the start of the second cycle and remained anergic.
EXAMPLE 3
Generation of HER-2neu Specific CTLs Capable of Lysing Breast &
Ovarian Tumor Cells
[0217] We were interested in applying our CTL-generation technology
to other tumor types to determine if all forms of cancer can be
targeted with this approach. HER-2/neu is a proto-oncogne with
homology to EGFR that is amplified and over-expressed in many human
cancers, largely adenocarcinomas of the breast, ovary and colon. It
is often associated with aggressive disease and can be an indicator
of a poor prognosis. It has been studied in several clinical trials
as a possible target for these types of cancers.
[0218] In the early 1990's HER-2/neu HLA-A2.1 restricted peptide
epitopes were identified either by computer-assisted peptide
binding algorithms or by mapping CTLs isolated from ascites of
ovarian cancer patients (Table 3).
9TABLE 3 HLA-A2.1-Restricted HER-2/neu Peptides HER-2/neu Sequence
Peptides PRI # Other ID# Location (SEQ ID NO) Reference 48-56 827
D113 EC HLYQGCQVV Disis et al., 1994 (SEQ ID NO: 13) 369-377 835
E75 EC KIFGSLAFL Fisk et al., 1995 (SEQ ID NO: 8) 650-658 838 GP1
TM PLTSIISAV Fisk et al., 1995 (SEQ ID NO: 15) 654-662 837 GP2 TM
IISAVVGIL Peoples et al., 1995 (SEQ ID NO: 14) 773-782 861 N/A IC
VMAGVGSPYV Lustgarten et al., 1997 (SEQ ID NO: 16) 789-797 826 E90
IC CLTSTVQLV Disis et al., 1994 (SEQ ID NO: 7) 851-859 862 E89 IC
VLVKSPNHV Disis et al., 1994 (SEQ ID NO: 17) 971-979 863 C85 IC
ELVSEFSRM Fisk et al., 1995 (SEQ ID NO: 18)
[0219] All of the peptides were synthesized, given an
identification number (PRI#) and evaluated for the ability to
generate CTLs ex vivo utilizing the same method we employed for
melanoma-associated, T cell peptide epitopes. CD8 cells were
isolated from normal donors to determine the ability to routinely
generate CTLs ex vivo with Drosophila cells loaded with known CTL
peptide epitopes. Peptides 826, 835, 861 and 863 had the highest
frequency of CTL generation (Table 4).
10TABLE 4 Frequency of HER-2/neu CTL Generation in Normal Donors
Donor 826 827 835 837 838 861 862 863 193 + + 194 + - + - - + - +
195 + + + + 196 + - + - - + 197 + - + - + + - + 198 - - + - + + - +
207 + + + + 212 + + + + 218 + + + + 232 - + + - 233 + + + + 241 + +
243 + +
[0220] While transfected Drosophila cells have the unique ability
to present up to ten different peptide epitopes (FIG. 10), we
selected the four HER-2 peptides 826, 835, 861 and 863 due to the
frequency of generating CTLs to these peptides ex vivo. These four
different HER-2 peptides represent weak to moderate binders to the
HLA-A2.1 molecule presented on the surface of the transfected
Drosophila cells. We tend to include A2 binders that are weak, as
our experience in with melanoma-associated peptides suggests that
weak class I binders generally generate potent CTLs which recognize
tumor cells, if indeed they represent native T cell epitopes. The
majority of the tumor-associated proteins that we target are
self-antigens and such would be expected to have the high affinity
for the class I molecule that is seen with viral peptides. The low
to moderate binders generally generate CTLs that lyse the tumor
very efficiently. This was demonstrated with the MART-1 peptide
which is a low affinity binder on the Drosophila cells (FIG. 3),
yet represents an epitope that routinely generate potent CTLs
capable of lysing both peptide-loaded target cells (T2), or more
importantly, melanoma cells (Malme3M) (FIG. 12).
[0221] HER-2/neu is a member of the EGF-R family and functions as a
growth factor receptor. HER-2 protein is expressed during fetal
development in humans. In adults, the protein is weakly detectable
in epithelial cells of many normal tissues. In normal cells the
HER-2 gene is present as a single copy. Amplification of the gene
and/or over-expression of the associated protein has been
identified in many human cancers including breast, ovarian,
uterine, stomach and adenocarcinoma of the lung. Sequence
differences between HER-2 and EGF-R receptor are noted in Table 5.
Three of the four HER-2 peptides we have evaluated have three or
more amino acids changes between the two proteins. A single amino
acid change is sufficient to discriminate between the two
proteins.
11TABLE 5 HER-2/neu Versus EGF-R SEQUENCE PROTEIN PEPTIDE # (SEQ ID
NO) # CHANGES HER - 2/neu 835 KIFGSLAFL 5 EGFR (SEQ ID NO: 8)
SISGDLHII (SEQ ID NO: 37) HER - 2/neu 861 VMAGVGSPYV 5 EGFR (SEQ ID
NO: 16) VAASVDNPHV (SEQ ID NO: 38) HER - 2/neu 863 ELVSEFSRM 3 EGFR
(SEQ ID NO: 18) ELIIEFSKM (SEQ ID NO: 39) HER - 2/neu 826 CLTSTVQLV
1 EGFR (SEQ ID NO: 7) CLTSTVQLI (SEQ ID NO: 40) HER - 2/neu 689-697
RLLQETELV 1 EGFR (SEQ ID NO: 41) RLLQERELV (SEQ ID NO: 42)
[0222] Once the CTLs have been generated after the four-week ex
vivo stimulation protocol, we evaluated whether peptide specific
cells were present using HLA-A2.1 tetrameric molecules prepared
with the immunizing peptides. As demonstrated in FIG. 13, the
ability to generate peptide-specific CTLs was donor-dependent. In
Panel A (donor 261), the donor made a strong CTL response to
peptide 835 (37.55%). In Panel B (donor 262), peptide-specific CTLs
can be detected with both the 835 and 861 tetrameric molecules
(3.6% and 15.1%, respectively). This supports the use of multiple
peptides to guarantee peptide-specific CTLs at the end of the
stimulation protocol. This ex vivo protocol allows one to generate
multiple-specific CTLs relatively easily.
[0223] Anti-Peptide and Anti-Tumor Responses
[0224] After the completion of the full ex vivo protocol, the CTLs
generated were evaluated for antigen-specificity. To generate the
CTLs, on Day 0 Drosophila cells were loaded with a combination of
the four HER-2 peptides. At the end of the four-week ex vivo
stimulation protocol, the bulk CD8 culture was evaluated for
antigen-specificity. T2 cells loaded with each of the immunizing
peptides were used as target cells. In FIG. 14, a typical response
is depicted. The bulk culture contains specificity for each of the
four HER-2 peptides. The anti-tumor response was assessed on an
ovarian tumor cell line (ATCC; HTB-77). When a target cell line is
not HLA-A2.1-restricted, we transfected the cell line to have a +/-
assay system. When HLA-A2.1 was transfected into the HTB-77 line,
an enhanced killing by CD8 effector cells was noted (FIG. 15,
Panels A to D). HER-2 specific effectors, representing the
individual peptides were evaluated to confirm the presentation of
each of the peptide epitopes on this tumor cell line.
[0225] A breast adenocarcinoma cell line (ATCC; HTB-131),
transfected with HLA-A2.1 was also evaluated for the ability to
demonstrate tumor lysis with the HER-2 specific peptide effectors.
CTLs specific for peptide 861 could lysis this tumor cell line when
transfected with HLA-A2.1 (FIG. 16).
[0226] IFN.gamma. Treatment Reguired for Tumor Cell Lysis
[0227] The HTB-77/A2.1 cell line requires a pretreatment with
IFN.gamma. to demonstrate peptide-specific lysis. The cells were
treated with 500 U/ml of IFN.gamma. (specific activity of 25 ng/ml)
for twenty-four hours prior to the initiation of the
.sup.51Cr-release assay. In FIG. 17, the addition of the IFN.gamma.
resulted in enhanced lysis of the HLA-A2.1 transfected cell line.
To determine the effect of this dose of IFNg on the surface
expression of both HLA-A2.1 and HER-2, a FACS analysis was
performed to determine the levels of these molecules after both
twenty-four and forty-eight hours of induction. FIG. 18, Panels A
and B depict the FACS analysis results. In Panel A, there was no
enhancement of the HER-2 molecule on the surface of the HTB-77
cells at twenty-four and forty-eight hours after induction with
IFNg. In the HLA-A2.1 transfected cells, neither HER-2 nor HLA-A2.1
demonstrated an increase in surface level of expression after a
similar treatment protocol. What was noted was an increase in the
level of TAP-1 expression, as well as HLA-DM and -DR, Cathepsin S
and D and Caspase 5, when the mRNA levels were evaluated by
microarrary DNA chip analysis (FIG. 19). This would explain why
there is an enhance killing of the HTB-77/A2.1 cells in the
presence of IFN.gamma.. An up-regulation of this particular
molecule would result in more efficient processing of the HER-2
molecule, allowing better presentation of the peptides of
interest.
[0228] Peptides
[0229] Synthetic peptides were made by standard Fmoc chemistry
using a peptide synthesizer (Gilson Company, Inc.) All peptides
were purified to >95% purity by reverse-phase HPLC on a C-8
column. Purity and identity were established using a mass
spectrometer with electrospray ionization. Melanoma-associated
peptides included: peptide 819 was MART-1 specific (AAGIGILTV SEQ
ID NO:6), 817 and 853 were both gp100 peptides (ITDQVPFSV SEQ ID
NO:4 and KTWGQYWQV SEQ ID NO:5, respectively), tyrosinase-specific
peptides were 689 and 792, with 792 representing the post
translational modified version (YMDGTMSQV SEQ ID NO:2) of the
native sequence (YMNGTMSQV SEQ ID NO:1) represented by peptide 689.
Peptides 826 ( CLTSTVQLV SEQ ID NO:7) and 835 (KIFGSLAFL SEQ ID
NO:8) represented HER-2/neu sequences from the intracellular and
extracellular domains, respectively of the p185 protein.
Pec60.sub.20 (ALALAALLVV SEQ ID NO:10) Pec60.sub.25 (ALLVVDREV SEQ
ID NO:11) were overlapping sequences representing a mucinous
protein detected in ovarian tumor lines. C-lectin also was a
protein detected in ovarian tumor cell lines and a peptide from its
sequence (C-lectin.sub.8) is represented by KMASRSMRL SEQ ID
NO:9.
[0230] In Vitro Cytotoxicity Assay
[0231] Standard .sup.51Cr-release assays were performed to
determine CTL effector cell recognition of melanoma-associated
peptide epitopes loaded onto T2 cells. Harvest 3.times.106 T2 cells
were grown in RPMI+10% FBS (media). 0.1 mCi of .sup.51Cr was added
and incubated at 37.degree. C. in a water bath. Labeled cells were
added to 10 ml of 4% wash (RPMI+4% FBS) and pellet, washed two
additional times, and re-suspended in media to a final
concentration of 0.2.times.106/mL to record radioactivity of
spontaneous versus detergent lysed cells. The cells were pulsed
with the appropriate peptide(s) at 20 .mu.g/mL for thirty minutes.
50 .mu.L was added to each 96-well plate each containing CD8
effector cells at 10, 2, 0.4, and 0.08.times.10.sup.6/mL, which was
incubated at 37.degree. C. for six hours, spun and harvested for
supernatant.
[0232] Flow Cytometry and Tetramer Staining
[0233] The cells were labeled with FITC- or PE conjugated
monoclonal antibodies by incubation at 4.degree. C. for 30 minutes
in FACS buffer (1% BSA, 0.02% NaN.sub.3 in PBS), followed by a wash
in the same buffer. Cells were fixed in 0.5% formaldehyde prior to
data acquisition and analysis on a FACScan flow cytometer (Becton
Dickinson) with its CellQuest software. Nonspecific staining was
measured with the same secondary antibody used to label purified
primary antibodies, or an isotype-matched control when the primary
antibodies were directly labeled. Tetrameric staining was performed
with HLA-A2.1 specific HIVgag tetrameric molecules (Beckman
Coulter) harboring the sequence SLYVTVATL SEQ ID NO:43 as a
negative control. HER-2 specific tetramers were made with the
sequences CLTSTVQLV (826 SEQ ID NO:7), KIFGSLAFL (835 SEQ ID NO:8),
or VMAGVGFSPYV (861 SEQ ID NO:16) peptides. PE-labeled tetrameric
HLA-A2.1-peptide complexes were used in conjunction with
fluorescein isothicyante (FITC)-labeled anti-human CD8a (BD
PharMagin) monoclonal antibodies to stain epitope-specific CD8+ T
cells as described in package insert. Samples were analyzed by
two-color flow cytometry on a Becton Disckenson FACScan, and gated
CD8+ T cells were examined for staining with tetrameric
HLA-A2.1-peptide complexes.
EXAMPLE 4
Generation of Additional Breast and Ovarian Specific CTLs with This
Ex Vivo Stimulation Protocol
[0234] We have demonstrated the ability to generate CTL responses
to all known HLA-A2.1-restricted peptide epitopes for several tumor
antigens of different tumor origins. Our initial studies focused on
melanoma where we were able to demonstrate objective clinical
responses in patients treated with CTLs specific for four different
peptide epitopes specific for the MART-1, gp100 and tyrosinase
melanoma-associated proteins [Richards et al., Amer. Soc. Clin.
Oncol., San Francisco, Calif. (2001, May)].
[0235] To extend the ability to raise CTLs to other tumor antigens
present in a wide variety of other cancers we have selected
published and novel sequences to tumor antigens common to several
different tumor types. These include AES, MUC-1, CEA, FBP,
C-Lectin, NY-ESO-1, Pec60, CA-125, MAGE-3, telomerase and G250.
Table 7 describes these antigens, the frequency of expression and
the cancers, which express them. The frequency of response to these
peptides with our ex vivo stimulation protocol is listed in Table
6.
12TABLE 6 Frequency of Response to Breast and Ovarian Peptide
Epitopes in Normal Donors Donor 879 893 894 899 900 901 902 903 906
907 908 909 910 911 912 913 914 248 + + + 249 + + + 250 + - + 251 -
- + 252 + - + 253 - - + + 254 + - + + 255 - + + - + + + + 256 + - +
+ + + - + 257 + + + 259 + - - 260 - - - - + 261 + + + + 262 + + - +
- 265 + + + + - - - - + + + -
[0236]
13TABLE 6 Tumor Antigen Descriptions Antigen Description CA-125
Cancer Antigen 125 is an epithelial cell marker expressed by
ovarian tumors and some ovarian cell lines. About 85% of ovarian
cancer patients have an increased serum CA125 and is therefore
commonly used as a serum tumor marker. (Cancer Letters (1999, Oct.)
145(1-2) pg. 133-141) MUC-1 Mucin is a transmembrane glycoprotein
expressed on both normal and malignant epithelium. The
underglycosylated form of MUC-1 over-expressed on the cell surface
of many human adenocarcinomas such as breast and ovarian cancer, as
well as hematological malignancies including multiple myeloma and
B-cell Lymphoma. (Blood (1999, June) 93(12) pg. 4309-4317) G250 A
renal cell carcinoma associated antigen expressed in 85% of RCC's
but not normal kidney tissue. It is identical to the
tumor-associated antigen MN/CAIX which is expressed in about 50% of
invasive breast cancers. (Cancer Research (1999, Nov.) ; 59(21) pg.
5554-5559) FBP Folate binding protein is a receptor involved in
folate transport. It is over-expressed in over 90% of ovarian
tumors and 20-50% of breast cancers. (Anticancer Research (1999
Jul-Aug) 19(4B) pg. 2907-2916) HER-2/neu A proto-oncogene (HER-2)
encoding a transmembrane protein similar in sequence and structure
to EGF-R. HER-2/neu is over-expressed as much as 200 fold over
normal tissues in breast and ovarian tumors. It has also been
identified in renal cell and lung carcinomas. (J. Exp. Med. (1995,
June) Vol. 181, pg. 2109-2117) NY-ESO-1 A cancer-testes antigen
found in 30% of breast, prostate and ovarian cancers, lung cancer,
bladder cancer, head and neck cancer and melanoma. Patients who
have cancers with tumors expressing this antigen usually have
circulating antibodies against it as well. (J. Immunology (2000)
vol. 165 pg. 948-955) CEA Carcinoembryonic antigen is a
tumor-associated antigen frequently expressed in epithelial tumors
(colon, breast, lung). CEA levels in the serum can correlate with
disease stage and is used to monitor treatment and reoccurrence of
disease. (Human Immunology (1998) vol. 59 pg. 1-14) MAGE-3 A
cancer-testis antigen expressed on 70-80% of metastatic melanoma
lesions and cell lines. It is a member of the family of melanoma
associated or MAGE proteins. In addition, MAGE-3 has been found in
20-60% of epithelial tumors (colon, breast, lung, gastric
carcinomas). (Human Immunology (1998) vol. 59 pg. 1-14) AES The
amino enhancer of split protein is part of a set of transcriptional
repressors encoded by the Enhancer of split genes. This tumor
antigen was identified in tumor- associated lymphocytes of ovarian
and breast tumors. (Molecular Immunology (1998) 35(17) pg.
1121-1133) HTR Telomerase(hTR) is a specialized type of reverse
transcriptase (hTRT or hTERT) that catalyzes the synthesis and
extension of telomeric DNA. The activity of this enzyme is elevated
in about 90% of all human tumors including cancers of the breast,
thyroid, bladder, cervix, prostate, colon, pancreas and stomach.
(Cancer Research (2001, Dec) 61(23) pg. 8366-8370)
[0237]
Sequence CWU 1
1
42 1 9 PRT Homo sapiens 1 Tyr Met Asn Gly Thr Met Ser Gln Val 1 5 2
9 PRT Homo sapiens 2 Tyr Met Asp Gly Thr Met Ser Gln Val 1 5 3 10
PRT Homo sapiens 3 Phe Leu Pro Trp His Arg Leu Phe Leu Leu 1 5 10 4
9 PRT Homo sapiens 4 Ile Asp Thr Gln Val Pro Phe Ser Val 1 5 5 9
PRT Homo sapiens 5 Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5 6 8 PRT
Homo sapiens 6 Ala Ala Gly Ile Gly Leu Thr Val 1 5 7 9 PRT Homo
sapiens 7 Cys Leu Thr Ser Thr Val Gln Leu Val 1 5 8 9 PRT Homo
sapiens 8 Lys Ile Phe Gly Ser Leu Ala Phe Leu 1 5 9 9 PRT Homo
sapiens 9 Lys Met Ala Ser Arg Ser Met Arg Leu 1 5 10 10 PRT Homo
sapiens 10 Ala Leu Ala Leu Ala Ala Leu Leu Val Val 1 5 10 11 9 PRT
Homo sapiens 11 Ala Leu Leu Val Val Asp Arg Glu Val 1 5 12 12 PRT
Homo sapiens 12 Ala Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly 1 5
10 13 9 PRT Homo sapiens 13 His Leu Tyr Gln Gly Cys Gln Val Val 1 5
14 9 PRT Homo sapiens 14 Ile Ile Ser Ala Val Val Gly Ile Leu 1 5 15
9 PRT Homo sapiens 15 Pro Leu Thr Ser Ile Ile Ser Ala Val 1 5 16 10
PRT Homo sapiens 16 Val Met Ala Gly Val Gly Ser Pro Tyr Val 1 5 10
17 9 PRT Homo sapiens 17 Val Leu Val Lys Ser Pro Asn His Val 1 5 18
9 PRT Homo sapiens 18 Glu Leu Val Ser Glu Phe Ser Arg Met 1 5 19 8
PRT Homo sapiens 19 Gly Pro Leu Thr Pro Leu Pro Val 1 5 20 8 PRT
Homo sapiens 20 Ser Thr Ala Pro Val His Asn Val 1 5 21 9 PRT Homo
sapiens 21 Tyr Leu Ser Gly Ala Asn Leu Asn Leu 1 5 22 9 PRT Homo
sapiens 22 Glu Ile Trp Thr His Ser Tyr Lys Val 1 5 23 10 PRT Homo
sapiens 23 Ser Ile Leu Ser Leu Lys Glu Ala Ser Thr 1 5 10 24 9 PRT
Homo sapiens 24 Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5 25 9 PRT
Homo sapiens 25 Ser Leu Leu Met Trp Ile Thr Gln Val 1 5 26 9 PRT
Homo sapiens 26 Gln Leu Ser Leu Leu Met Trp Ile Thr 1 5 27 9 PRT
Homo sapiens 27 Tyr Leu Glu Thr Phe Arg Glu Gln Val 1 5 28 9 PRT
Homo sapiens 28 Val Leu Leu Lys Leu Arg Arg Pro Val 1 5 29 9 PRT
Homo sapiens 29 Gly Leu Gln Ser Pro Lys Ser Pro Leu 1 5 30 9 PRT
Homo sapiens 30 Glu Leu Tyr Ile Pro Ser Val Asp Leu 1 5 31 9 PRT
Homo sapiens 31 Lys Ala Leu Phe Ala Gly Pro Pro Val 1 5 32 9 PRT
Homo sapiens 32 Phe Met Trp Gly Asn Leu Thr Leu Ala 1 5 33 9 PRT
Homo sapiens 33 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 34 9 PRT
Homo sapiens 34 Ile Leu Ala Lys Phe Leu His Trp Leu 1 5 35 9 PRT
Homo sapiens 35 Arg Leu Val Asp Asp Phe Leu Leu Val 1 5 36 9 PRT
Homo sapiens 36 His Leu Ser Thr Ala Phe Ala Arg Val 1 5 37 9 PRT
Homo sapiens 37 Ser Ile Ser Gly Asp Leu His Ile Ile 1 5 38 10 PRT
Homo sapiens 38 Val Ala Ala Ser Val Asp Asn Pro His Val 1 5 10 39 9
PRT Homo sapiens 39 Glu Leu Ile Ile Glu Phe Ser Lys Met 1 5 40 9
PRT Homo sapiens 40 Arg Leu Leu Gln Glu Thr Glu Leu Val 1 5 41 9
PRT Homo sapiens 41 Arg Leu Leu Gln Glu Thr Glu Leu Val 1 5 42 9
PRT Homo sapiens 42 Arg Leu Leu Gln Glu Arg Glu Leu Val 1 5
* * * * *