U.S. patent application number 14/247372 was filed with the patent office on 2014-07-31 for methods and compositions for detecting immune responses.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Kelledy Manson.
Application Number | 20140213479 14/247372 |
Document ID | / |
Family ID | 37076299 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140213479 |
Kind Code |
A1 |
Manson; Kelledy |
July 31, 2014 |
METHODS AND COMPOSITIONS FOR DETECTING IMMUNE RESPONSES
Abstract
Methods and compositions for detecting immune responses and
antigen-specific cells are described herein.
Inventors: |
Manson; Kelledy; (Boylston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
37076299 |
Appl. No.: |
14/247372 |
Filed: |
April 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11913583 |
Jul 11, 2008 |
8703484 |
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PCT/US2006/017765 |
May 5, 2006 |
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14247372 |
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60678329 |
May 5, 2005 |
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Current U.S.
Class: |
506/9 ; 435/34;
435/7.92; 506/10 |
Current CPC
Class: |
G01N 33/56977 20130101;
C07K 14/4748 20130101; G01N 33/6866 20130101; G01N 33/56972
20130101; G01N 33/505 20130101 |
Class at
Publication: |
506/9 ; 435/34;
506/10; 435/7.92 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/68 20060101 G01N033/68; G01N 33/50 20060101
G01N033/50 |
Claims
1.-42. (canceled)
43. A method of evaluating treatment of a subject, the method
comprising: (a) obtaining a sample comprising hematopoietic cells
from a subject of a first species, wherein the subject is
undergoing or being evaluated for an immunotherapeutic treatment
for a disease or condition; (b) providing a target cell of a second
species, wherein the second species is different from the first
species, wherein the second species is a macaque species, wherein
the target cell comprises an antigen, and wherein previous
determination of the target cell MHC genotype is not required; (c)
contacting the target cell with the sample; (d) detecting
expression of an immune activation marker or activity in the
sample, wherein an increase in expression of the immune activation
marker or activity in the sample, relative to a control, is an
indication that the sample comprises an antigen-specific
hematopoietic cell; and wherein a cell essentially identical to the
target cell but lacking the antigen does not stimulate expression
of the immune activation marker or activity in hematopoietic cells
of the first species, thereby evaluating treatment of the subject
or evaluating the subject.
44. The method of claim 43, further comprising: (e) transmitting
the result from the detecting of step (d) to a caregiver.
45. The method of claim 44, wherein the caregiver evaluates a
further treatment of the subject as a function of the result of the
detecting of step (d).
46. The method of claim 43, wherein the macaque species is rhesus
macaque.
47. The method of claim 43, wherein the target cell is the cell of
a cell line.
48. The method of claim 47, wherein the cell line is an epithelial
cell line.
49. The method of claim 48, wherein the epithelial cell line is a
mammary epithelial cell line.
50. The method of claim 49, wherein the cell line is CMMT
110/C1.
51. The method of claim 43, wherein the antigen is a
tumor-associated antigen (TAA) or a microbial antigen.
52. The method of claim 51, wherein the antigen is a
carcinoembryonic antigen (CEA) or a mucin-1 (MUC-1).
53. The method of claim 43, wherein the sample comprises peripheral
blood mononuclear cells (PBMC).
54. The method of claim 43, wherein the immune activation marker is
selected from the group consisting of a cytokine, a chemokine, a
cytotoxin, and a cell surface marker for activated hematopoietic
cells.
55. The method of claim 54, wherein the immune activation marker is
a cytokine selected from IFN-.gamma., TGF-.beta., TNF-.alpha.,
TNF-.beta., GM-CSF, G-CSF, interleukin-2 (IL-2), IL-3, IL-4, IL-5,
IL-6, IL-10, IL-12, and IL-15.
56. The method of claim 54, wherein the immune activation marker is
a chemokine selected from CCL3/MIP-1.alpha., MIP-1.beta.,
CCL5/RANTES, XCL1/lymphotactin, and CXCL10/IP-10.
57. The method of claim 54, wherein the immune activation marker is
granzyme.
58. The method of claim 54, wherein the immune activation marker is
a costimulatory molecule.
59. The method of claim 54, wherein the immune activation marker is
selected from B7.1, B7.2, CD152 (CTLA-4), CD28, CD40, CD40 ligand
(CD40L), and CD69.
60. The method of claim 43, wherein the immune activation activity
is proliferation of the hematopoietic cells or lysis of target
cells.
62. The method of claim 43, wherein the target cell is infected
with a virus that encodes the antigen.
63. The method of claim 62, wherein the virus is a DNA virus or a
poxvirus.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S. Ser.
No. 60/678,329, filed May 5, 2005, the contents of which are hereby
incorporated by reference in their entirety.
TECHNICAL FIELD
[0002] This invention relates to methods and compositions for
detecting immune responses, and more particularly to methods and
compositions for detecting cellular immune responses.
BACKGROUND
[0003] Methods for monitoring antigen-specific immune responses are
important for evaluating the efficacy of immunotherapies such as
cancer vaccines. Detection of tumor-specific immune responses can
be particularly difficult because tumor antigens are typically
self-antigens which are poorly immunogenic. Also, assays in which
cell-mediated immune responses are detected in vitro require the
use of antigen presenting cells (APC) that are immunologically
compatible with the test cells. In other words, in vitro assays to
detect antigen-dependent activation of lymphocytes such as T cells
employ APC (i) that display major histocompatibility complex
antigens (MHC) recognized by antigen receptors expressed on the
lymphocyte and (ii) that do not elicit non-specific responses from
the lymphocytes.
SUMMARY
[0004] Methods and compositions for detecting antigen-specific
hematopoietic cells (e.g., antigen-specific lymphocytes) and
evaluating immune responses in a subject are provided herein.
[0005] In one aspect, the invention features a method for detecting
an antigen-specific hematopoietic cell (e.g., an antigen-specific T
cell) in a biological sample. The method includes, for example: (a)
providing a biological sample comprising a hematopoietic cell of a
first species; (b) providing a target cell of a second species,
wherein the target cell comprises the antigen; (c) contacting the
target cell with the sample; and (d) detecting expression of an
immune activation marker or activity in the sample, wherein an
increase in expression of the immune activation marker or activity
in the sample, relative to a control, is an indication that the
sample includes an antigen-specific hematopoietic cell; and wherein
a cell essentially identical to the target cell but lacking the
antigen does not stimulate expression of the immune activation
marker or activity in hematopoietic cells of the first species. The
control can be a cell identical to the target cell but which does
not comprise the antigen, or a reference value (e.g., a reference
level of expression of the immune activation marker or
activity).
[0006] The antigen can be a tumor-associated antigen (TAA) (e.g., a
carcinoembryonic antigen (CEA) or a mucin-1 (MUC-1); a microbial
antigen (e.g., a viral, fungal, or bacterial antigen); or a
self-antigen associated with an autoimmune condition. In some
embodiments, the TAA is a TAA of the first species.
[0007] The biological sample can be a sample that includes
peripheral blood mononuclear cells (PBMC) or purified subsets
thereof (e.g., lymphocytes, e.g., T cells).
[0008] In various embodiments, the first species is human and the
second species is a primate species (e.g., a macaque species such
as rhesus macaque). The target cell can be a cell of a cell line,
e.g., an epithelial cell line such as a mammary epithelial cell
line.
[0009] In some embodiments, the immune activation marker is a
cytokine, e.g., selected from IFN-.gamma., TGF-.beta., TNF-.alpha.,
TNF-.beta., GM-CSF, G-CSF, interleukin-2 (IL-2), IL-3, IL-4, IL-5,
IL-6, IL-10, IL-12, and IL-15. In some embodiments, the immune
activation marker is a chemokine, e.g., selected from
CCL3/MIP-1.alpha., MIP-1.beta., CCL5/RANTES, XCL1/lymphotactin, and
CXCL10/IP-10. In other embodiments, the immune activation marker is
a cytotoxin such as granzyme. In other embodiments, the immune
activation marker is a cell surface marker for activated
hematopoietic cells, e.g.; a costimulatory molecule, e.g., B7.1,
B7.2, CD152 (CTLA-4), CD28, CD40, CD40 ligand (CD40L), and
CD69.
[0010] The immune activation marker can be detected with an
antibody-dependent assay such as an enzyme-linked immunosorbent
assay (ELISA) or an enzyme-linked immunosorbent spot (ELISPOT)
assay.
[0011] An activity associated with immune activation (e.g., cell
proliferation or cytotoxic cell lysis) can be detected in the
methods described herein.
[0012] In various embodiments, the target cell is infected with a
virus that encodes the antigen, e.g., a DNA virus such as a
poxvirus. The poxvirus can be an orthopox (e.g., vaccinia or MVA)
or avipox (e.g., fowlpox or canarypox).
[0013] In various embodiments, the virus further encodes one or
more costimulatory molecules of the first species, e.g., one or
more of B7.1, LFA-3, and ICAM-1, or all three.
[0014] In various embodiments, the target cell is transfected with
a nucleic acid that encodes the antigen.
[0015] The sample and the target cell can be incubated together at
step (c) for at least 24, 48, or 72 hours, or less than 7 days.
[0016] In another aspect, the invention features a method for
detecting an antigen-specific hematopoietic cell in a biological
sample, including: (a) providing a biological sample comprising a
hematopoietic cell; (b) providing a target cell of the same species
as the hematopoietic cell in (a), wherein the target cell comprises
the antigen and wherein the target cell does not express MHC class
I molecules; (c) contacting the target cell with the sample; and
(d) detecting an immune activation marker or activity in the
sample, wherein an increase in expression of the immune activation
marker in the sample, relative to a control, is an indication that
the sample comprises an antigen-specific hematopoietic cell; and
wherein a cell essentially identical to the target cell but lacking
the antigen does not stimulate expression of the immune activation
marker in hematopoietic cells of the first species. In various
embodiments, the target cell does not express MHC class II
molecules and/or costimulatory molecules. The control can be a cell
identical to the target cell but which does not comprise the
antigen, or a reference value (e.g., a reference level of
expression of the immune activation marker or activity). The method
can further include other features described herein.
[0017] In another aspect, the invention features a method of
evaluating treatment of a subject. The method includes, for
example: (a) obtaining a sample comprising hematopoietic cells from
a subject of a first species, wherein the subject is undergoing or
being evaluated for an immunotherapeutic treatment for a disease or
condition; (b) providing a target cell of a second species, wherein
the target cell comprises the antigen; (c) contacting the target
cell with the sample; (d) detecting expression of an immune
activation marker or activity in the sample, wherein an increase in
expression of the immune activation marker or activity in the
sample, relative to a control, is an indication that the sample
comprises an antigen-specific hematopoietic cell; and wherein a
cell essentially identical to the target cell but lacking the
antigen does not stimulate expression of the immune activation
marker or activity in hematopoietic cells of the first species,
thereby evaluating treatment of the subject. The method can further
include transmitting the result from the detecting of step (d) to a
caregiver. In one embodiment, the caregiver evaluates a further
treatment of the subject as a function of the result of the
detecting of step (d).
[0018] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0019] The term "epitope" refers to the portion of a macromolecule
that is specifically recognized by a component of the immune
system, e.g., an antibody or T-cell antigen receptor. The term
"tumor-associated antigen" or "TAA" refers to a molecule that is
differentially expressed in tumor cells relative to non-tumor cells
of the same cell type. As used herein, "tumor-associated antigen"
includes not only complete tumor-associated antigens, but also
epitope-comprising portions (fragments) thereof. A TAA may be one
found in nature, or may be a synthetic version of a TAA found in
nature, or may be a variant of a naturally-occurring TAA, e.g., a
variant that has enhanced immunogenic properties (see, e.g., U.S.
Pat. No. 6,756,038 and WO 03/047506).
[0020] A "biological sample" encompasses a variety of sample types
that are obtained from a subject and can be used in an assay
described herein. The term encompasses blood and other liquid
samples of biological origin, bone marrow, solid tissue samples
such as a biopsy specimen or tissue cultures or cells derived
therefrom. The term also includes samples that have been
manipulated in any way after their procurement, such as by washing,
lysis, fractionation, or treatment with reagents or enrichment for
certain cell populations, such as CD8.sup.+ T lymphocytes,
macrophages, tumor cells, or PBMC.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an HLA-A2.sup.+ patient (002-007) 70
days after initiation of treatment with the PANVAC-VF regimen PBMC
were stimulated in the presence of media, vaccinia lysate, C1R-A2
cells alone or pulsed with CAP-1-6D peptide or the MUC-1 T2L
peptide, CMMT 110/C1 cells alone or transfected with TBC-FPV,
rF-CEA, or rF-MUC-1.
[0022] FIG. 2 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an HLA-A2.sup.+ patient (002-007)
and an HLA-A2.sup.- patient (002-006) 70 days after initiation of
treatment with the PANVAC-VF regimen, and by PBMC from a
non-vaccinated patient (Naive donor). PBMC were stimulated in the
presence of CMMT 110/C1 cells infected with the following
recombinant viruses (either at MOI=10 or MOI=40, as indicated):
TBC-FPV, rF-CEA, rF-MUC-1, PANVAC-F (sample 1), and PANVAC-F
(sample 2).
[0023] FIG. 3 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an HLA-A2.sup.+ patient (002-007)
and an HLA-A2.sup.- patient (002-006) 70 days after initiation of
treatment with the PANVAC-VF regimen, and by PBMC from a
non-vaccinated patient (Naive Donor). PBMC were stimulated in the
presence of CMMT 110/C1 cells infected with the following
recombinant viruses (either at MOI=10 or MOI-40, as indicated):
TBC-FPV, rF-TRICOM, rF-CEA, rF-MUC-1, PANVAC-F (sample 1), and
PANVAC-F (sample 2).
[0024] FIG. 4 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an HLA-A2.sup.- patient (002-006) 70
days after initiation of treatment with the PANVAC-VF regimen. PBMC
were stimulated in the presence of CMMT 110/C1 cells uninfected
with virus or infected with TBC-FPV or PANVAC-F. Cells were
incubated at ratios of 1:1, 1:2, or 1:10 CMMT 110/C1: PBMC and
supernatants were sampled for IFN-.gamma. ELISA at 6 hours, and 1,
2, 3, 4, 5, 6, and 7 days after co-culture.
[0025] FIG. 5 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an HLA-A2.sup.- patient (002-006) 70
days after initiation of treatment with the PANVAC-VF regimen PBMC
were stimulated in the presence of CMMT 110/C1 cells infected with
TBC-FPV or PANVAC-F. Cells were incubated at ratios of 1:1, 1:2, or
1:10 CMMT 110/C1: PBMC and supernatants were sampled for
IFN-.gamma. ELISA at 3, 4, 5, 6, and 7 days after co-culture.
[0026] FIG. 6 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an HLA-2.sup.- (002-006) patient 70
days after initiation of treatment with the PANVAC-VF regimen. PBMC
were stimulated in the presence of CMMT 110/C1 cells infected with
TBC-FPV or PANVAC-F. Cells were incubated at ratios of 1:1, 1:2, or
1:10 CMMT 110/C1: PBMC and supernatants were sampled for
IFN-.gamma. ELISA at 3, 4, 5, 6, and 7 days after co-culture.
[0027] FIG. 7 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an HLA-A2.sup.+ patient (002-007) 70
days after initiation of treatment with the PANVAC-VF regimen. PBMC
were stimulated in the presence of CMMT 110/C1 cells infected with
TBC-FPV or PANVAC-F. Cells were incubated at ratios of 1:1, 1:2, or
1:10 CMMT 110/C1: PBMC and supernatants were sampled for
IFN-.gamma. ELISA at 3, 4, 5, 6, and 7 days after co-culture.
[0028] FIG. 8 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC obtained from an unvaccinated patient (Naive
Donor). PBMC were stimulated in the presence of CMMT 110/C1 cells
infected with TBC-FPV or PANVAC-F. Cells were incubated at ratios
of 1:1, 1:2, or 1:10 CMMT 110/C1: PBMC and supernatants were
sampled for IFN-.gamma. ELISA at 3, 4, 5, 6, and 7 days after
co-culture.
[0029] FIG. 9 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC and subsets of cells thereof in response to
stimulation in the presence of CMMT 110/C1 cells infected with
TBC-FPV or PANVAC-F. Two sets of PBMC used in this experiment were
obtained from an HLA-A2.sup.+ patient (002-007) and an HLA-A2.sup.-
patient (002-006) 70 days after initiation of treatment with the
PANVAC-VF regimen. The third set was obtained from a non-vaccinated
patient (Naive Donor). The cell subsets tested in this assay were
PBMC, CD3.sup.+, CD4.sup.+, CD8.sup.+, and NK.sup.+ cells.
[0030] FIG. 10 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC from an HLA-A2.sup.- (002-006) patient 0 and 70
days after initiation of treatment with the PANVAC-VF regimen. PBMC
were tested for secretion in response to incubation with media,
uninfected CMMT 110/C1 cells, and CMMT 110/C1 cells infected with
one of the following recombinant viruses: TBC-FPV, rF-TRICOM,
PANVAC-F, PROSTVAC.RTM.-F.
[0031] FIG. 11 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC from two HLA-A2.sup.- individuals (002-006 and
001-011). PBMC were taken from one patient 28, 42, 70 days, 2
months plus 70 days, and 3 months plus 70 days after initiation of
treatment with the PANVAC-VF regimen; from the a second patient 0,
14, 28, 42, 70 days, and 2 months plus 70 days after initiation of
treatment with the PANVAC-VF regimen. PBMC were tested for
secretion in response to incubation with media, uninfected CMMT
110/C1 cells, and CMMT 110/C1 cells infected with one of the
following recombinant viruses: TBC-FPV, rF-TRICOM, rF-IF, PANVAC-F,
and PROSTVAC.RTM.-F.
[0032] FIG. 12 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC from two HLA-A2.sup.- patients (002-001 and
001-011) 0, 14, 28, 42, 70 days, 70 days plus 1 month, and 70 days
plus 2 months after initiation of treatment with the PANVAC-VF
regimen (or a subset of those time points). PBMC were tested for
secretion in response to incubation with media and CMMT 110/C1
cells infected with one of the following recombinant viruses:
rF-TRICOM, PANVAC-F, PROSTVAC.RTM.-F.
[0033] FIG. 13 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC from an HLA-A2.sup.+ patient (002-007) and an
HLA-AT patient (002-006) 70 days after initiation of treatment with
the PANVAC-VF regimen, and by PBMC from a non-vaccinated patient
(Naive Donor). PBMC were stimulated in the presence of CMMT 110/C1
cells infected with the following recombinant viruses: rF-TRICOM,
rF-CEA(6D), rF-CEA(6D)/TRICOM, rF-MUC-1, rF-MUC-1/TRICOM, and
PANVAC-F.
[0034] FIG. 14 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC from three different patients (#12, #13, and #14)
taken 0, 14, 28, 37, 42, and 63 days (or a subset thereof), after
initiation of treatment with the PANVAC-VF regimen. PBMC were
stimulated in the presence of uninfected CMMT 110/C1 cells and CMMT
110/C1 cells infected with the following recombinant viruses:
rF-TRICOM, PROSTVAC.RTM.-F, and PANVAC-F.
[0035] FIG. 15 is a bar graph depicting levels of IFN-.gamma.
secreted by PBMC from an HLA-A2.sup.- patient (002-007) taken 70
days, and 70 days plus 4, 5, 6, 7, 8, and 9 months after initiation
of treatment with the PANVAC-VF regimen. PBMC were stimulated in
the presence of uninfected CMMT 110/C1 cells and CMMT 110/C1 cells
infected with the following recombinant viruses: rF-TRICOM,
PROSTVAC.RTM.-F, and PANVAC-F.
[0036] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0037] The methods described herein are based, in part, on the
discovery that APC (also referred to as "target cells") expressing
an antigen can elicit cell-mediated immune responses in in vitro
assays in an MHC-non-restricted manner. The methods employ an APC
which does not elicit a non-specific immune response from
hematopoietic cells of a subject in the absence of antigen (or, if
an immune response is elicited, it is low or undetectable). The APC
is treated so as to express the antigen of interest (e.g., by
infection or transfection with a recombinant virus or nucleic acid
encoding the antigen) and incubated with hematopoietic cells (e.g.,
PBMC, lymphocytes) in vitro. Responsiveness of the hematopoietic
cells to the APC is then evaluated.
[0038] Use of APC (and antigens) that elicit responses in an
MHC-non-restricted manner in assays are advantageous because the
assays do not require previous determination of a subject's MHC
genotype. These assays provide an alternative to MHC-restricted
cellular assays that employ a target cell (i.e., an APC) of a
given, pre-defined MHC genotype. For example, C1R cells are
transformed human plasma leukemia cells which do not express
endogenous human leukocyte antigen-A (HLA-A) or HLA-B antigens.
C1R-A2 cells are stably transfected with a genomic clone of HLA-A2
(described in Arlen et al., Cancer Immunol. Immunother.,
49:517-529, 2000). C1R-A2 cells are frequently used as APC in
assays to measure patient responses to cancer vaccines. However,
these cells are generally used only for testing cells from patients
who express HLA-A2, because a patient who does not express HLA-A2
will not exhibit HLA-A2-restricted T cell responses.
[0039] It has been discovered that antigen-presenting cells having
certain characteristics stimulate antigen-specific responses,
regardless of the HLA genotype of the donor. One type of cell which
can be used with human hematopoietic cells is CMMT 110/C1 cells.
CMMT 110/C1 cells were derived from rhesus monkey mammary gland
carcinoma cells obtained from the American Type Culture Collection.
As described in further detail in the Examples below, these cells
do not elicit immune responses in the absence of antigen. In other
words, the MHC, costimulatory molecules (if any) and endogenous
antigens expressed by these cells fail to elicit detectable levels
of IFN-.gamma. secretion from human PBMC in culture. In this sense,
the cells are immunologically "naked" to human PBMC in vitro. CMMT
110/C1 cells infected with recombinant viruses expressing tumor
antigens stimulate IFN-.gamma. secretion from PBMC from individuals
that have been vaccinated with the same recombinant viruses.
Assay Conditions
[0040] Test Cells.
[0041] Hematopoietic cells of any subject may be examined by the
methods described herein. Mammalian subjects include primate (e.g.,
human, chimpanzee, macaque), rodent (e.g., mouse, rat, guinea pig,
hamster), rabbit, and porcine subjects. PBMC are typically examined
in in vitro assays that detect cell-mediated immune (CMI)
responses. Other types of samples that may be examined include
cells isolated from bone marrow, tissues (e.g., tumor tissues or
tissues of a particular organ, e.g., liver), spleen, lymph,
cerebrospinal fluid, peritoneum, gut, lung, and secondary lymphoid
organs. Purified cells may be provided, such as T cells or natural
killer (NK) cells, e.g., isolated from PBMC. Kits for isolation of
hematopoietic cell subsets are commercially available. Cells that
have been transformed, frozen, and/or propagated in vitro may be
assayed as well.
[0042] APC/Target Cells.
[0043] APC can include any cell type that does not elicit a
response (or that elicits a low level of response) from the test
cells of interest in the absence of antigen. In one embodiment, the
APC can be cells that elicit a response from an antigen-specific
lymphocyte in vitro that is at least four times increased in the
presence of antigen compared to the response to the APC in the
absence of antigen. For example, an APC used to evaluate
IFN-.gamma. release by T cells will elicit secretion of quantities
of IFN-.gamma. by an antigen-specific T cell that are at least four
times greater when the APC expresses the antigen as compared to
when the APC which does not express the antigen, e.g., as measured
by ELISA for bulk IFN-.gamma. secretion into culture
supernatants.
[0044] The APC may be from the same species as the test cells, or
from a different species. In general, APC of the same species as
the test cells that can be used in the methods will express low
levels of or no costimulatory molecules. In some embodiments, the
APC are negative for expression of MHC molecules (MHC class I and
MHC class II molecules). In various embodiments the APC are
negative for expression of a costimulatory molecule (e.g., B7.1,
B7.2, CD28, CD40, CD40L) and/or are also negative for expression of
MHC molecules. The APC also will not express endogenous antigens
which are likely to elicit from the test cells a non-specific
response that will interfere with detection of the antigen-specific
response of interest. For example, APC will be negative for
expression of a virus common in the test cell population, wherein
the test cell population will include cells that react to the
virus. Epstein-Barr Virus (EBV) is common in humans. Therefore, in
various embodiments, APC expressing EBV antigens are not be used to
evaluate TAA-specific responses in human PBMC, because responses of
cells in the PBMC to the EBV antigens will interfere with detection
of TAA-specific responses.
[0045] APC of a species other than that of the test cells (i.e.,
xenogeneic APC) can also be used. In, one embodiment, xenogeneic.
APC express low levels or no endogenous MHC and costimulatory
molecules. In another embodiment, xenogeneic APC express MHC and
costimulatory molecules that do not elicit a non-specific immune
response (or elicit a very low level of response) by the test cells
in vitro in the absence of antigen (e.g., the response to the APC
in the presence of antigen is at least four times as great as the
response in the absence of antigen).
[0046] In one embodiment, the test cells are human and the APC are
from a non-human primate (e.g., ape such as chimpanzee, gorilla,
baboon, or monkey such as macaque (e.g., rhesus macaque), lemur,
green monkey, or New World monkey species). In one embodiment, the
test cells are human and the APC are from a non-primate mammal
(e.g., mouse, rat, hamster, rabbit, pig).
[0047] The APC used in the present methods may be adherent (e.g.,
epithelial, fibroblastoid) or non-adherent (e.g., lymphoid) cells.
For embodiments in which virus infection will be employed as the
means of introducing a DNA encoding a foreign antigen into the APC;
the APC must be a cell type permissive for infection with the
virus. Transfection of APC with nucleic acid encoding MHC, or
otherwise loaded with MHC polypeptides, is not required for the
methods described herein.
[0048] Antigens.
[0049] The methods described herein can be used to detect immune
responsiveness, e.g., to TAA or to viral, fungal, parasitic or
bacterial antigens. In various embodiments, the antigen is an
antigen that includes tandem repeats (such as mucin-1 or
carcinoembryonic antigen). These methods can also be used to
monitor a subject's immune response against a self-antigen other
than a TAA. For example, the methods can be used to detect and/or
monitor the progression or prognosis of an autoimmune condition in
a subject. Examples of autoimmune conditions include systemic lupus
erythematosus, insulin-dependent diabetes mellitus, rheumatoid
arthritis, multiple sclerosis, autoimmune hemolytic anemia,
autoimmune thrombocytopenic purpura, Goodpasture's syndrome,
pemphigus vulgaris, acute rheumatic fever, ankylosing spondylitis,
uveitis, Grave's disease, myasthenia gravis, and Hashimoto's
thyroiditis. Self antigens associated with these diseases include
acetylcholine receptors, thyroid stimulating hormone receptors,
platelets, desmoglein-3, Ro protein, insulin receptors, myelin
basic protein, proteolipid protein, myelin oligodendrocyte protein,
cadherins, Rh blood group antigens, and collagens.
[0050] Exemplary TAA include but are not limited to the following:
mucin-1 (MUC-1); carcinoembryonic antigen (CEA); prostate-specific
antigen (PSA); 707 alanine proline (707-AP); alpha
(.alpha.)-fetoprotein (AFP); adenocarcinoma antigen recognized by T
cells 4 (ART-4); B antigen (BAGE); .beta.-catenin/mutated;
breakpoint cluster region-Abelson (Bcr-abi); CTL-recognized antigen
on melanoma (CAMEL); carcinoembryonic antigen peptide-1 (CAP-1);
caspase-8 (CASP-8); cell-division cycle 27 mutated (CDC27m);
cycline-dependent kinase 4 mutated CDK4/m); cancer/testis (CT)
antigen; cyclophilin B (Cyp-B); differentiation antigen melanoma
(DAM-6, also known as MAGE-B2, and DAM-10, also known as MAGE-B1);
elongation factor 2 mutated (ELF2M); Ets variant gene 6/acute
myeloid leukemia 1 gene ETS (ETV6-AML1); glycoprotein 250 (G250); G
antigen (GAGE); N-acetylglucosaminyltransferase V (GnT-V);
glycoprotein 100 kD (GnT-V); helicase antigen (HAGE); human
epidermal receptor-2/neurological (HER-2/neu); HLA-A*0201-R170I
(HLA-A*0201 having an arginine (R) to isoleucine exchange at
residue 170 of the .alpha.-helix of the .alpha.2-domain in the
HLA-A2 gene); human papilloma virus E7 (HPV-E7); human papilloma
virus E6 (HPV-E6); heat shock protein 70-2 mutated (HSP70-2M);
human signet ring tumor-2 (HST-2); human telomerase reverse
transcriptase (hTERT or hTRT); intestinal carboxyl esterase (iCE);
KIAA0205; L antigen (LAGE); low density lipid
receptor/GDP-L-fucose: .beta.-D-galactosidase
2-.alpha.-L-fucosyltransferase (LDLR/FUT); melanoma antigen (MAGE);
melanoma antigen recognized by T cells-1/Melanoma antigen A
(MART-1/Melan-A); melanocortin 1 receptor (MC1R); myosin mutated
(Myosin/m); melanoma ubiquitous mutated 1 (MUM-1), melanoma
ubiquitous mutated 2 (MUM-2), melanoma ubiquitous mutated 3
(MUM-3); New York-esophagus 1 (NY-ESO-1); protein 15 (P15); protein
of 190KD bcr-abl (p190 minor bcr-abl); promyelocytic
leukaemia/retinoic acid receptor .alpha. (Pml/RAR.alpha.)
preferentially expressed antigen of melanoma (PRAME);
prostate-specific membrane antigen (PSM); renal antigen (RAGE);
renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2); sarcoma antigen
(SAGE); SART-1; SART-3; translocation Ets-family leukemia/acute
myeloid leukemia 1 (TEL/AML1); triosephosphate isomerase mutated
(TPI/m); tyrosinase related protein 1 (TRP-1 or gp75); tyrosinase
related protein 2 (TRP2); TRP-2/intron 2 (TRP-2/INT2); Wilms' tumor
gene (WT-1).
[0051] The antigens used in the methods described herein include
fragments of full-length polypeptides and/or polypeptides that
include mutations relative to the antigen as it is naturally
expressed. In one embodiment, the antigen is a CEA (or fragment
thereof) which includes an amino acid substitution of Asn to Asp at
position 6 in the CAP-1 peptide of CEA. The CAP-1 peptide has the
following sequence: YLSGANLNL (SEQ ID NO:1)(Zaremba et al., Cancer
Res., 57(20):4570-7, 1997). In one embodiment, the antigen is MUC-1
T2L which includes an amino acid substitution of Thr to Leu at
amino acid position 2 of the following fragment of the MUC-1
sequence: ATWGQDVTSV (SEQ ID NO:2)(Tsang et al., Clin Cancer Res.,
10(6):2139-49, 2004).
[0052] Exemplary bacterial antigens include those associated with
human and animal bacterial pathogens including but not limited to
Mycobacterium spp. (e.g., Mycobacterium tuberculosis, Mycobacterium
leprae), Streptococcus spp. (e.g., Streptococcus pneumoniae,
Streptococcus pyogenes), Staphylococcus spp. (e.g., Staphylococcus
aureus), Treponema (e.g., Treponema pallidum), Chlamydia spp.,
Vibrio spp. (e.g., Vibrio cholerae), Bacillus spp. (e.g., Bacillus
subtilis, Bacillus anthracis), Yersinia spp. (e.g., Yersinia
pestis), Neisseria spp. (e.g., Neisseria meningitides, Neisseria
gonorrhoeae), Legionella spp., Bordetella spp. (e.g., Bordetella
pertussis), Shigella spp., Campylobacter spp., Pseudomonas spp.
(e.g., Pseudomonas aeruginosa), Brucella spp., Clostridium spp.
(e.g., Clostridium tetani, Clostridium botulinum, Clostridium
perfringens), Salmonella spp. (e.g., Salmonella typhi), Borrelia
spp. (e.g., Borrelia burgdorferi), Rickettsia spp. (e.g.,
Rickettsia prowazeki), Mycoplasma spp. (e.g., Mycoplasma
pneumoniae), Haemophilus spp. (e.g., Haemophilus influenzae),
Branhamella spp. (e.g., Branhamella catarrhalis), Corynebacteria
spp. (e.g., Corynebacteria diphtheriae), Klebsiella spp. (e.g.,
Klebsiella pneumoniae), Escherichia spp. (e.g., Escherichia coli),
and Listeria spp. (e.g., Listeria monocytogenes). Functional
fragments and variants of polypeptides encoded by such pathogens
are known in the art and can be expressed by the APC in the methods
described herein.
[0053] Fungal antigens can be those derived from fungi including
but not limited to Candida spp. (e.g., albicans), Cryptococcus spp.
(e.g., neoformans), Blastomyces spp. (e.g., dermatitidis),
Histoplasma spp. (e.g., capsulatum), Coccidroides spp. (e.g.,
immitis), Paracoccidroides spp. (e.g., brasiliensis), and
Aspergillus spp. A bacterial antigen can be an antigen or fragment
or variant thereof derived from, e.g., the listed organisms.
[0054] Parasitic antigens can be derived from organisms that
include but are not limited to Plasmodium spp., Eimeria spp.,
Schistosoma spp., Trypanosoma spp., Babesia spp., Leishmania spp.,
Cryptosporidia spp., Toxoplasma spp., Pneumocystis spp., Entamoeba
histolytica, Giardia spp., Plasmodium spp., Cryptosporidium spp.,
Trichuris trichura, Trichinella spiralis, Enterobium vermicularis,
Ascaris lumbricoides, Ancylostoma spp., Stongyloides spp., Filaria
spp., and Schistosoma spp. A parasitic antigen can be an antigen or
fragment or variant thereof derived from, e.g., the listed
organisms.
[0055] Expression of Antigens in APC/Target Cells.
[0056] The antigen of interest may be expressed by the APC via
transfection with a nucleic acid encoding the antigen or via
infection with a recombinant virus encoding the antigen. In various
embodiments, a recombinant poxvirus (e.g., orthopox such as
vaccinia or Modified Vaccinia Ankara (MVA), or an avipox such as
fowlpox or canarypox) is used to express the antigen. In various
embodiments, the recombinant virus further expresses one or more
costimulatory molecules (e.g., costimulatory molecules that
interact with ligands on the test cells, e.g., B7.1, LFA-3, or
ICAM-1). The combination of B7.1, LFA-3, and ICAM-1 is also
referred to as TRICOM.TM.. The conditions for expression by viral
infection will depend on the particular APC cell type. Typically,
APC will be infected 10-72 hours, e.g., 24 hours, prior to
incubation with test cells and washed before being plated with the
test cells.
[0057] Incubation of Test Cells and APC/Target Cells.
[0058] A sample containing test cells is incubated with APC,
typically in multiwell culture plates (e.g., 24-well, 48-well, or
96-well plates), in media, under culture conditions appropriate for
the cells (e.g., 37.degree. C., 5% CO.sub.2) and for an amount of
time sufficient for expression of a detectable amount of an immune
activation marker by antigen-specific cells, or for an
antigen-dependent biological event (e.g., cytotoxic lysis of APC),
in the sample (e.g., 6 hours, 12 hours, 1 day, 2 days, 3 days, 4
days, 5 days, 6 days, 7 days, 10 days, or longer). Optimal ratios
of test cells:APC can be determined empirically, and will generally
be in the range of 1:1, 2:1, 5:1, 10:1, or 25:1. Cells and/or
culture supernatants are then examined to evaluate the quantity
and/or quality of immune activation markers. Additional preparation
steps may be required for assays in which cytotoxic lysis or
ELISPOT analyses are to be performed. These are discussed in the
section below.
Detection of Immune Responses
[0059] Antigen-specific immune responses elicited in the assays can
be detected by art-known methods. Expression of immune activation
markers on test cells or in the culture supernatants can be
examined. Alternatively, or in addition, immune responsiveness is
examined by evaluating cytotoxic lysis of antigen presenting
cells.
[0060] Immune Activation Markers.
[0061] Many molecules have been identified as indicative of
antigen-specific immune activation. These molecules, or markers,
include soluble mediators (e.g., cytokines, chemokines),
cell-surface molecules that are upregulated under conditions of
immune activation (e.g., costimulatory molecules, adhesion
molecules), and toxic mediators produced by immune effector cells
(e.g., cytotoxins such as granzymes). Cytokines produced by T
cells, NK cells, and other immune effector cells (e.g.,
macrophages, monocytes, B cells, neutrophils, eosinophils,
basophils) which may be assayed in the present methods include:
IFN-.gamma., TGF-.beta., TNF-.alpha., TNF-.beta., GM-CSF, G-CSF,
interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, and
IL-15. Chemokines that may be assayed include: CCL3/MIP-1.alpha.,
MIP-1.beta., CCL5/RANTES, XCL1/lymphotactin, and CXCL10/IP-10. Cell
surface markers indicative of activation include B7.1, B7.2, CD152
(CTLA-4), CD28, CD40, CD40 ligand (CD40L), and CD69.
[0062] ELISA.
[0063] Enzyme-linked immunosorbent assays (ELISA) are useful for
detecting soluble mediators such as cytokines. In one embodiment,
supernatants are collected from test cell/APC incubation assay
samples and levels of cytokine released in the supernatants are
quantitated by ELISA. For a description of ELISA methods for
detecting cytokines see, e.g., U.S. Pat. No. 6,218,132.
[0064] ELISPOT.
[0065] Enzyme-linked immunosorbent spot (ELISPOT) assays detect
cytokine release on a single-cell basis. Typically, test cells and
APC are incubated in a culture plate that has been coated with
antibody that specifically binds to the cytokine of interest. After
test cells and APC are incubated together for the desired length of
time, cells are lysed in the wells. A second antibody that
recognizes the cytokine of interest is added to the plates to bind
to cytokine that has been captured by the plate-bound antibody, and
is subsequently detected by secondary reagents, that reveal spots
where individual cells secreted cytokine. For a detailed protocol
for detecting cytokine secretion by ELISPOT, see, e.g., Arlen et
al., Cancer Immunol. Immunother., 49:517-529, 2000.
[0066] Proliferative and Cytotoxic Cellular Responses.
[0067] Cytotoxic responses (e.g., T cell- or NK-cell mediated
cytotoxic responses) are one type of immune activity that can be
evaluated in the methods described herein. These can be measured by
any suitable technique, e.g., chromium release assays.
Proliferative responses of hematopoietic cells can also be
evaluated, e.g.; by a mixed lymphocyte reaction (MLR). Briefly,
target cells are irradiated and co-cultured with test cells in
microtiter culture plates for .about.5 days. During the last 8
hours of the culture period, the cells are pulsed with 1
.mu.Ci/well of .sup.3H-thymidine, and the cells are harvested for
counting onto filter paper by a cell harvester. .sup.3H-thymidine
incorporation is measured by standard techniques. Proliferation of
cells in such assays is expressed as the mean counts per minute
(cpm) read for the tested wells. Limiting dilution analysis (LDA)
is another method by which to evaluate the frequency of
antigen-specific lymphocytes in a sample. Guidance and principles
related to T cell proliferation assays are described in, e.g.,
Plebanski and Burtles, J. Immunol. Meth. 170:15, 1994; Sprent et
al., Philos. Trans R. Soc. Lond. B. Biol. Sci., 355(1395):317-22,
2000; and Messele et al., Clin. Diagn. Lab. Immunol., 7(4):687-92,
2000. LDA is described in, e.g., Sharrock et al., Immunol. Today,
11:281-286, 1990. Other lymphocyte analytical techniques are
described in Hartel et al., Scand. J. Immunol., 49(6):649-54, 1999
and Parish et al., J. Immunol. Methods, 58(1-2):225-37, 1983.
Diagnostics and Patient Care
[0068] The methods described herein can be used for diagnostic
purposes, e.g., in patient care. For example, the methods can be
used in evaluating a subject. The subject can be healthy or
suffering from a disease. In various embodiments, the subject is a
patient undergoing treatment (e.g., vaccination and/or
immunotherapy) for a malignant disease, e.g., a cancer such as a
pancreatic cancer or a prostate cancer. In one embodiment, the
method includes: obtaining a sample comprising hematopoietic cells
from a subject, e.g., from a caregiver, e.g., a caregiver who
obtains the sample from the subject; determining whether the sample
includes antigen-specific hematopoietic cells, e.g., by a method
described herein. The subject can be a subject that has been
exposed to an antigen (either naturally exposed or exposed via
vaccination or immunotherapy), or a subject who is naive to the
antigen. Samples can be obtained from the subject one, two, three
or more times over a period of time in order to evaluate
responsiveness to a vaccine or immunotherapy regimen. Data obtained
from the methods in which the samples are evaluated can be compared
with other data regarding the subject, e.g., a clinical parameter
such as tumor progression or incidence of an infectious disease or
a symptom associated with an infectious disease.
[0069] In various embodiments, data obtained from a method in which
a sample is taken from a subject and evaluated for immune
responsiveness is transmitted to a caregiver. The caregiver may
make a decision regarding further treatment of the subject based on
the data transmitted.
EXAMPLES
Example 1
Comparison of ELISPOT and Cytokine Secretion Assays to Detect
Tumor-Specific Immune Cells
[0070] The following assays were performed to test a patient's
responsiveness to treatment with the PANVAC-VF regimen, a
therapeutic vaccination regimen for pancreatic cancer patients. The
regimen involves the sequential use of a recombinant vaccinia virus
(PANVAC-V) and a recombinant fowlpox virus (PANVAC-F) as vaccines.
Each of the two recombinant viruses contains sequences encoding
five proteins: CEA, mucin-1 (MUC-1), B7.1, Intercellular Adhesion
Molecule-1 (ICAM-1), and Lymphocyte Function-associated Antigen-3
(LFA-3). Patients treated with the PANVAC-VF regimen received a
priming dose of 2.times.10.sup.8 plaque forming units (pfu) of
PANVAC-V subcutaneously on day 0, followed by a boost dose of
1.times.10.sup.9 pfu PANVAC-F subcutaneously on days 14, 28, and
42. Recombinant GM-CSF (100 .mu.g) was administered at the
injection site on the day of each vaccine administration and for
three consecutive days thereafter. PBMC were collected from
patients at time points after initiation of treatment and frozen
for assays.
[0071] The patient tested in this assay, patient #7 (002-007),
expresses HLA-A2. Therefore patient #7's PBMC were tested for
responsiveness to target cells expressing HLA-A2. The
HLA-A2-expressing target cells chosen for this assay were C1R-A2
cells. PBMC were also tested in a non-restricted assay in which
CMMT 110/C1 cells were used as target cells.
[0072] To prepare the patient's PBMC for the assays, they were
thawed, resuspended in Animal Media (RPMI with 10% fetal bovine
serum, L-glutamine, antibiotics, and .beta.-mercaptoethanol),
counted, and incubated overnight at 37.degree. C., 5% CO.sub.2.
Next, PBMC were incubated in culture in 24-well plates with target
cells and/or antigen for 72 hours. A 1:1 ratio of PBMC:target cells
(1.times.10.sup.6 PBMC:1.times.10.sup.6 target cells per well) was
used for all culture conditions. Incubations with the following
combinations of PBMC and target cells and/or antigens were
performed: [0073] PBMC+Media [0074] PBMC+Con A [0075] PBMC+Vaccinia
Lysate (TBC-Wyeth) [0076] PBMC+C1R-A2 [0077] PBMC+C1R-A2 pulsed
with CAP-1-6D [0078] PBMC+C1R-A2 pulsed with MUC-1 T2L peptide
[0079] PBMC+CMMT 110/C1 [0080] PBMC+CMMT 110/C1 infected with
TBC-FPV (MOI=10) [0081] PBMC+CMMT 110/C1 infected with
rF-CEA/TRICOM (MOI=10) [0082] PBMC+CMMT 110/C1 infected with
rF-MUC-1/TRICOM (MOI=10)
[0083] Con A is concanavalin A, a polyclonal T cell mitogen derived
from Jack beans. CAP-1-6D is a peptide from human carcinoembryonic
antigen (CEA)(American Peptide Co., cat. no. 30341) with the
following sequence: YSGADLNL (SEQ ID NO:3). The MUC-1 T2L peptide
has the following sequence: ALWGQDVTSV (SEQ ID NO:4)(American
Peptide Co.). This peptide has a Thr to Leu substitution at
position 2 relative to the corresponding peptide within the
wild-type MUC-1 sequence. C1R-A2 cells were pulsed with 10 .mu.g/ml
of each peptide.
[0084] TBC-FPV is a fowlpox virus that does not encode any tumor
antigens or human costimulatory molecules. rF-CEA/TRICOM is a
recombinant fowlpox virus derived from TBC-FPV and that expresses
CEA and TRICOM. rF-MUC-1/TRICOM is a recombinant fowlpox virus
derived from TBC-FPV and that expresses MUC-1 and TRICOM.
[0085] CMMT 110/C1 cells were infected 24 hours before culture with
PBMC and washed immediately before plating with PBMC. After the
incubation, cells were harvested and counted to perform ELISPOT
assays as described in Arlen et al., Cancer Immunol. Immunother.,
49:517-529, 2000. Briefly, harvested cells were counted and plated
(2.times.10.sup.5 total cells/well) on an ELISPOT assay plate
pre-blocked with complete (i.e., serum-containing) medium. Cells
were incubated overnight at 37.degree. C. for 34 hours; plates were
washed six times with phosphate buffered saline-Tween20
(PBS-Tween20); and 100 .mu.l of biotinylated anti-IFN-.gamma.
antibody (2 .mu.g/ml; Pharmingen, cat. no. 554550) in PBS with 1%
bovine serum albumin (BSA) was added to each well. Plates were
incubated at 4.degree. C. overnight, washed three times with PBS,
and incubated with avidin alkaline phosphatase (Southern
Biotechnology Assoc., cat. no. 7100-04) at a 1:2000 dilution for
two hours at room temperature. Plates were washed three times with
PBS, incubated with KPL BCIP/NBT
(5-Bromo-6-Chloro-3-indoylphosphate p-Toluidine Salt/Nitro-Blue
Tetrazolium Chloride) phosphatase substrate for 30-60 minutes and
washed with distilled, deionized water. The plastic bottoms were
removed from the plates, rinsed and dried, and spots were
counted.
[0086] The Con A and vaccinia lysate conditions produced high
numbers of spots in the assays, as expected. The media condition
displayed few or no spots. High levels of spots were observed in
all wells in which C1R-A2 cells were used, indicating lack of
specificity of a response. Very few spots were seen in samples
containing CMMT 110/C1 cells.
[0087] IFN-.gamma. secretion was determined by performing
IFN-.gamma. ELISA assays using a Human IFN-.gamma. Immunoassay Kit
(R&D Systems Inc., Minneapolis, Minn.; catalog no. DIF50 or
SIF50). Culture supernatants were examined by ELISA both neat and
at 1:100 dilution. The results from the ELISA assays with undiluted
supernatants are plotted in FIG. 1. ConA samples were excluded from
the graph because the levels of IFN-.gamma. were too high to be
accurately measured. As shown in FIG. 1, all conditions other than
those that included C1R-A2 cells showed low or negative responses.
The non-specific responsiveness to C1R-A2 cells is thought to be
due to reaction of the PBMC to EBV antigens expressed by the C1R-A2
cells.
Example 2
HLA-A2.sup.+ and HLA-A2.sup.- Cells Respond to Antigens Presented
by Macaque Cells
[0088] The following assays were performed to examine
responsiveness of two patients receiving the PANVAC-VF regimen; an
HLA-A2.sup.+ patient, #7, and an HLA-A2.sup.- patient, #6
(002-006). PBMC of a non-vaccinated individual (Naive Donor) were
examined in parallel. The assays were performed as described in
Example 1, above. Incubations with the following combinations of
PBMC and target cells and/or antigens were performed: [0089]
PBMCs+Media [0090] PBMCs+Con A [0091] PBMCs+Vaccinia Lysate [0092]
PBMCs+C1R-A2s [0093] PBMCs+C1R-A2s pulsed with CAP-1-6D peptide
[0094] PBMCs+C1R-A2s pulsed with MUC-1 T2L peptide [0095]
PBMCs+CMMT 110/C1 [0096] PBMCs+CMMT 110/C1 infected with TBC-FPV
[0097] PBMCs+CMMT 110/C1 infected with rF-CEA/TRICOM [0098]
PBMCs+CMMT 110/C1 infected with rF-MUC-1/TRICOM [0099] PBMCs+CMMT
110/C1 infected with PANVAC-F (sample 1) [0100] PBMCs+CMMT 110/C1
infected with PANVAC-F (sample 2)
[0101] CMMT 110/C1 cells were infected with viruses at a MOI of 40.
For certain conditions, CMMT 110/C1 cells were also infected at a
MOI of 10. CMMT 110/C1 cells infected with two different samples of
PANVAC-F.
[0102] ELISPOT assays were performed as described in Example 1. The
results of the vaccinia, Con A, and media conditions in which
patient #7 and #6 PBMC samples were used were positive, positive,
and negative, respectively, as expected. The results in which
C1R-A2 cells were used exhibited high levels of background compared
to assays in which C1R-A2 cells were incubated alone, as a negative
control. No uniform results were observed in CMMT 110/C1 assays
when measured by ELISPOT.
[0103] ELISA assays for IFN-.gamma. assays were performed as
described in Example 1 above. Undiluted culture supernatants were
assayed. The results are shown in FIG. 2. Con A results are
excluded because the levels of IFN-.gamma. were too high to be
measured accurately. The results for conditions in which C1R-A2
cells were used are not graphed because there was very little
difference between the control and peptide-pulsed C1R-A2
samples.
[0104] As shown in FIG. 2, very little or no IFN-.gamma. was
produced by PBMC from the unvaccinated donor at all conditions
tested. Neither patient #7 nor 6 samples responded to CMMT 110/C1
cells infected with TBC-FPV and rF-CEA/TRICOM when the target cells
had been infected at a MOI of 10. The response to
rF-MUC-1/TRICOM-infected cells was more robust. Patients #7 and 6
PBMC responded to TBC-FPV, rF-CEA/TRICOM, rF-MUC-1/TRICOM, and
PANVAC-F infected cells prepared with a MOI of 40.
[0105] Responses of PBMC from vaccinated donors were generally
higher in the presence of cells infected with a virus expressing a
tumor antigen and were higher than those of the non-vaccinated
donor, suggesting that the responses are antigen-specific. The fact
that PBMC of both patients #6 and #7 exhibited responses indicate
that cells of both HLA-A2.sup.+ and an HLA-A2.sup.- donors can be
measured in this assay.
Example 3
Comparison of Immune Responsiveness of Vaccinated and
Non-Vaccinated Donors
[0106] The assays using CMMT 110/C1 target cells were repeated with
PBMC of patient #7, #6, and anon-vaccinated donor. In these
experiments, additional conditions were tested using CMMT 110/C1
cells infected with a fowlpox virus encoding human B7.1, LFA-3, and
ICAM-1 (rF-TRICOM). Cells were infected at a MOI of both 10 and 40
for all conditions tested. The following combinations of PBMC and
target cell/antigen conditions were tested: [0107] PBMCs+Media
[0108] PBMCs+Con A [0109] PBMCs+Vaccinia Lysate [0110] PBMCs+CMMT
110/C1 [0111] PBMCs+CMMT 110/C1 infected with TBC-FPV [0112]
PBMCs+CMMT 110/C1 infected with rF-CEA/TRICOM [0113] PBMCs+CMMT
110/C1 infected with rF-MUC-1/TRICOM [0114] PBMCs+CMMT 110/C1
infected with PANVAC-F (sample 1) [0115] PBMCs+CMMT 110/C1 infected
with PANVAC-F (sample 2)
[0116] ELISPOT assays were performed with cells after harvest, as
described in Example 1. Once again, the vaccinia, Con A, and media
controls gave the expected positive, positive, and negative
signals, respectively. Spots were detected from samples for subsets
of conditions with PBMC of patients #7 and #6, but no clear pattern
of response was detected between samples when measured by this
method.
[0117] IFN-.gamma. ELISA assays were performed with culture
supernatants as described in Example 1. The results are depicted in
FIG. 3, which shows that the levels of IFN-.gamma. were higher in
conditions employing CMMT 110/C1 cells infected at the higher MOI.
Con A results are not plotted in FIG. 3. The order of responses
under different sample conditions, ordered from highest response to
lowest, was the same for both vaccinated donors: PANVAC-F (sample
1)>PANVAC-F (sample
2)>rF-CEA/TRICOM>rF-MUC-1/TRICOM>rF-TRICOM>TBC-FPV. The
responses of PBMC from the non-vaccinated donor were equivalent at
both MOI of 10 and 40. The low levels of IFN-.gamma. secreted by
the non-vaccinated PBMC suggested a lack of antigen-specific
response in the non-vaccinated donor.
Example 4
Optimization of Incubation Time and Target:PBMC Ratios
[0118] The following assay was performed using PBMC from one
vaccinated patient, #6, undergoing treatment with the PANVAC-VF
regimen. This assay was designed to examine IFN-.gamma. levels
released over a one-week incubation period. Culture supernatants
were collected after 6 hours, 1 day, 2 days, 3 days, 4 days, 5
days, 6 days, and 7 days. The conditions were as follows: [0119]
PBMCs+Uninfected CMMT 110/C1 [0120] PBMCs+CMMT 110/C1 infected with
TBC-FPV [0121] PBMCs+CMMT 110/C1 infected with PANVAC-F (sample
1)
[0122] In addition, three different ratios of PBMC:CMMT 110/C1
cells for each condition above were tested: 1:1; 2:1; and 10:1. All
CMMT 110/C1 cells were infected at a MOI of 40. Levels of
IFN-.gamma. secretion in culture supernatants were quantitated by
ELISA as described in Example 1. The results are plotted in FIG. 4,
which shows that responses against uninfected and TBC-FPV were
negative at all days and cell ratios tested. Responses against
PANVAC-F were observed at 1:1, 2:1, and 10:1 PBMC: CMMT 110/C1
ratios. Levels of IFN-.gamma. increased with each additional day of
culture, with the highest levels observed at day 7.
[0123] The assay was repeated with PBMC from the same vaccinated
donor. Supernatants were collected after 3, 4, 5, 6, and 7 days of
incubation and IFN-.gamma. levels were quantitated by ELISA. The
results are depicted in FIG. 5. The 1:1 ratio of cells gave the
highest level of response to TBC-FPV-infected cells. The 10:1 ratio
produced the lowest level of response to TBC-FPV-infected cells.
Levels of responsiveness to TBC-FPV-infected cells appeared to
increase over time. The 2:1 and 10:1 ratios elicited the highest
levels of response to PANVAC-F-infected cells. Day 7 supernatants
contained the highest levels of IFN-.gamma. to PANVAC-F-infected
cells at all three ratios.
[0124] The assay conditions were repeated with PBMC from the same
vaccinated donor, #6, from a second vaccinated donor, #7, and a
non-vaccinated donor. CMMT 110/C1 cells were infected at a MOI of
40 with either TBC-FPV or PANVAC-F. Supernatants were collected
after 3, 4, 5, 6, and 7 days of incubation. Levels of IFN-.gamma.
secretion in culture supernatants were quantitated by ELISA. The
results for patients #6, 7, and non-vaccinated PBMC are depicted in
FIGS. 6, 7, and 8, respectively. The standard curves for this ELISA
did not produce accurate values, therefore the concentrations shown
in FIGS. 6, 7, and 8 may not reflect the actual concentration, but
trends are apparent. The relatives levels of IFN-.gamma. observed
for the different conditions in FIG. 6 for patient #6 are similar
to those obtained in FIG. 5. In general, levels of IFN-.gamma.
increased with each day of incubation. PANVAC-F induced levels of
IFN-.gamma. higher than those induced by TBC-FPV, indicative of a
specific, response to antigens expressed by PANVAC-F. This trend
was also observed in the assays with PBMC from patient 7 (FIG. 7).
Levels of IFN-.gamma. produced by PBMC from the non-vaccinated
donor were low at all conditions tested (FIG. 8).
Example 5
Identification of Responsive Cell Subsets
[0125] In order to examine the cell types responsible for
IFN-.gamma. production in response to stimulation with CMMT 110/C1
cells, PBMC from two PANVAC-vaccinated donors (patient #6 and #7)
and one non-vaccinated donor, were separated into subsets by cell
type and assayed for responsiveness to incubation with PANVAC-F or
TBC-FPV-infected CMMT 110/C1 cells. Dynal Negative Isolation kits
were used to prepare subsets. The subsets analyzed were total PBMC,
CD3.sup.+, CD4.sup.+, CD8.sup.+, and NK.sup.+ cells. Incubations
were performed with a ratio of 10:1 cell subsets: CMMT 110/C1 cells
using 5.times.10.sup.5:5.times.10.sup.4 cells due to low numbers of
cells recovered after separation. Supernatants were collected after
3 and 6 days in culture.
[0126] The subsets of cells which elicited an IFN-.gamma. response
by cells from vaccinated donors were PBMC (as expected), CD8.sup.+
cells, and CD3.sup.+ cells from samples exposed to
PANVAC-F-infected CMMT 110/C1 cells (FIG. 9). CD8.sup.+ cells from
patient #6 elicited the highest levels, followed by PBMC and
CD3.sup.+ cells of this patient. PBMC from patient #7 elicited the
highest IFN-.gamma. response, followed by CD8.sup.+ and CD3.sup.+
cells. All values obtained for the unvaccinated donor cells were
negative, except for positive responses from CD8.sup.+ cells
exposed to PANVAC-F-infected CMMT 110/C1 cells. The reason for this
response is unknown.
Example 6
Optimization of Plate Size and Cell Number and Comparison of
Patient Responsiveness Before and after Vaccination
[0127] The non-restricted assay was repeated with a 24-well plate
rather than a 48-well plate (as used in the assays described in
Examples 1-5). A 10:1 ratio of PBMC:CMMT 110/C1 cells were used,
with cell quantities of 1.times.10.sup.6 and 1.times.10.sup.5.
Supernatant was removed for IFN-.gamma. quantitation after 3 days
of incubation.
[0128] Patient responsiveness to treatment with the PANVAC-VF
regimen was also examined in this assay by comparing PBMC obtained
from a patient (#6) at day 0 or treatment and day 70 of treatment.
Another specificity control was added by adding a condition in
which CMMT 110/C1 cells were infected with PROSTVAC.RTM.-F.
(PROSTVAC.RTM.-F is a recombinant fowlpox that expresses the TAA
prostate-specific antigen (PSA) and TRICOM and does not express the
TAAs present in PANVAC-VF). The results are shown in FIG. 10. There
were no IFN-.gamma. responses in any of the day 0 conditions. The
day 70 PBMC from this patient also showed no response in the media
and uninfected CMMT 110/C1 conditions, and small responses in the
TBC-FPV, rF-TRICOM and PROSTVAC.RTM.-F conditions, possibly due to
responses against fowlpox antigens. The day 70 PBMC exhibited a
much greater (.about.5 times greater) response to PANVAC-F than to
any other conditions, suggestive of a TAA-specific response.
Example 7
Patient Responses at Various Time Points after Treatment
[0129] PBMC samples from two patients treated with the PANVAC-VF
regimen, patient #7 and patient #11 (001-011; an HLA-A2.sup.-
patient), were examined. PBMC taken 28, 42, 70 days, 70 days plus
one month, 70 days plus 2 months, and 70 days plus 3 months after
initiation of the PANVAC-VF regimen were tested. PBMC from these
time points were incubated in the following combinations: this was
the order in which to do the assay, depending on the number of
cells recovered. At some timepoints not all conditions were tested.
[0130] Each patient PBMCs (from all time-points)+CMMT 110/C1
infected with PANVAC-F [0131] Each patient PBMCs (from all
time-points)+CMMT 110/C1 infected with rF-TRICOM [0132] Each
patient PBMCs (from all time-points)+CMMT 110/C1 infected with
PROSTVAC.RTM.-F [0133] Each patient PBMCs (from all
time-points)+CMMT 110/C1 infected with rF-IF [0134] Each patient
PBMCs (from all time-points)+Uninfected CMMT 110/C1 cells [0135]
Each patient PBMCs (from all time-points)+CMMT 110/C1 infected with
TBC-FPV
[0136] PROSTVAC.RTM.-F -infected CMMT 110/C1 cells were used as a
control. Sets of target cells infected with rF-IF, a recombinant
fowlpox virus expressing an influenza antigen, were also used.
Cells were incubated in 24-well plates at 10:1 ratios. Supernatants
were collected after 3 days in culture and IFN-.gamma. levels were
quantitated by ELISA.
[0137] As shown in FIG. 11, PBMC from patient #6 exhibited the
highest responses to PANVAC-F-infected target cells at all time
points examined, releasing quantities of IFN-.gamma. 1.5-4 times
higher than those at all other conditions. The cells did not
respond to uninfected and TBC-FPV-infected targets. Low levels of
responses were seen to targets infected with rF-TRICOM, rF-IF, and
PROSTVAC.RTM.-F at day 70 and month 3. Responses by patient #11
PBMC were much lower than those of patient #6 PBMC, with the
highest responses appearing at day 42, day 70, and 70 days plus 2
months in cells stimulated with PANVAC-F-infected targets. Lower
levels of responses to rF-TRICOM were also observed. Overall, the
most robust responses in both patients were to cells infected with
PANVAC-F.
[0138] PBMC samples from two more patients receiving treatment with
the PANVAC regimen, patients #1 and #11 (both of whom are
HLA-A2.sup.-), were examined in the non-restricted assay. The assay
was performed in a 24-well plate with a 10:1 ratio of cells. PBMCs
from the following days after treatment were used in this
assay:
i. #1--Day 0, 14, 28, 42, 70, Month #1RD and Month #2
ii. #11--Day 14, 28, 42
[0139] PBMC were incubated with CMMT 110/C1 cells as follows, where
numbers of patient PBMC cells permitted: [0140] PBMCs (from all
time-points)+CMMT 110/C1 infected with PANVAC-F [0141] PBMCs (from
all time-points)+CMMT 110/C1 infected with rF-TRICOM [0142] PBMCs
(from all time-points)+CMMT 110/C1 infected with PROSTVAC.RTM.-F
[0143] PBMCs (from all time-points)+Con A in media at 2.5 .mu.g/ml
[0144] PBMCs (from all time-points)+Uninfected CMMT 110/C1
cells
[0145] Con A-containing samples exhibited very high levels of
IFN-.gamma. (.about.800 pg/ml or greater) in many assay conditions
and are not plotted on FIG. 12. Patient #1's cells were not healthy
once thawed, so for many time points, there were only enough cells
to perform one assay condition. As shown in FIG. 12, PBMC of all
patients exhibited the greatest responsiveness to PANVAC-F infected
cells at many of the time points tested. No PANVAC-F
-responsiveness was observed at day 0 in any of the samples.
Example 8
Responsiveness to Target Cells Expressing Tumor Antigens
[0146] The following assay was performed with PBMC collected 70
days after treatment with the PANVAC-VF regimen was initiated in
patients #7 and #6. PBMC of a non-vaccinated donor were also
tested. The non-restricted assay was performed in 24-well plates
with a 10:1 ratio of cells (1.times.10.sup.6:1.times.10.sup.5). The
following conditions were used: [0147] PBMCs+CMMT 110/C1 infected
with PANVAC-F [0148] Each patient PBMCs+CMMT 110/C1 infected with
rF-TRICOM [0149] Each patient PBMCs+CMMT 110/C1 infected with
rF-CEA(6D) [0150] Each patient PBMCs+CMMT 110/C1 infected with
rF-MUC-1 [0151] Each patient PBMCs+CMMT 110/C1 infected with
rF-CEA(6D)/TRICOM [0152] Each patient PBMCs+CMMT 110/C1 infected
with rF-MUC-1/TRICOM
[0153] Supernatants were removed after three days and IFN-.gamma.
levels were quantitated. The results are depicted in FIG. 13. No
responses were observed in PBMC from the non-vaccinated individual.
All samples from patients #6 and #7 responded in this assay.
rF-CEA(6D)/TRICOM elicited the highest response. PANVAC-F elicited
the next-highest response. rF-CEA (6D) elicited the next highest
response. rF-MUC-1 responses were low in these assays, which was
not expected.
Example 9
Analysis of Additional Patient PBMC Responses to Tumor Antigens
[0154] In this assay, PBMC from three different patients (#12, #13,
and #14) taken 0, 14, 28, 37, 42, and 63 days (or a subset thereof)
after initiation of PANVAC-VF treatment were incubated under the
following conditions: [0155] Each patient PBMCs (from all
time-points)+CMMT 110/C1 infected with PANVAC-F [0156] Each patient
PBMCs (from all time-points)+CMMT 110/C1 infected with rF-TRICOM
[0157] Each patient PBMCs (from all time-points)+CMMT 110/C1
infected with PROSTVAC.RTM.-F [0158] Each patient PBMCs (from all
time-points)+Con A in media at 2.5 .mu.g/ml [0159] Each patient
PBMCs (from all time-points)+Uninfected CMMT 110/C1 cells
[0160] The results are depicted in FIG. 14. Patient #13 PBMC
exhibited low/no responses under all conditions. High levels of
IFN-.gamma. secretion were observed in samples from patient #13 in
response to cells infected with all three viruses and in response
to ConA stimulation. Patient #14 PBMC responded to Con A at time
points from days 42 and 63 only, and did not exhibit responses to
infected cells at any time points.
Example 10
Long-Range Responsiveness in Vaccinated Individuals
[0161] In this assay, cells from patient 7 undergoing PANVAC-VF
treatment were incubated with CMMT 110/C1 cells infected with
various viral recombinants. PBMC samples obtained from the patient
at various times after initiation of treatment with the PANVAC-VF
regimen were tested. PBMC isolated from patient #7 at 4, 5, 7, and
9 months after initiation of PANVAC-VF treatment responded, whereas
cells from 70 days, 6 months, and 8 months after treatment did not
(FIG. 15). PANVAC-F elicited the greatest response (in those
samples from the time point in which a response was observed). The
responses elicited by PROSTVAC.RTM.-F and rF-TRICOM were roughly
equivalent, suggesting that the cells are responding to fowlpox
rather than PSA expression.
[0162] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
419PRTHomo sapiens 1Tyr Leu Ser Gly Ala Asn Leu Asn Leu 1 5
210PRTHomo sapiensmisc_featureSynthetically generated peptide 2Ala
Thr Trp Gly Gln Asp Val Thr Ser Val 1 5 10 38PRTHomo sapiens 3Tyr
Ser Gly Ala Asp Leu Asn Leu 1 5 410PRTHomo sapiens 4Ala Leu Trp Gly
Gln Asp Val Thr Ser Val 1 5 10
* * * * *