Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MSI-specific frameshift peptides (FSP) for prevention and
treatment of cancer
The present invention provides a vaccine for prevention and
treatment of cancer characterized by microsatellite
instability (MSI). The vaccine contains an MSI-specific
frameshift peptide (FSP) generating humoral and cellular
responses against tumor cells or a nucleic acid encoding
said FSP.
Human tumors develop through two major pathways of genome
instability, chromosomal instability and microsatellite
instability (MSI) that results from defects in the DNA
mismatch repair system. MSI is encountered in 15% of
colorectal cancers and a variety of extracolonic
malignancies showing a deficient DNA mismatch repair
system, including endometrial cancers, gastric cancers,
small bowel cancers and tumors of other organs. MSI cancers
may develop sporadically or in the context of a hereditary
tumor syndrome, hereditary non-polyposis colorectal cancer
(HNPCC) or Lynch syndrome.
MSI colorectal cancers are characterized by a high
immunogenicity that results from the generation of numerous
frameshift peptides (FSP) during the development of MSI
tumors as a direct result of mismatch repair deficiency
leading to alterations of the translational reading frame
when microsatellites in gene-encoding regions are affected
by mutation (Figure 1).
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The abundance of predictable MSI-specific FSP antigens and
the fact that they directly result from the malignant
transformation process render FSP highly promising targets
for immune therapy. It is believed that the human immune
system is a potential resource to eradicate tumor cells and
that effective treatment can be developed if the components
of the immune system are properly stimulated to recognize
and eliminate cancer cells. Thus, immunotherapy, which
comprises compositions and methods to activate the body's
immune system, either directly or indirectly, to shrink or
eradicate cancer, has been studied for many years as an
adjunct to conventional cancer therapy.
It is generally admitted that the growth and metastasis of
tumors depends largely on their capacity to evade host
immune surveillance. Most tumors express antigens that can
be recognized to a variable extent by the host immune
system, but in many cases, the immune response is
inadequate. Failure to elicit a strong activation of
effector T-cells may result from the weak immunogenicity of
tumor antigens or inappropriate or absent expression of co-
stimulatory molecules by tumor cells. For most T-cells,
production of IL-2 and proliferation require a co-
stimulatory signal simultaneous with TCR engagement,
otherwise, T-cells may enter a functionally unresponsive
state, known as clonal anergy.
In spite of the length of time that these therapies have
been investigated, there remains a need for improved
strategies for enhancing the immune response against the
tumor antigens.
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Nevertheless, there is a need in the art for safe and
effective compositions that can stimulate the immune system
as a cancer immunotherapy.
According to the invention, safe and effective stimulation
of the immune system as a cancer immunotherapy is achieved
by the subject matters defined in the claims. In vitro data
showed that FSP are highly immunogenic and can elicit
pronounced FSP-specific T cell responses in vitro
(Linnebacher et al. 2001, Ripberger et al. 2003, Schwitalle
et al. 2004). In further studies examining peripheral blood
drawn from patients with MSI colon cancer, a high frequency
of FSP-specific T cell responses was demonstrated
(Schwitalle et al. 2008). In spite of the high number of
FSP-specific T cells in the tumor and in the peripheral
blood, these patients showed no signs of autoimmunity,
suggesting that FSP vaccination approaches are not expected
to have side effects in terms of autoimmunity.
Immunological analyses in individuals carrying a germ line
mutation in DNA mismatch repair genes predisposing to
hereditary non-polyposis colorectal cancer (HNPCC) were
also found to exhibit cellular immune responses against
FSP, even in the absence of a clinically detectable tumor.
This suggests that FSP-specific immune responses may be
protective in HNPCC individuals, suggesting that FSP
vaccination may also be used in a preventive setting as the
first specific prevention approach in an inherited cancer
condition.
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In summary:
(a) Frameshift peptides (FSP) are MSI-specific and
directly result from MSI tumor pathogenesis;
(b) No clinically relevant side effects are expected;
(c) Combinations of FSPs are predicted to target all
tumors with MSI;
(d) FSP vaccination has been designed for therapy of 15%
of colon cancers and tumors of the endometrium, stomach,
small intestine and other organs;
(e) Molecular tumor analysis can identify patients that
may benefit from FSP vaccination (targeted therapy); and
(f) FSP vaccination may be used as a preventive
vaccination in high risk groups.
Brief description of the drawings
Figure 1: Schematic illustration of coding microsatellite
instability resulting from DNA mismatch repair deficiency
(Kloor et al., 2010)
Truncated proteins encompassing FSP sequences (red) are
generated when coding microsatellite mutations lead to
alterations of the translational reading frame (example:
TGFER2 protein).
Figure 2: Exemplary T cell responses against newly designed
FSPs in peripheral blood from three patients with MSI colon
cancer
Figure 3: Humoral immune responses against FSPs derived
from AIM2(-1), HT001(-1), TAF1B(-1), and TGFBR2(-1)
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ELISA revealed FSP-specific antibody responses directed
against neopeptides derived from AIM2(-1), HT001(-1),
TAF1B(-1), and TGFBR2(-1). Peptide specificity was
demonstrated by preabsorption of respective serum
antibodies as described previously (Reuschenbach et al.,
2008).
Figure 4: Cytotoxic responses as determined by CD107a
surface expression
(A) Significant FSP-specific responses were observed in
different healthy donors after stimulation of T cells with
the antigen for four weeks. Responses did only occur in the
presence of antigen-presenting B cells and the FSP antigen.
Representative responses are shown in bar graphs.
(B) representative FACS analysis of CD107a assay for T
cells incubated with B cells as antigen-presenting cells in
the absence (left panel) or presence (right panel) of the
FSP antigen HT001(-1).
Thus, the present invention provides a vaccine containing
an MSI tumor specific frameshift peptide (FSP), e.g.,
derived from TAF1B (Acc.No. L39061), HT001 (Acc.No.
AF113539), AIM2 (Acc.No. AF024714), or TGFBR2 (Acc.No.
N14_003242) or a nucleic acid encoding said FSP wherein said
FSP is capable of eliciting an immune response against
cancer showing MSI.
In a preferred embodiment, the vaccine of the present
invention contains
(a) an FSP comprising or consisting of the following amino
acid sequence:
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NTQIKALNRGLKKKTILKKAGIGMCVKVSSIFFINKQKP (TAF1B(-1));
EIFLPKGRSNSKKKGRRNRIPAVLRTEGEPLHTPSVGMRETTGLGC (HT001(-1));
HSTIKVIKAKKKHREVKRTNSSQLV (AIM2(-1));
or
AS PKC IMKEKKSLVRLSSCVPVALMSAMTTS S SQKNI TPA I LTCC ( TGFBR2 ( - 1 ) ) ;
(b) a functional equivalent of an FSP of (b); or
(c) a combination of the FSP of (a) and/or (b).
The term "functional equivalent" as used herein relates to,
e.g., variants or fragments of the FSP which are still
capable of eliciting an immune response against the cancer,
i.e., are still useful as an efficient vaccine. An immune
response is defined as a condition fulfilling at least one
of the following criteria: 1. The induction of CD8-positive
T cells, as detectable by cytotoxicity assays or IFN-gamma
secretion or perforin expression or granzyme B expression
or other cytokines that may be produced by CD8-positive T
cells, measurable as above background by ELISpot or
intracellular cytokine staining or cytokine ELISA or
equivalent methods. 2. The induction of CD4-positive T
cells, as detectable by cytokine secretion measurable as
above background by ELISpot or intracellular cytokine
staining or cytokine ELISA or equivalent methods. Cytokines
may comprise IFN-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-6,
IL-10, IL-12, IL-13, IL-17, TNF-alpha, TGF-beta or other
cytokines that may be produced by CD4-positive T cells. 3.
The induction of antibodies, as detectable by Western blot,
ELISA and equivalent or related methods. 4. The induction
of any kind of cellular Immune response not mediated by
CD8-positive or CD4-positive T cells as described in 1 and
2.
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The variants are characterized by amino acid deletions,
substitutions, and/or additions. Preferably, amino acid
differences are due to one or more conservative amino acid
substitutions. The term "conservative amino acid
substitutions" involves replacement of the aliphatic or
hydrophobic amino acids Ala, Val, Leu and Ile; replacement
of the hydroxyl residues Ser and Thr; replacement of the
acidic residues Asp and Glu; replacement of the amide
residues Asn and Gln, replacement of the basic residues
Lys, Arg, and His; replacement of the aromatic residues
Phe, Tyr, and Trp, and replacement of the small-sized amino
acids Ala, Ser, Thr, Met, and Gly.
For the generation of peptides showing a particular degree
of identity to the FSP, e.g., genetic engineering can be
used to introduce amino acid changes at specific positions
of a cloned DNA sequence to identify regions critical for
peptide function. For example, site directed mutagenesis or
alanine-scanning mutagenesis (introduction of single
alanine mutations at every residue in the molecule) can be
used (Cunningham and Wells, 1989). The resulting mutant
molecules can then be tested for immunogenicity using the
assay of Example 1.
Preferably, the variants are characterized by not more than
8 aa, more preferably by not more than 6 aa and, even more
preferably, by not more than 4 aa substitutions, deletions
and/or additions.
In the fragment of an FSP at least 5 contiguous aa,
preferably at least 10 contiguous aa, more preferably at
least contiguous 15 aa and even more preferably at least 20
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contiguous aa of the particular amino acid sequence are
left. The fragment is still capable of eliciting an immune
response.
In a more preferred embodiment, the vaccine of the present
invention additionally comprises an adjuvant and/or
immunostimulatory cytokine or chemokine.
Suitable adjuvants include an aluminium salt such as
aluminium hydroxide gel (alum) or aluminium phosphate, but
may also be a salt of calcium, iron or zinc, or may be an
insoluble suspension of acylated tyrosine, or acylated
sugars, cationically or anionicaily
derivatised
polysaccharides, or polyphosphazenes. Other known adjuvants
include CpG containing oligonucleotides. The
oligonucleotides are characterised in that the CpG
dinucleotide is unmethylated. Such oligonucleotides are
well known and are described in, for example WO 96/02555.
The use of immunostimulatory cytokines has become an
increasingly promising approach in cancer immunotherapy.
The major goal is the activation of tumour-specific T
lymphocytes capable of rejecting tumour cells from patients
with low tumour burden or to protect patients from a
recurrence of the disease. Strategies that provide high
levels of immunostimulatory cytokines locally at the site
of antigen have demonstrated pre-clinical and clinical
efficacy. Preferred immunostimulatory cytokines comprise
IL-2, IL-4, IL-7, IL-12, IFNs, GM-CSF and TNF-a.
Chemokines are small (7-16 kD), secreted, and structurally
related soluble proteins that are involved in leukocyte and
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dendritic cell chemotaxis, PIAN degranulation, and
angiogenesis. Chemokines are produced during the initial
phase of host response to injury, allergens, antigens, or
invading microorganisms. Chemokines selectively attract
leukocytes to inflammatory foci, inducing both cell
migration and activation. Chemokines may enhance innate or
specific host immunity against tumors and, thus may also be
useful in combination with an FSP.
The vaccine of the present invention might also contain a
nucleic acid encoding the FSP for DNA immunization, a
technique used to efficiently stimulate humoral and
cellular immune responses to protein antigens. The direct
injection of genetic material into a living host causes a
small amount of its cells to produce the introduced gene
products. This inappropriate gene expression within the
host has important immunological consequences, resulting in
the specific immune activation of the host against the gene
delivered antigen. Direct injection of naked plasmid DNA
induces strong immune responses to the antigen encoded by
the gene vaccine. Once the plasmid DNA construct is
injected the host cells take up the foreign DNA, expressing
the viral gene and producing the FSP inside the cell. This
form of antigen presentation and processing induces both
MHC and class I and class II restricted cellular and
humoral immune responses. The DNA vaccines are composed of
vectors normally containing two unites: the antigen
expression unit composed of promoter/enhancer sequences,
followed by antigen (FSP)-encoding and polyadenylation
sequences and the production unit composed of sequences
necessary for vector amplification and selection. The
construction of vectors with vaccine inserts is
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accomplished using recombinant DNA technology and the
person skilled in the art knows vectors that can be used
for this approach. The efficiency of DNA immunization can
be improved by stabilising DNA against degradation, and
increasing the efficiency of delivery of DNA into antigen
presenting cells. This has been demonstrated by coating
biodegradable cationic microparticles (such as
poly(lactide-co-glycolide) formulated
with
cetyltrimethylammonium bromide) with DNA. Such DNA-coated
microparticles can be as effective at raising CTL as
recombinant vaccinia viruses, especially when mixed with
alum. Particles 300 nm in diameter appear to be most
efficient for uptake by antigen presenting cells.
A variety of expression vectors, e.g., plasmids or viral
vectors, may be utilised to contain and express nucleic
acid sequences encoding an FSP of the present invention.
A preferred viral vector is a poxvirus, adenovirus,
retrovirus, herpesvirus or adeno-associated virus (AAV).
Particularly preferred poxviruses are a vaccinia virus,
NYVAC, avipox virus, canarypox virus, ALVAC, ALVAC(2),
fowlpox virus or TROVAC.
Recombinant alphavirus-based vectors have also been used to
improve DNA vaccination efficiency. The gene encoding the
FSP is inserted into the alphavirus replicon, replacing
structural genes but leaving non-structural replicase genes
intact. The Sindbis virus and Semliki Forest virus have
been used to build recombinant alphavirus replicons. Unlike
conventional DNA vaccinations, however, alphavirus vectors
are only transiently expressed. Alphavirus replicons raise
CA 02799803 2012-12-13
an immune response due to the high levels of protein
expressed by this vector, replicon-induced cytokine
responses, or replicon-induced apoptosis leading to
enhanced antigen uptake by dendritic cells.
In a further preferred embodiment, the FSP contains a Tag
sequence, preferably at the C-terminus which might be
useful for purification of a recombinantly produced FSP. A
preferred Tag sequence is a His-Tag. A particularly
preferred His-Tag consists of 6 His-residues.
The vaccine of the present invention is administered in an
amount suitable for immunization of an individual and,
preferably, additionally contains one or more common
auxiliary agents. The employed term "amount suitable for
immunization of an individual" comprises any amount of FSP
with which an individual can be immunized. The amount
depends on whether immunization is intended as a
prophylactic or therapeutic treatment. In addition, the
individual's age, sex and weight play a role for
determining the amount. Thus, the amount suitable for
immunization of an individual refers to amounts of the
active ingredients that are sufficient to affect the course
and the severity of the tumor, leading to the reduction or
remission of such pathology. An ""amount suitable for
immunization of an individual" may be determined using
methods known to one skilled in the art (see for example,
Fingl et al., 1975). The term "individual" as used herein
comprises an individual of any kind and being able to fall
ill with carcinomas. Examples of such individuals are
humans and animals as well as cells thereof.
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The administration of the vaccine by injection may be made
at various sites of the individual intramuscularly,
subcutaneously, intradermally or in any other form of
application. It may also be favourable to carry out one or
more "booster injections" having about equal amounts.
The employed term "common auxiliary agents" comprises any
auxiliary agents suitable for a vaccine to immunize an
individual. Such auxiliary agents are, e.g., buffered
common salt solutions, water, emulsions, such as oil/water
emulsions, wetting agents, sterile solutions, etc.
An FSP, nucleic acid sequence or vector of the present
invention can be present in the vaccine as such or in
combination with carriers. It is favourable for the
carriers in the individual not to be immunogenic. Such
carriers may be the individual's own proteins or foreign
proteins or fragments thereof. Carriers, such as serum
albumin, fibrinogen or transferrin or a fragment thereof
are preferred.
The vaccine of the present invention may be therapeutic,
that is, the compounds are administered to treat an
existing cancer, or to prevent the recurrence of a cancer,
or prophylactic, that is, the compounds are administered to
prevent or delay the development of cancer. If the
compositions are used therapeutically, they are
administered to cancer patients and are designed to elicit
an immune response to stabilize a tumor by preventing or
slowing the growth of the existing cancer, to prevent the
spread of a tumor or of metastases, to reduce the tumor
size, to prevent the recurrence of treated cancer, or to
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eliminate cancer cells not killed by earlier treatments. A
vaccine used as a prophylactic treatment is administered to
individuals who do not have cancer, and are designed to
elicit an immune response to target potential cancer cells.
The present invention also relates to the use of an FSP or
functional equivalent, nucleic acid sequence or vector as
defined above for the production of a vaccine for the
prevention of a carcinoma, e.g., preventive vaccination of
a high risk group, or treatment of a carcinoma. For
example, these may be a colorectal cancer, preferably a
hereditary non-polyposis colorectal cacner (BNPCC), an
endometrial cancer, a gastric cancer or small bowel cancer.
By means of the present invention it is possible to
immunize individuals, in particular humans and animals.
Immunization takes place by both induction of antibodies
and stimulation of C1J8' T cells. Thus, it is possible to
take prophylactic and therapeutic steps against carcinomas.
The below examples explain the invention in more detail.
Example 1
Detection of FSP-specific T cells in peripheral blood from
patients with MSI colon cancer and healthy HNPCC mutation
carriers
(A) Methods (ELISpot Assay)
Frequencies of FSP-specific peripheral T cells (pTc) were
quantified by determining the number of specific IFN-gamma-
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secreting Tc against newly designed FSPs derived from 3
cMS-containing candidate genes using ELISpot analysis.
ELISpot assays were performed using 96-well nitrocellulose
plates (Multiscreen; Millipore, Bedford, MA) coated
overnight with mouse anti-human IFN-gamma monoclonal
antibodies (mAb) (Mabtech, Nacka, Sweden) and blocked with
serum containing medium. PTc (day 0, lx105/well) were plated
6-fold with autologous CD40-activated B cells (4x104/well,
TiBc or pBc, respectively) as antigen-presenting cells in
200 pl IMDM with 10% human AB serum. Peptides were added at
a final concentration of 10 pg/mL. As a positive control,
pTc were treated with 20 nmol/L phorbol-12-myristate-13-
acetate in combination 350 nmol/L ionomycin. After
incubation for 24 hours at 37 C, plates were washed
thoroughly, incubated with biotinylated rabbit anti-human
IFN-gamma mAb for 4 hours, washed again, and incubated with
streptavidin-alkaline phosphatase for 2 hours, followed by
a final wash step. Spots were detected by incubation with
NBT/BCIP (Sigma-Aldrich) for 1 hour, reaction was stopped
with water, and, after drying, spots were counted
microscopically. Methods have been described in detail in
Schwitalle et al., 2008.
(B) Results
To examine whether FSP-specific T cell responses were
detectable in the peripheral blood of MSI-H CRC patients,
ELISpot analyses were performed. Autologous pBc, which
showed high expression of MHC class I and II, and
costimulators (CD40, CD80, and CD86) as well as B-cell-
specific antigens (CD19 and CD23) were used as antigen-
presenting cells.
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Pronounced reactivities were observed against newly
designed FSPs derived from AIM2(-1), HT001(-1), TAF1B(-1),
and TGFBR2(-1):
TAF1B (-1) NTQIKALNRGLKKKTILKKAGIGMCVKVSSIFFINKQKP
HT001(-1) EIFLPKGRSNSKKKGRRNRIPAVLRTEGEPLHTPSVGMRETTGLGC
AIM2 (-1) HSTIKVIKAKKKHREVKRTNSSQLV
TGFBR2 ( -1) ASPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC
(neopeptide sequences are underlined)
The results obtained from patients (n=8) are summarized in
Table 1. Representative ELISpot results are shown in Figure
2.
Table 1
FSP-specific T cell responses against FSPs
Patient ID no peptide TAF1B (-1) HT001 (-1) AIM2 (-1) TGFBR2 (-1)
MS101 4 8.17 5.83 3.5 6
MS102 1.5 4.83 7.33 7.17 1.2
MS103 11.83 28.17 31.83 23.33 10.16
MSI04 5.5 13 14.17 11.83 8.75
MSI05 3 7.75 16 10 2.8
MS106 3.17 12 12.33 13.83 5.2
MS107 6 17 16.83 14.5 9.33
MC01 1.17 3.5 3.5 3 8
MCO2 1.67 2.83 5.83 4 3.33
MC03 0.83 1.17 1.17 2.17 4.4
MC04 15.17 18.83 18.67 15.17 20.75
MC05 0.33 3.17 2.17 1.33 5.67
MC06 _ 30.5 37.83 37.8 34.83 39.5
Mean spot numbers from replicate analyses are given for
each peptide and tested individual. MSI01-MSIO7 - patients
CA 02799803 2012-12-13
with MSI-H CRC, MC01-MC06 - healthy HNPCC germ line
mutation carrier.
Example 2
Detection of FSP-specific humoral immune responses in
peripheral blood from patients with MSI colon cancer and
healthy HNPCC mutation carriers
(A) Methods (ELISA)
For enzyme-linked immunosorbent assay (ELISA), peptides
were coated to 96 well polystyrol microtiter plates
"Maxisorp"' (Nunc, Roskilde, Denmark) at a concentration of
40 pg/m1 in PBS overnight at 4 C. After coating, plates
were washed 4 times with PBS (0.05% Tween) and blocked for
1 h with 0.5% casein in PBS. Peptide binding to the
microtiter plates and optimal saturating peptide
concentration were assessed using an alkaline phosphatase¨
peptide competition assay. To monitor individual background
reactivity of each serum, a control peptide derived from
the p16INK4a protein (p16_76-105) was used, against which no
antibody reactivity was found in a large cohort of
individuals (Reuschenbach et al., 2008). Each serum was
diluted 1:100 in blocking buffer (0.5% casein in PBS) and
tested in duplicates for the presence of antibodies against
all FSPs and the control peptide. As a reference for inter-
plate variance, one control serum was included on every
plate, and peptide specific ODs of the control serum were
used for normalization. Diluted sera (50 pl /well) were
incubated for 1 h, and after a wash step plates were
incubated with HRP-labeled rabbit anti-human-IgG antibody
(Jackson Immunoresearch, West Grove, PA; 1:10,000 in
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blocking buffer) for 1 h. After washing, 50 pl /well of TMB
substrate (Sigma, Deisenhof en, Germany) was added and the
enzyme reaction was stopped after 30 minutes by adding 50
pl /well of 1 N H2SO4. Absorption was measured at 450 nm
(reference wavelength 595 nm). Pre-absorption of serum
antibodies for specificity control was done according to
the method described in detail in Reuschenbach et al.,
2008.
(B) Results
To examine whether FSP-specific antibodies were detectable
in the peripheral blood of MSI-H CRC patients, healthy
Lynch syndrome mutation carriers, and healthy controls
ELISA analyses were performed. Pronounced reactivities were
observed against newly designed FSPs derived from AIM2(-1),
HT001(-1), TAF1B(-1), and TGFBR2(-1). ELISA results are
shown in Figure 3.
Example 3
Detection of FSP-specific cytotoxic T cell responses
CD107a surface expression on T effector cells upon
stimulation with the clinical FSP antigens was measured.
CD107a assays are used to demonstrate secretion of
cytotoxic granula containing perforin/granzyme B from
effector cells. CD107a molecules are expressed on the
surface of cytotoxic granula and become detectable on the
cell surface if granula are released in the context of a
cytotoxic T cell response.
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To determine the potential of the FSP peptides to induce a
cytotoxic cellular immune response, blood was drawn from
healthy donors, and T cells were stimulated with the FSPs
using dendritic cells as antigen-presenting cells.
Stimulation was repeated weekly and over a time span of
four weeks. After four weeks, T cells were harvested and
coincubated with target cells and FSPs CD107a assay was
used to analyze peptide-specific induction of a cytotoxic T
cell response.
Cytotoxic responses as determined by CD107a surface
expression were observed for T cells coincubated with B
cells as antigen-presenting cells in the presence of the
antigenic FSP (Figure 4A). Significant responses were
observed in different healthy donors after stimulation of T
cells with the antigen for four weeks. Representative
responses are shown in bar graphs. Figure 4B shows
representative FACS analysis of CD107a assay for T cells
incubated with B cells as antigen-presenting cells in the
absence (left panel) or presence (right panel) of the FSP
antigen HT001(-1).
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