and Immunomodulatory xenobiotics) .For modulation of Th cell responses, peptides offere several advantages over intact Ags as immunogens or tolerogens (either as altered peptide ligands (APLs), competitors, or vaccines). First, peptides require less stringent degradative conditions than native Ags. Second, with a smaller determinant, there is less likelihood of cross-reactivity between the peptide and other self-proteins. And third, peptides offer exquisite specificity over native Ags. Despite these advantages, the use of peptides has remained fairly limited, because they are rapidly cleared from the circulation and poorly taken-up by APCs (by pinocytosis only) : effectiveness of taking up can be increased by coupling peptides to ligands (eg. transferrin (Tf)) specific for cell surface receptors found on APCs (eg. CD71 / TfR).
) are
used for chronic infections and cancers
vaccine : if given within 4 days of exposure to virus, it
halts the disease's advance.
vaccine : intramuscular
(2.5 IU = 1 mL) or intradermic (0.1 mL) injections of
at day 0, 3, 7, 14, 28 and 90. Effective
for 2-3 years. The treatment of rabies exposure has come a
long way since the time a person had to take 21 shots in
the stomach. Today, most people need 5 rabies vaccinations
in the arm spaced out over a 28-day period, as well as a
one-time dose of rabies
immune globulin (RIG).
The WHO recommends 2 economical intradermal regimens for post-exposure
rabies
prophylaxis (PEP) :;
purified
chick
embryo cell vaccine (PCECV)
and purified
duck
embryo vaccine (PDEV).
The other recommended vaccine, purified
vero
cell rabies vaccine (PVRV),
has only 0.5 ml per ampoule and it has not been tested with
the 8-site regimen, but if it were used the logical dose
would be 0.05 ml per site, or alternatively 0.1 ml in half
the number of sites. (A whole ampoule of vaccine must be
used on day 0 in every case). The 2-site id regimen was
designed for use with PVRV. It requires a dose of 0.1 ml per
site for PVRV and 0.2 ml per site for the other 3 vaccines.
Full details
of
the economical regimens and practical advice are
availableon the WHO website. The advantages of the 8-site
method are a rapid induction of neutralising antibody, making it the treatment of choice
when no rabies immunoglobulin is available (>95% of
post-exposure treatments in the third world); a wide margin
of safety, because if up to half of the 8 id injections on
day 0 are not truly id, the immune response will still be
adequate; and using a wholeampoule on day 0, which avoids
the need to share ampoules of vaccine between patients
during the emergency treatment and eliminates any risk of
contamination or wastage of vaccine. Finally, giving a large
antigenic stimulus on day 0 gives the best chance of
survival to patients who are 'low responders' to the vaccine
and to those who fail to return on time for subsequent
doses, a common problem in the rural tropics. The complexity
of these treatments demonstrates the urgent need for
animproved, single, simplified regimen that is suitable for
use with all the WHO recommended vaccines. Efforts are being
made to achieve this goal. Meanwhile there is reluctance to
use these vaccines economically. This deprives many patients
of excellent treatment and perpetuates double standards of treatment in the third world.
Under current CDC guidelines, post-exposure prophylaxis for
persons not pre-exposure vaccinated currently consists of a vaccine
: V-1 Immunitor
(V1) (Remune©; Immune Response Corp.,
Carlsbad, CA, USA, and Trinity Medical Group Co., Ltd. of
Bangkok, Thailand) is a low-cost,
polyvalent therapeutic mucosal
AIDS vaccine against core antigens
based on the Salk Immunogen, which is derived from an HIV
isolate which has been inactivated by chemical depletion
of gp120 (clade A/G) : it is composed of an HIV-1 isolate
(HZ321) from serum collected from a patient in Zaire in
1976. This virus has been sequenced and classified as
clade A envelope and clade G Gag and grown in the Hut 78
T-cell lineref1,
ref2,
ref3.
It was developed and manufactured in Thailand and
has been licensed by the Thai FDA as a orally available dietary supplement
and investigational R&D drug. It
has shown effectiveness against wasting syndromeref1,
ref2,
ref3.
A phase II double-blind controlled clinical trial using
HIV-1 immunogen was undertaken in Thailand in 1995 in
conjunction with phase III approval in the USA. In most
instances, immune response to the virus was induced. The
results from this study led to the extension of further
trials over the following 4 years. At present, the Thai
Food and Drug Administration (FDA) is in the process of
reviewing the files on all information to assess the
effectiveness of this therapeutic vaccine as well as
reviewing the proposal to conduct a phase III trial to
further confirm previous efficacy results from the phase
II trialsref.
vaccine : the incidence
and severity of herpes zoster and postherpetic neuralgia
increase with age in association with a progressive
decline in cell-mediated immunity to VZV. 38,546 adults
> 60 years of age were enrolled in a randomized,
double-blind, placebo-controlled trial of an
investigational live attenuated Oka/Merck VZV vaccine
("zoster vaccine"). Herpes zoster was diagnosed according
to clinical and laboratory criteria. The pain and
discomfort associated with herpes zoster were measured
repeatedly for 6 months. The primary end point was the
burden of illness due to herpes zoster, a measure affected
by the incidence, severity, and duration of the associated
pain and discomfort. The secondary end point was the
incidence of postherpetic neuralgia. > 95% of the
subjects continued in the study to its completion, with a
median of 3.12 years of surveillance for herpes zoster. A
total of 957 confirmed cases of herpes zoster (315 among
vaccine recipients and 642 among placebo recipients) and
107 cases of postherpetic neuralgia (27 among vaccine
recipients and 80 among placebo recipients) were included
in the efficacy analysis. The use of the zoster vaccine
reduced the burden of illness due to herpes zoster by
61.1%, reduced the incidence of postherpetic neuralgia by
66.5% (P<0.001), and reduced the incidence of herpes
zoster by 51.3%. Reactions at the injection site were more
frequent among vaccine recipients but were generally mild.
The zoster vaccine markedly reduced morbidity from herpes
zoster and postherpetic neuralgia among older adults.) : on the
basis of the successes of attenuated pathogen vaccines and
owing to the initial lack of defined tumour antigens, the
first cancer vaccines were composed of whole cancer cells
removed during surgery, killed by irradiation and
re-administered together with non-specific adjuvants.
(modified tumour cells / transfectoma) : hapten-treated autologous melanoma
cells,
then killed by irradiation and administered intradermallyref
(good responses)ref1,
ref2,
ref3,
ref4
(good responses)ref1,
ref2ref1,
ref2ref in patients with recurrent
prostate
adenocarcinoma.
A single-institution phase I/II trial was done in hormone
therapy-naive patients with PSA relapse following
radical prostatectomy and absence of radiologic
metastases. Treatments were administered weekly via
intradermal injections of 1.2 x 108 GM-CSF
gene-transduced, irradiated, cancer cells (6 x 107
LNCaP cells and 6 x 107 PC-3 cells) for 8
weeks. 21 patients were enrolled and treated. Toxicities
included local injection-site reactions, pruritus, and
flu-like symptoms. 1 patient had a partial PSA response of
7-month duration. At 20 weeks post first treatment, 16 of
21 (76%) patients showed a statistically significant
decrease in PSA velocity (slope) compared with
prevaccination (P < 0.001). Injection site biopsies
showed intradermal infiltrates consisting of CD1a+
dendritic cells and CD68+ macrophages, similar
to previous clinical trials using autologous
GM-CSF-transduced cancer cells. Posttreatment, patients
developed new oligoclonal antibodies reactive against at
least five identified antigens present in LNCaP or PC-3
cells. A high-titer antibody response against a 250-kDa
antigen expressed on normal prostate epithelial cells was
induced in a patient with partial PSA remission; titers of
this antibody decreased when treatment ended, and
subsequent PSA relapse occurredrefref1,
ref2 is an allogeneic melanoma tumor cell lysate
combined with the adjuvant Detox-PC, often in regimens
containing pretreatment with low-dose cyclophosphamide
and IFN-a2bref1,
ref2
: phase I and II trials in stage IV patients showed a
10-20% response rate (clearing of some metastatic sites)
and in another 10-20% of patients disease was stabilized
(no progression for various periods of time of tumours
that were growing at the start of the vaccine protocol.
It may have its greatest benefit in a large subset of
melanoma patients who express either the HLA-A2 and/or
HLA-C3ref.
and colon cancer : irradiated allogeneic cells induce IgG
and IgM immune responses to glycoprotein
melanoma-associated antigen (TA90) after surgical
resection, but only the IgM response is correlated with
improved survival : endogenous immune responses to TA90
have also been reportedref.
It is a polyvalent vaccine (PV) containing > 20 tumor
antigens. In vitro cellular immune response to
Canvaxin cells directly correlates with survival after
subsequent initiation of immunotherapy for AJCC stage III
melanomaref.
Canvaxin is currently undergoing a multi-centre phase III
randomized clinical trials for melanoma and a phase II
trial for colorectal
carcinoma.
and colorectal
carcinomas. as adjuvants would
activate DCs to prime immunity to many autoantigens (otherwise
subject to peripheral tolerance) other than the tumor antigens :
the use of whole tumour cells or complex mixtures o
tumour-derived material undermines one unique advantage that
immunotherapy has over other forms of therapy - that is,
specificity. Recently, in spite of the availability of
well-defined tumour antigens, development in the cancer-vaccine
field has focused again on the use of whole tumour cells or
whole cell lysates as antigens : the reason being that these
complex mixtures will contain unique tumour antigens
that are expressed only by an individual tumour that, by analogy
to unique antigens of mouse carcinogen-induced tumours might be
more immunogenic and promote a better anti-tumour immune
response. But experiments carried out in mice transgenic for shared tumour antigenshave
shown that these antigens can elicit equally strong antitumour
immunity and tumour rejection.
vaccine : conventional
envelope-based antibody-inducing vaccines do not appear to
hold promise, and broadly-neutralizing antibodies are now
being searched as an alternative to the failed approach with
subunit vaccines. The current consensus is that cellular
immune responses, especially those mediated by CD8+
CTL and CD4+ Th lymphocytes, are
needed to control HIV. Vaccines capable of inducing
cell-mediated responses are, therefore, considered critical
for controlling the spread of HIV. DNA-based vaccines
triggering CTL reaction are currently thought to be an
answer, but will they fulfill the promise?
vaccine : P.
insidiosum-antigen (PIA) 100-200 ml of PIA (2.0 mg/ml), at a 14-day
intervalref1,
ref2,
ref3-antigen therapeutic vaccines
(see also immunooncology).
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has developed as a powerful technique to select ligands to
essentially any chosen target from diverse repertoires of
peptides, proteins, or Abs displayed on the surface of a
phage particle. Previous application has mainly focused on
Ab libraries and constrained or linear peptide libraries.
This technology has also been used to identify immunogenic
targets or epitopes recognized by Abs; for example,
selection of peptide libraries on mAbs and complex sera of
patients with disease has led to the isolation of
immunoreactive peptide epitopes. Although mapping sera with
peptide phage libraries has had some success with small
viral genomes such as HBV,
the selection of peptide repertoires appears not to be a
suitable approach when profiling the complexity of the
immune response in the context of the human genome. One of
the drawbacks is that often predominantly Ag mimotopes
are recovered, for which further complicated analysis is
required to recover the original Ag that elicited the immune
response. A more successful approach is to display cDNA
expression libraries on filamentous phage. In contrast to
the use of peptide display repertoires, there have been
significantly fewer reports on cDNA display due to technical
challenges in their construction and expression. For
example, due to stop codons inherent to cDNA, cDNA display
libraries cannot be fused to the N terminus of the popular
phage anchor protein pIII. As such, anchoring of the cDNA
product on the phage coat has to be performed via indirect
linkage to pIII or by direct fusion to the C terminus of
another phage coat protein, pVI. In the case of low titer
IgG response that predominate in immunosuppressed conditions
such as cancer, early studies using autologous selection of
phage-displayed primary tumor cDNA repertoires were
unsuccessful, although selections with both mAbs and
polyclonal rabbit sera did recover specific ligands.
Selections performed with immobilized autologous cancer
patient IgG using and anti-human IgG capture Ab resulted in
the preferential recovery of only IgG transcripts that were
present due to tumor-infiltrating B cells. Mimotopes,
mimetics of T cell epitopes, have been derived from known
epitopes by sequence modification, or developed de novo
using combinatorial peptide libraries to scan the entire
sequence space for peptides that induce the desired T cell
responsesref.-associated
tumors provide target Ags for immunotherapy
and Hodgkin's
lymphoma
that have a restricted pattern of EBV Ag expression
(type II latency) is under investigation. Potential
EBV Ag targets for T cell therapy expressed by these
tumors include LMP1 and LMP2. A broad panel of
epitopes must be identified from these target Ags to
optimize vaccination strategies and facilitate
monitoring of tumor-specific T cell populations after
immunotherapeutic interventions. To date, LMP2
epitopes have been identified for only a limited
number of HLA alleles. Using a peptide library
spanning the entire LMP2 sequence, 25 CTL lines from
patients with EBV+ malignancies expressing
type II latency were screened for the presence of
LMP2-specific T cell populations. In 21 of 25 lines, T
cell responses against 1 to 5 LMP2 epitopes were
identified. These included responses to previously
described epitopes as well as to newly identified
HLA-A*0206-, A*0204/17-, A29-, A68-, B*1402-, B27-,
B*3501-, B53-, and HLA-DR-restricted epitopes. 7 of
the 9 newly identified epitopes were antigenically
conserved among virus isolates from nasopharyngeal
carcinoma tumors. These new LMP2 epitopes broaden the
diversity of HLA alleles with available epitopes, and,
in particular, those epitopes conserved between EBV
strains provide valuable tools for immunotherapy and
immune monitoringref., lung,
bladder,
ovarian
carcinomas,
hepatocellular
carcinoma
(HCC)
(MAGE-1, SSX-1, CTp11 and HCA587 => polyvalent
vaccinationsref),
breast
carcinomas
and multiple
myeloma.
T-cell immune responses to CGAg have been identified
in patients with solid tumors and multiple myelomaref.
They do not belong to the immunological self because
they are expressed only in immune-privileged
sites.
Since germ line cells do not carry HLA molecules on
their surface they cannot present antigens to T cells.
Their immunogenicity can be enhanced if they are
expressed (together with both allogeneic and
(transfected) syngeneic and MHC determinants) by
highly antigenic (e.g. non-self) cells after DNA
transfection.
Supporting the role of DNA methylation in generating
the intratumor heterogeneity of CTA, the DNA
hypomethylating agent 5-aza-2'-deoxycytidine
(5-AZA-dCyd) invariably induced their expression in
all CTA-negative clones of human cutaneous melanomaref) : a MAGE-3 antigen
presented by HLA-A1 has been used in several
vaccination trials on metastatic melanoma
patients. Only a small minority of patients have
shown evidence of tumor regression. Attempts to
correlate the tumor rejections with the
cytotoxic T lymphocyte (CTL) response against
the vaccine have been hampered by the low level
of these responses. In noncancerous individuals,
the frequency of the T cell precursors against
antigen MAGE-3.A1 is approximately 4 x 10-7
CD8 T cells. The diversity of the TcR repertoire
of these anti-MAGE-3.A1 precursors was analyzed
in one individual. The results indicate that it
is very likely that the repertoire comprises
>100 clonotypes. On this basis, it is
possible to use not only the frequency of CTL
precursors in the blood but also the presence of
dominant clonotypes to ascertain in patients the
existence of anti-MAGE-3.A1 responses as low as
10-6 of CD8. With this approach, we
observed a correlation between tumor regression
and anti-MAGE-3.A1 CTL responses in patients
vaccinated with a recombinant virus encoding the
antigen and also in patients vaccinated with
peptide-pulsed dendritic cells. In contrast, for
patients showing tumor regression after
vaccination with peptide alone, CTL responses
were almost never observed. It is possible that
even those CTL responses that are below our
present detection level can trigger a sequence
of events that leads to tumor regressionref.
MAGE-A3 peptides presented by HLA class I
molecules have been identified using CD8
lymphocytes stimulated with cells that either
expressed gene MAGE-A3 or were pulsed with
candidate peptides. One antigen identified with
the latter method is peptide MAGE-A3195-203
IMPKAGLLI, presented by HLA-A24 molecules. It
has been used to vaccinate advanced cancer
patients. HLA/peptide tetramers were used to
detect T cells recognizing this peptide. Their
frequency was estimated to be 2 x 10-8
of the blood CD8 cells in non-cancerous HLA-A24+
individuals, which is 10-fold lower than the
reported frequencies of T cells against other
MAGE peptides. In the blood of a patient
vaccinated with MAGE-A3, the estimated frequency
was 5 x 10-7. Anti-MAGE-3.A24
cytolytic T cell clones were derived, that lysed
peptide-pulsed cells with half-maximal effect at
the low concentration of 500 pM. However, these
CTL did not recognize a panel of HLA-A24+
tumor cells that expressed MAGE-A3 at levels
similar to those found in HLA-A1+
tumor cells recognized by anti-MAGE-3.A1 CTLs.
Furthermore, 293-EBNA cells transfected with
MAGE-A3 and HLA-A24 constructs were hardly
recognized by the anti-MAGE-3.A24 CTL clones.
These results suggest that peptide MAGE-A3195-203
is poorly processed and is not an appropriate
target for cancer immunotherapyref., lung, bladder, and head
and
neck cancers) : the peptide
NYKRCFPVI, which corresponds to amino acids 143
to151 of the MAGE-4 protein, can be presented by
HLA-A24 molecules, which are widely expressed in
different ethnic groups. A cytolytic T cell
clone that recognized the MAGE-4 peptide was
isolated from the blood cells of a donor without
cancer and lysed specifically A24 carcinoma
cells expressing MAGE-4. The antigenic peptide
is processed more efficiently in tumor cells
pre-treated with IFN-gref., melanoma, breast cancer,
prostate cancer, lung cancer, ovarian cancer and
bladder cancer; several clinical trials are in
progress with NY-ESO-1 peptides, protein,
recombinant poxviruses, and dendritic cells pulsed
with peptides. 3 immunodominant regions of the
protein are located within 3 distinct clusters of
MHC class I binding sequences that co-localize
with previously defined clusters of MHC class II
binding sequences, are predicted to be hydrophobic
and undergo efficient proteasomal processingref.
PowderMed
Ltd. has produced a DNA Plasmid (pPJV7611),
which encodes the NY-ESO-1 protein. PPJV7611 is
precipitated onto the surface of gold particles 1
to 3 µM in diameter and will be administered to
patients by particle mediated epidermal delivery
(PMED). Access to purified protein both for
vaccine formulations and for monitoring
antigen-specific immune responses is vital to
vaccine development. Currently available
recombinant Escherichia coli-derived NY-ESO-1 is
isolated from inclusion bodies as a complex
protein mixture and efforts to improve the purity
of this antigen are required, especially for
later-stage clinical trials. Using yeast cell
surface display and fluorescence activated cell
sorting techniques, an NY-ESO-1 variant
(NY-ESO-L5; C(75)A C(76)A C(78)A L(153)H) was
engineered with a 100x improved display level on
yeast compared to the wild-type protein. This
mutant can be effectively produced as an
Aga2p-fusion and purified in soluble form directly
from the yeast cell wall. In the process, we have
identified the epitope recognized by anti-NY-ESO-1
mAb E978 (79-87, GARGPESRL). The availability of
an alternative expression host for this important
antigen will help avoid artifactual false positive
tests of patient immune response due to reaction
against expression-host-specific contaminantsref.
MAGE-A4 and NY-ESO-1 were expressed in 40 of 141
(28.4%) and 13 of 157 (8.3%) NSCLC respectively. Both CT
antigens were more frequently expressed in
squamous cell carcinoma (SCC) than in
adenocarcinoma. An inverse correlation was found
between MAGE-A4 expression and patient survival in
advanced stage cancers. Combined infiltration of
both CD4+ and CD8+ T cells into tumor nest
predicted better survival. There was no
correlation, however, between lymphocyte
infiltration and antigen expression in the tumor.
MAGE-A4 expression in advanced group and T cell
infiltration may provide prognostic information.
Lastly, these CT antigens, especially MAGE-A4, may
represent potential targets for cancer
immunotherapy in patients with NSCLCref and acute
myeloid
leukemias)ref)
is a highly immunogenic protein and the observation
that > 90% of vasectomized males develop immunity against
Sp17 suggests the opportunity and safety of Sp17 for
tumor vaccines. Although Sp17 transcripts could be
detected by RT-PCR in some normal tissues, the
levels of expression were <2% of those in normal
testis. In contrast, Sp17+ myeloma cells
expressed 3-18% of normal testis levels of Sp17
transcript. IHC using 2 Sp17 murine mAbs, each
directed at a non-overlapping B-cell epitope, showed
Sp17 protein to be expressed only in testis and not
any other normal tissues. Specificity of binding of
the antibodies to testis was also confirmed in
competitive binding assaysref.
Recent works by other workers suggest a low level of
expression of Sp17 in some normal tissues, and
investigators have questioned whether Sp17 is in
fact a suitable target for immunotherapyref.
Sp17-specific HLA class I-restricted CTL generation
was successful in all 4 patients, irrespective of
the Sp17 status of their tumors : most importantly,
the CTLs were able to lyse autologous tumor cells
that expressed Sp17ref.
Sp17-specific HLA class I-restricted CTLs can also
be easily generated from the peripheral blood of
healthy donor. Because CTLs are able to kill
HLA-matched fresh myeloma cells, it may be possible
to generate and administer myeloma-specific donor T
cells to MM patients following allogeneic stem cell
transplantation to enhance graft-versus-myeloma
(GVM) without inducing graft-versus-host disease
(GVHD)ref1,
ref2.
Sp17 was found to be expressed in the primary tumor
cells from 70% of the patients with ovarian
carcinoma. HLA class I-restricted Sp17
specific CTLs were generated successfully from the
peripheral blood of 3 patients with ovarian
carcinoma at the time of disease presentation. These
CTLs were able to lyse autologous EBV-transformed
lymphoblastoid cells in a Sp17-dependent manner.
Target lysis was HLA class I-dependent and could be
blocked by antibodies against monomorphic HLA class
I but not HLA class II molecules. The CTLs also
lysed Sp17+ autologous tumor cells,
suggesting that Sp17 is processed and presented in
association with the HLA class I molecules in Sp17+
tumor cells in a concentration and configuration
that could be recognized by recombinant
protein-primed CTLs. Tumor cell killing by the CTLs
appeared to be mediated through the perforin pathwayref, 3 of 11 chronic
lymphocytic
leukemia (CLL), 4 of 14 chronic
myeloid
leukemia, and 6 of 17 multiple
myeloma. In contrast, they were not
detected in corresponding specimens from any healthy
donors. SLLP1 exhibited a very restricted normal
tissue expression, being found only in
testis/spermatozoa. SLLP1 was expressed in some
tumor cells at a level of >25%. High titer IgG
antibodies against SLLP1 were also detected in the
sera of some of these patients. CONCLUSIONS: SLLP1
is a novel cancer-testis antigen in hematologic
malignancies and is capable of eliciting B-cell
immune responses in vivo in cancer-bearing
individualsref and other hematologic
malignancies such as chronic
lymphocytic
leukemia (CLL), chronic
myeloid
leukemia, and acute
myeloid
leukemia. It is not expressed in any
normal tissue except testis. High-titer IgG3
and IgG2 against SPAN-Xb were
detectable in the sera of these patients. In
contrast, SPAN-Xb mRNA or antibodies could not be
detected in any of the healthy donors. There was a
good correlation between SPAN-Xb gene expression and
B-cell immune responses. These results suggest the
in vivo immunogenicity of the SPAN-Xb protein.
The presence of high-titer IgG responses suggests
that the B-cell responses are likely to have been
generated with CD4 T-cell cognitive helpref)ref have antibody responses to
SCP-1, but not to other cancer testis antigens
(GAGE, LAGE-1a,-1b, CT-7, NY-ESO-1, SSX-1-5).
Antibody response correlated with the expression of
SCP-1 in the primary tumor of the respective patient
as shown by RT-PCR, IHC and Western blot. In
contrast, no serological response to CTAgs was
observed in healthy donors. The humoral immune
response against SCP-1 was associated with the size
of tumor, but not with other clinico-pathological
parameters such as histology, stage, presence of
lymph node metastases, grading, age, gender or
gemcitabine treatmentrefref
and multiple
myelomaref
immunotherapy is highly expressed in 80%
of hepatocellular
carcinoma
(HCC)ref1,
ref2,
ref3,
ref4,
ref5,
but AFP immunization alone still resulted in lower
levels of specific response and poorly reproducible
protective immunityref1,
ref2,
ref3,
ref4,
ref5
: the immunogenicity of AFP could be improved by
presenting to APCs through HSP70 moleculesref1,
ref2,
ref3,
ref4,
ref5.
Sequential immunization by i.m. injection of a
recombinant DNA plasmid vaccine encoding AFP and
HSP70 fragments could generate effective
AFP-specific T cell responses and induce definite
antitumor effects on AFP-producing tumors, which may
be suitable for some clinical testing as a vaccine
for HCCref.
The recombinant expression vectors PEGF-N3/AFP1 and
PEGF-N3/AFP2 with or without signal peptide were
constructed successfully. AFP2/DCs can specifically
activate T lymphocytes in vitro and
effectively induce antitumor immune responseref.
Chimeric AFP-Mt.HSP70 DNA vaccine was constructed by
molecular clone techniques. Spleen cells derived
from mice immunized twice were induced to secrete
IFNr and were assayed using ELISA. The activity of
the cytotoxic lymphocytes (CTL) derived from spleen
cells was assayed using LDH. 4 x 106
Hepa1-6 cells/200 ml
were injected subcutaneously into the right axilla
of each mouse bearing the tumor. The anti-tumor
effect of the recombinant DNA vaccine was evaluated
by measuring tumor sizes of the mice. RESULTS:
AFP-specific CTL reaction was induced by our
chimeric DNA vaccine and Mt.HSP70 enhanced this
effect. The CTL activity was about 32% at E/T=50:1.
The IFNr secreted by spleen cells of mice immunized
with chimeric plasmids was about 200 pg/ml. It was
higher than those in the other groups. Tumor sizes
of mice immunized with fused plasmids were smaller
than those in the other groups. Survival times of
mice immunized with the fused plasmids were
prolongedref.
Anti-HCC effects were achieved in a therapeutic
setting with a DNA vaccine encoding mouse AFP and
co-expressing HSP70 gene. This vaccine elicited a
marked and highly effective AFP specific CTL
response against AFP+ target cells. This
vaccine also induced the prolongation of life span
in mice bearing the tumor and the eradication of HCCref : C57BL/6 and BALB/c mice
were immunized with 2 different plasmids (p91023B
and pKCEA66) encoding different forms of the TAA
CEA. The wild type form of CEA (p91023B), which is
expressed at the cell surface, induces stronger
anti-CEA IgG response after DNA-plasmid
immunizations than the modified intracellular form
of CEA (pKCEA66), which was designed to mount strong
cellular responses. Boosting with recombinant CEA
(rCEA) increased the anti-CEA IgG response
significantly. In the tumor protection model used,
where SCID mice are challenged with human tumor
cells mixed with splenocytes from immunized mice,
both innate and specific immune responses are
responsible for the protective effectref.
Exosomes from heat-stressed CEA+ tumor
cells (CEA+/HS-Exo) contained CEA and
more HSP70 and MHC-I and significantly induced
dendritic cell maturation. Immunization of
HLA-A2.1/K(b) transgenic mice with CEA+/HS-Exo
was more efficient in priming a CEA-specific CTL,
and the CTL showed antitumor effect when adoptively
transferred to SW480-bearing nude mice. Moreover, in
vitro incubation of lymphocytes from
HLA-A*0201+ healthy donors and HLA-A*0201+CEA+
cancer patients with CEA+/HS-Exo-pulsed
autologous dendritic cells induces
HLA-A*0201-restricted and CEA-specific CTL response.
CEA+/HS-Exo has superior immunogenicity
than CEA+/Exo in inducing CEA-specific
CTL responseref.
CEA, the most widely used human tumor marker, is a
heavily glycosylated protein over-expressed by a
wide range of tumors. It has been indicated that CEA
might be a useful target for human anti-tumor
immunotherapy. CEA assay for research as well as
clinical trials demands a continuous source of CEA
protein preparations. In a multi-purpose research
program to provide a reliable source for large
production of CEA, the membrane-bound
carcinoembryonic antigen was converted into a
secretory protein by site-specific mutagenesis. The
secretory CEA protein was made by introducing a new
stop codon at 99 bp upstream of the original stop
codon in CEA cDNA by PCR-based mutagenesis. The
glycosylation of recombinant CEA proteins,
especially those destined for administration to
human trials is crucially important. To produce CEA
with the same glycosylation pattern and
immunogenicity as the native CEA expressed by human
tumors in vivo, the truncated CEA cDNA which
does not encode the last C-terminal 33-amino acid
hydrophobic domain was transfected into HT29, a
human colon carcinoma cell line by the calcium
phosphate method. Stable transfectants were selected
and pooled. CEA secretion from the cells was
verified by analysis of the transfectant culture
supernatant for CEA protein. As determined by ELISA,
16 mg/L of recombinant
CEA was secreted per 106 transfectants
within 48 hrs, an increase over 40 times relative to
the untransfected cells. The size of the recombinant
CEA secreted by HT29 transfectants in our experiment
is identical to that of reference CEA secreted from
tumors and is fully antigenic. It seems that the
C-terminal truncation does not affect CEA
glycosylation in HT29 cells. It is predicted that
human cancer immunotherapy using recombinant CEA
expressed in this system would be more effective
than the commercial protein which is usually
prepared from bacterial or other heterologous
expression systemsref.
The existence of a murine model transgenic for human
CEA allows CEA vaccination efficacy to be studied in
a physiologically tolerant context. Immunization of
CEA-transgenic mice with an adenoviral vector coding
for CEA induced a significant CD8+ T-cell
response specific to CEA but failed to induce
CEA-specific CD4+ T cells and antibodies.
To overcome CD4+ T-cell tolerance, the
effect of adjuvants inducing in vivo DC
maturation were explored. 2 different TLR ligands,
monophosphoryl lipid A (MPL) and CpG
motif-containing oligodeoxynucleotides (CpG-ODN),
were tested. CD4+-mediated IFN-g production was induced in
the CEA-transgenic mice only when the genetic
immunization was performed in the presence of these
adjuvants. Moreover, CpG-ODN had a greater effect
than MPL in inducing CD4+ T-cell response
and enabling anti-CEA antibody productionref. in juvenile
myelomonocytic
leukemia : after identifying amino acid
residues specific to PLAP and distinct from other
close PLAP homologs, sequence-based
immunoinformatics tools (BIMAS and SYFPEITHI) were
used and molecular modeling studies were conducted
using InsightII to investigate the binding affinity
of the epitopes containing the unique residues with
respective MHC class I molecules. Promiscuous
epitopes binding to different alleles of different
class I HLA loci were analyzed to get a population
coverage that is widespread. Binding affinity
deduced from the modeling studies corroborated the
status of most of the epitopes scoring high in BIMAS
and SYFPEITHI. Specific epitopes from PLAP that have
a potential for binding to their respective MHC
class I alleles with high affinity were thus
identifiedref
are, again, unfolded. Characteristically, expression
follows the same tissue presentation as during
embryogenesis. Clinical paternal disomies, i.e. trophoblastic
diseases,
and their maternal counterpart, i.e. ovarian teratomas, are
associated with apparent relaxation of imprinting once
they turn malignant. Paediatric neoplasms, like Wilms'
tumor
(WT)
and rhabdomyosarcoma,
often express IGF2 and H19. Recently H19 expression has
been found in invasive urothelial cancer. Evidently,
imprinted genes display an oncodevelopmental mode of
expression, very much like the classical oncofetal
proteins AFP and CEA.
for adenocarcinomas (certain
breast
carcinomas,
...) is overexpressed and modified by tumor cells in
> 50% of all cancer cases. Animal data show that it
can be a target for immune-mediated tumor rejection,
and finally, preliminary clinical results show that
vaccine-based immunotherapy with MUC1 does have an
impact on the therapy of cancerref.
BLP25 liposome vaccine (Stimuvax®;
source : Biomira)
is a cancer vaccine that targets the exposed core
peptide of the MUC1 tumor-associated antigen. Studies
in advanced-stage NSCLC demonstrate that L-BLP25
vaccine has the potential to extend the survival of
patients with Stage IIIB locoregional NSCLC and
maintain quality of life for longer. The global Phase
III clinical trial, START
(Stimulating Targeted Antigenic Responses To NSCLC),
assessing the efficacy and safety of Stimuvax®
as a potential treatment for patients with
unresectable stage III NSCLC is open for patient
enrollment. The trial is being conducted by Merck KGaA
of Darmstadt, Germany, and its U.S. affiliate EMD
Pharmaceuticals, Inc. under the provision of a
collaboration agreement between Biomira and Merck.
Biomira is entitled to a milestone payment upon
enrollment of the first patient in the Phase III
trial, which is anticipated to occur in January 2007.
Enrollment in the trial, which is expected to include
> 1,300 patients in approximately 30 countries, is
now open to patients in 12 countries, including the
U.S., and will subsequently expand to additional
countries. Currently, there are no approved
maintenance therapies for patients responding to
first-line treatment for unresectable stage III NSCLC.
L-BLP25 vaccine also shows promise for prostate
cancer
patients, having the potential to prolong PSA doubling
time in men with biochemical failure post
prostatectomy. These clinically meaningful results
with a relatively nontoxic therapeutic vaccine are
very encouraging and suggest potential for L-BLP25 to
fulfill an unmet medical needref. patients received Tn(c)-KLH
vaccine containing either 3, 7, or 15 mg of Tn(c) per
vaccination. 10 patients received 100 mg of Tn(c)-palmitic acid
(PAM). QS21 was included in all vaccines. 5
vaccinations were administered subcutaneously
during 26 weeks with an additional booster vaccine
at week 50. Tn(c), when given with the carrier
molecule KLH and QS21, stimulated the production
of high-titer IgM and IgG antibodies. Inferior
antibody responses were seen with T(c)-PAM. There
was no evidence of enhanced immunogenicity with
increasing doses of vaccine. An antitumor effect
in the form of a decline in posttreatment versus
pretreatment PSA slopes was also observed. A safe
synthetic conjugate vaccine in a trimer formation
was developed that can break immunologic tolerance
by inducing specific humoral responses. It seemed
to affect the biochemical progression of the
disease as determined by a change in PSA log sloperef. and MUC-1
(pNGVL-hFLex-MUC-1) is able to induce
antigen-specific CTL immunity in vivo,
resulting in a potent anti-tumour response, and the
Flt-3L component is essential to the efficacy of the
DNA vaccine. Moreover, the route of immunization is
critical in determining the type of immune response
generated; intramuscular (i.m.) immunization with
pNGVL-hFLex-MUC-1 conferred tumour protection in
contrast to poor response with hydrodynamic-based
intravenous delivery. Post-i.m. immunization, a
massive infiltration of mononuclear cells to the
injection site was observed, comprised predominantly
of CD11c+/CD8a-
DC. Flt-3L acts as an adjuvant to recruit DC,
thereby priming the anti-tumour response. However,
systemic expansion of DC prior to immunization did
not enhance the specific cellular response,
suggesting that it is in situ recruitment or
expansion of DC that is critical for
pNGVL-hFLex-MUC-1 potencyrefref1,
ref2
(expressed in > 85% of human cancers) : the main
risk of targeting dominant TERT epitopes is induction
of autoimmunity in activated B cells. Immunity against
low-affinity epitopes is induced with heteroclitical
variants. The CTL repertoire against high-affinity
epitopes of murine TERT (mTERT) is partially
tolerized, while that against low-affinity epitopes is
composed of frequent CTLs with high avidity. The
high-affinity p797 and p545 mTERT epitopes are not
able to protect mice from a lethal challenge with the
mTERT-expressing EL4-HHD tumor. In contrast, mice
developing CTL responses against the p572 and p988
low-affinity epitopes exhibit potent antitumor
immunity and no sign of autoimmune reactivity against
TERT-expressing normal tissuesref.
Endogenous in vivo priming of T cells against
hTERT is a rare event in multiple myeloma
patients. These results suggest that strategies
targeting hTERT may have to utilize techniques to
induce T cell responses from a naive precursor
frequencyref.
In an attempt to develop an effective vaccine against
most human cancers, a chemotactic-hTERT vaccine has
been constructed. 2 hTERT fragments encoding multiple
cytotoxic T lymphocyte and T helper cell epitopes were
fused as a tumor antigen (named Te). The plasmid based
DNA vaccine (pCCL21-Te-Fc) was constructed by linking
human CCL21 and IgG Fc gene sequences to each end of
Te. In poorly immunogenic B16F10 mouse melanoma model,
DNA (pCCL21-Te-Fc) vaccination significantly inhibited
tumor growth and all of the mice were dead by day 52.
The immunization with pCCL21-Te-Fc-modified tumor
cells (B16/CCL21-Te-Fc) resulted in a higher antitumor
effect than DNA vaccination and 25% of tumor-bearing
mice achieved long-term survival (>120 days). The
combined therapy of B16/CCL21-Te-Fc plus anti-4-1BB
MAbs further enhanced the immune response, resulting
in 75% of tumor-bearing mice achieved long-term
survival (>120 days) in subcutaneous model and few
lung nodules in pulmonary metastasis model.
Rechallenge experiment showed that a persistent memory
response was successfully induced by the combined
therapy. In vivo depletion of lymphocytes
indicated that CD8+ T cells were essential
in the antitumor activity induced by B16/CCL21-Te-Fc +
anti-4-1BB MAbs, whereas NK cells and CD4+
T cells played substantial roles. The CTL activity
induced by pCCL21-Te-Fc-transfected PBMCs specifically
lysed a variety of HLA-matched and hTERT-positive
human tumor cells, suggesting pCCL21-Te-Fc could serve
as a vaccine against most human cancersref.
HLA-B*0702-restricted peptides from hTERT were
selected by using a method of epitope prediction and
tested for their immunogenicity in human (in vitro)
and HLA-B*0702 transgenic mice (in vivo). All
the 6 hTERT peptides that were predicted to bind to
HLA-B*0702 molecule were found to induce primary human
CTL responses in vitro. The peptide-specific CD8+
CTL lines were tested against various hTERT+tumor
cells. Although differences were observed according to
the tumor origin, only 3 CTL lines specific for p277,
p342, and p351 peptides exhibited cytotoxicity against
tumor cells in a HLA-B*0702-restricted manner. In
addition, this cytotoxicity was inhibited by the
addition of peptide-loaded cold target cells and
indicated that these epitopes are naturally processed
and presented on the tumor cells. Further, in vivo
studies using humanized HLA-B*0702 transgenic mice
showed that all the candidate peptides were able to
induce CTL responses after peptide immunization.
Furthermore, vaccination with a plasmid DNA encoding
full-length hTERT elicited peptide-specific CTL
responses, indicating that these epitopes are
efficiently processed in vivo. Together with
previously reported hTERT epitopes, the identification
of new CTL epitopes presented by HLA-B*0702 increases
the applicability of hTERT-based immunotherapy to
treating cancerref.
A study evaluated the safety of the optimized cryptic
peptide TERT(572Y) in pretreated patients with
advanced cancer. 19 patients with progressive and
chemotherapy-refractory tumors received escalated
doses (2-6 mg) of 2 subcutaneous injections of the
optimized TERT(572Y) peptide followed by 4
subcutaneous injections of the native TERT(572)
peptide every 3 weeks. Both TERT peptides were
coinjected with adjuvant Montanide ISA51. Toxicity was
evaluated every 3 weeks and peptide-specific CD8+
cells were detected by flow cytometry using TERT(572Y)
tetramers. 14 out of 19 patients completed the
vaccination program. No grade III/IV toxicity was
observed. Grade I anemia was observed in 4 patients
and local skin reaction at the injection site in 11
patients. Other nonhematologic toxicities were mild,
and no late toxicity was observed after a median
postvaccination follow-up period of 10.7 months. There
was no dose-limiting toxicity. Peripheral blood
TERT(572Y)-specific CD8+ lymphocytes were
detected in 13 out of 14 evaluable patients after 2
injections with the optimized TERT(572Y) peptide.
There was no complete or partial response, but 4
patients (21%) with persistent TERT(572Y)-specific CD8+
experienced stable disease for a median of 10.5
months. TERT(572Y) peptide vaccine is well tolerated
and effective in eliciting specific TERT(572Y) CD8+
lymphocytes in pretreated cancer patients,
demonstrating that cryptic peptides could be used in
cancer immunotherapyref.
Human dendritic cells transduced with rAd-hTERT could
effectively induce hTERT-specific CTLs in vitro against
various tumor cell lines, which were hTERT+
and HLA-A2 matched. These hTERT-specific CTLs could
not lyse autologous lymphocytes with low telomerase
activity. Further studies revealed that rAd-hTERT
transduced DCs could increase secretion of IFN-g by effector cells when they
were co-cultured with hTERT-positive and HLA-A2
matched tumor cell lines. These data suggest that an
hTERT vaccine can induce anti-tumor immunity against
various tumor cells expressing hTERT in a
HLA-A2-restricted fashion in vitro. The
transduction of DCs with rAd-hTERT offers a great
opportunity in cancer immunotherapyref. :
mutations leading to functional inactivation of the
p53 tumour suppressor protein occurs in ~50% of human
cancers. Subsequent accumulation of mutant p53 in
nuclei and cytopasm is associated with high-level
presentation of p53-derived peptides by MHC class I
molecules on malignant cells. Due to the diversity of
p53 mutations in different tumours, peptides
representative of wild type, as opposed to mutant p53
sequences, were proposed to serve as a universal TAA
for a CTL-based immunotherapy of cancer. Increased
susceptibility of malignant cells to lysis by wt
p53-specific CTLs has been demonstrated to be linked
rather to a high turnover rate than to a high
steady-state level of p53 expression. A major barrier
to the design of broad-spectrum p53-specific
immunotherapeutics, however, has been the observation
that low-level expression of wt p53 peptides by
non-transformed tissues and cells results in
self-tolerance of T lymphocytes with high avidity for
self-MHC class I/self-p53-peptide complex. Although
the peripheral T cell repertoire is mostly devoid of
such high-avidity wt p53 epitope-specific CTLs due to
self-tolerance, this does not necessarily preclude the
possibility of developing wt p53-directed vaccination
strategies. Low-avidity and even residual high-avidity
CTLs have been demonstrated to partially escape the
induction of wt p53-specific self-tolerance : these
can be primed and amplified in vivo. p53
mutations are found in up to 30% of breast cancers and
peptides derived from over-expressed p53 protein are
presented by class I HLA molecules and may act as
tumor-associated epitopes in cancer vaccines. A
dendritic cell (DC) based p53 targeting vaccine was
analyzed in HLA-A2+ patients with progressive advanced
breast cancer. DCs were loaded with 3 wild-type and 3
P2 anchor modified HLA-A2 binding p53 peptides.
Patients received up to 10 sc vaccinations with 5 x 106
p53-peptide loaded DC with 1-2 weeks interval.
Concomitantly, 6 MIU/m2 IL-2 was
administered sc. Results from a phase II trial
including 26 patients with verified progressive breast
cancer are presented. 7 patients discontinued
treatment after only 2-3 vaccination weeks due to
rapid disease progression or death. 19 patients were
available for first evaluation after 6 vaccinations;
8/19 evaluable patients attained stable disease (SD)
or minor regression while 11/19 patients had
progressive disease (PD), indicating an effect of
p53-specific immune therapy. This was supported by:
(1) a positive correlation between p53 expression of
tumor and observed SD, (2) therapy induced p53
specific T cells in 4/7 patients with SD but only in
2/9 patients with PD, and (3) significant response
associated changes in serum YKL-40 and IL-6 levels
identifying these biomarkers as possible candidates
for monitoring of response in connection with DC based
cancer immunotherapy. In conclusion, a significant
fraction of breast cancer patients obtained SD during
p53-targeting DC therapy. Data encourage initiation of
a randomized trial in p53 positive patients evaluating
the impact on progression free survivalref
for breast
carcinomas
and oral
carcinomas.
HER-2 gene is highly overexpressed in approximately
25% to 30% of invasive breast carcinomasref1,
ref2,
and in comparison to normal breast epithelial cells,
tumor cells may have up to a 100-fold greater
expression of HER-2ref, with
both dominant-negative and a gain-of-function
properties, display early aggressive and
metastasizing parotid
tumors. Multiple acinar and ductal
hyperplasia foci overexpressing the HER-2/neu gene
product are evident at wk 5 and progress to poorly
differentiated carcinoma by wk 7. Mice die before wk
18 with invasive carcinomas and multiple metastases
that no longer express HER-2/neu. A combination of
repeated electroporations of plasmids coding for the
extracellular and transmembrane domains of the rat
HER-2/neu receptor with systemic IL-12
administrations started when the parotids that
present diffuse hyperplasia protected all female and
50% of the male mice until the close of the
experiment at wk 40. This combined treatment began
when multifocal in situ carcinomas that
were already present cured 33% of the females and
25% of the males. The most prominent immunologic
features associated with the antitumor protection
were the production of high titers of anti-HER-2/neu
Abs and the nonappearance of cell-mediated cytotoxic
reactivity. In conclusion, anti-HER-2/neu
vaccination combined with systemic IL-12 control
parotid carcinomas as far as p53 mutation makes
their growth independent of HER-2/neu expressionref.
and melanoma in humans, and it was useful
as a novel tumor marker. The preimmunization of BALB/c
mice with dendritic cells pulsed with the H-2Kd-restricted
mouse GPC3298-306 (EYILSLEEL) peptide
prevented the growth of tumor-expressing mouse GPC3.
Because of similarities in the peptide binding motifs
between H-2Kd and HLA-A24 (A*2402), the
GPC3298-306 peptide therefore seemed to be
useful for the immunotherapy of HLA-A24+
patients with HCC and melanoma. The GPC3(298-306)
peptide could induce GPC3-reactive CTLs from the
peripheral blood mononuclear cells (PBMC) of HLA-A24
(A*2402)+ HCC patients. In addition,
HLA-A2.1 (HHD) transgenic mice were used to identify
the HLA-A2 (A*0201)-restricted GPC3 epitopes to expand
the applications of GPC3-based immunotherapy to the
HLA-A2+ HCC patients. The GPC3144-152
(FVGEFFTDV) peptide could induce peptide-reactive CTLs
in HLA-A2.1 (HHD) transgenic mice without inducing
autoimmunity. In 5 out of 8 HLA-A2+ GPC3+
HCC patients, the GPC3144-152
peptide-reactive CTLs were generated from PBMCs by in
vitro stimulation with the peptide and the GPC3298-306
peptide-reactive CTLs were also generated from PBMCs
in 4 of 6 HLA-A24+ GPC3+ HCC
patients. The inoculation of these CTLs reduced the
human HCC tumor mass implanted into nonobese
diabetic/severe combined immunodeficiency miceref.
HLA-A2 or HLA-A24 restricted GPC3 epitope peptide, and
investigated whether these peptides could induce GPC3
reactive CTLs from the peripheral blood mononuclear
cells (PBMCs) of HLA-A2+ or HLA-A24+
HCC patients. HLA-A2.1 (HHD) transgenic mice (Tgm) to
identify the HLA-A2-restricted GPC3 epitopes. These
epitope peptides could induce peptide-reactive CTLs in
about 50% of HLA A2+ or HLA-A24+,
and GPC3+ HCC patientsref.
is expressed and extremely sensitive, easily
detectable and highly correlated with breast cancer. A
gene vaccine was developed using a plasmid vector to
deliver the six copies of 10-amino acid residues of b-hCG 109-118 and b hCG C-terminal 37-amino acid
(CTP37). BALB/c female mice were immunized with a
combination of pCR-HBc-X6-betahCGCTP37 DNA vaccine and
HSP-X6-betahCGCTP37 protein vaccine.
pCR-HBc-X6-betahCGCTP37 DNA vaccine were injected
intramuscularly 3 times, on days -46,-25 and -11 and
HSP-X6-bhCGCTP37 protein
were applied 2 times, 21 and 14 days before tumor cell
challenge. A combined DNA and protein vaccine was
assessed for its effect of against murine EMT6 mammary
tumor cells. In this study, animals vaccinated DNA
vaccination boosting with the repeat b-hCG C-terminal peptide
carried by mycobacterial heat-shock protein HSP65
induced higher avidity antibodies and effectively
inhibited the growth of tumor, compared with treatment
using DNA alone or BCG priming HSP-X6-bhCGCTP37 protein boosting. The
data presented demonstrate that improve immunogenicity
of DNA vaccination by boosting with the repeat b-hCG C-terminal peptide
carried by mycobacterial heat-shock protein HSP65,
which should prove useful in the development of new
DNA vaccine against growth factors for cancer
immunotherapyref is
overexpressed in a variety of solid tumors :
HLA-A*0201-binding peptides and a naturally processed
and presented T-cell epitope capable of inducing
CYP1B1-specific CTLs in HLA-A2 transgenic miceref.
Immunization of HLA-A2/Kb transgenic mice
with human CYP1B1 encoding plasmid DNA formulated in
poly(lactide-co-glycolide) (PLG) microparticles
elicits CD8+ T cells that respond to human
CYP1B1+ target cells. PLG-encapsulated DNA
therapeutic elicits durable immune responses to
CYP1B1, the responses are dependent on repeat
immunization, and that the formulation is well
toleratedref.
Endogenous in vivo priming of T cells against
CYP1B1 is a rare event in multiple myeloma
patients. These results suggest that strategies
targeting CYP1B1 may have to utilize techniques to
induce T cell responses from a naive precursor
frequencyref
cells but not in normal cells. To explore the
potential of HM1.24 as a target for cellular
immunotherapy, 4 HM1.24-derived peptides that possess
binding motifs for HLA-A2 or HLA-A24 were selected by
using 2 computer-based algorithms. The ability of
these peptides to generate cytotoxic T lymphocytes
(CTLs) was examined in 20 healthy donors and 6
patients with MM by a reverse immunologic approach.
Dendritic cells (DCs) were induced from
peripheral-blood mononuclear cells of healthy donors
or peripheral-blood stem-cell (PBSC) harvests from
patients with MM, and autologous CD8+ T
cells were stimulated with HM1.24 peptide-pulsed DCs.
Both IFN-g-producing and
cytotoxic responses were observed after stimulation
with either HM1.24-126 or HM1.24-165 peptides in
HLA-A2 or HLA-A24 individuals. The peptide-specific
recognition of these CTLs was further confirmed by
tetramer assay and cold target inhibition assay.
Importantly, HM1.24-specific CTLs were also induced
from PBSC harvests from patients with MM and these
CTLs were able to kill MM cells in an HLA-restricted
manner. These results indicate the existence of
functional DCs and HM1.24-specific CTL precursors
within PBSC harvests and provide the basis for
cellular immunotherapy in combination with autologous
PBSC transplantation in MMref.
The HM1.24 sequence was scanned for immunogenic
peptides using the HLA-binding prediction software
SYFPEITHI and BIMAS. Peripheral blood mononuclear
cells from HLA-A2+ healthy volunteers/blood
donors (ND) were stimulated with autologous
HM1.24-peptide-loaded dendritic cells, and expanded in
vitro. Activation of T cells was assessed by
ELISpot and cytotoxicity by 51Cr-release
assays. T2-cells pulsed with irrelevant peptide, the
HM1.24-/HLA-A2+ breast carcinoma
cell line MCF-7 and the HM1.24+/HLA-A2-
myeloma cell line RPMI-8226 were used as controls.
Expression of the HM1.24 gene (BST2) was assessed
using purified plasma cells and Affymetrix-U133A+B
microarrays. Frequency of peptide-specific CD8+
T cells was detected using the flow-cytometric
tetramer technique. Of 8 nona-peptides with the
highest probability of binding to HLA-A2, the HM1.24
aa22-30 peptide (LLLGIGILV) showed the most
frequent activation of CD8+ T cells in
healthy volunteers (specific activation in 8 of 11
[73%] ND; compared with 5-19% for the 7 other HM1.24
peptides). Antigen recognition by the HM1.24
aa22-30-specific CD8+ T cells was
HLA-A2-restricted (ELISpot with HLA-A2-blocking
antibodies: median, 15; range, 14-18 spots/well;
isotype-control antibodies: median, 47; range, 44-48).
HM1.24-aa22-30-specific CD8+ T cells lysed
HLA-A2+ myeloma-derived cell lines (51Cr-release
assay:
XG-1 vs MCF-7, 91% vs 0%; U266 vs MCF-7, 38% vs 4.2%;
IM-9 vs RPMI-8226, 22% vs 0%). Using the
cross-reactive Neisseria meningitidis peptide
LLSLGIGILV-specific CD8+ T cells
recognizing target cells loaded with the HM1.24
aa22-30 peptide (LLLGIGILV) as well as the
myeloma-derived cell line U266 could be expanded from
MM patients. The HM1.24 gene was expressed at
comparable levels by plasma cells from 65 MM patients,
7 patients with MGUS, and 7 ND. HM1.24 aa22-30 is a
newly identified HLA-A2-restricted T-cell epitope that
is processed and presented by major histocompatibility
complex class I. Specifically activated CD8+
T cells are able to lyse MM cell lines. HM1.24 aa22-30
represents a suitable candidate target for a specific
peptide-based immunotherapy of MMref.
offers an attractive commercial opportunity for the
development of TroVax. Studies show that the 5T4 antigen
is present on > 90% of RCC samples; current treatment
options are ineffective or have serious side effects;
and TroVax could benefit from orphan drug and fast track
designations in this indication. A Phase II trial in RCC
with TroVax alongside high dose interleukin-2 is
underway in the United States. Preliminary immunology
data are expected around mid-2005. In breast cancer, the
Southwest Oncology Group, which is a US clinical trials
consortium, is expected to start a Phase II trial in
late stage patients, in 2005. Clinical response data
from Oxford BioMedica’s Phase II trials are unaudited.
They are based on patients that are evaluable and use
the industry standard Response Evaluation Criteria in
Solid Tumours (RECIST). The data from the trials of the
chemotherapy agents alone, cited in this press release,
are derived from evaluable patients, using clinical
response criteria set by the World Health Organisation
(WHO). The criteria defined by RECIST and WHO are
similar but not identical. TroVax has been given to 150
patients so far in clinical trials : in a kidney cancer
trial it was administered together with IL-2 and caused
a complete regression in 1 patient. Another 2 patients
have seen their tumours shrink, and a further 15 have
been stable for > 3 months : among them one has been
stable for 46 weeks. A further 700 patients will now
receive the treatment as the vaccine undergoes further
study. The Phase III trial will compare TroVax +
standard kidney cancer treatment and a dummy version
plus standard treatmentref.
cells and is expressed at low levels in normal cells.
Therefore, mesothelin represents a potential target
antigen for ovarian cancer vaccine development. DNA
vaccines employing single-chain trimers (SCT) have
been shown to bypass antigen processing and
presentation and result in significant enhancement of
DNA vaccine potency. In the current study, a DNA
vaccine employing an SCT targeting human mesothelin
was created and characterized the ensuing
antigen-specific CD8+ T cell-mediated
immune responses and anti-tumor effects against human
mesothelin-expressing tumors in HLA-A2 transgenic
mice. Vaccination with DNA employing an SCT of HLA-A2
linked to human mesothelin epitope aa540-549
(pcDNA3-Hmeso540-b2m-A2)
generated
strong human mesothelin peptide (aa540-549)-specific
CD8+ T cell immune responses in HLA-A2
transgenic mice. Vaccination with pcDNA3-Hmeso540-b2m-A2 prevented the
growth of HLA-A2+ human
mesothelin-expressing tumor cell lines in HLA-A2
transgenic mice in contrast to vaccination with DNA
encoding SCT linked to OVA CTL epitope. Thus, the
employment of SCT of HLA-A2 linked to the human
mesothelin epitope aa540-549 represents a potential
opportunity for the clinical translation of DNA
vaccines against human mesothelin-expressing tumors,
particularly ovarian cancer cellsref-melanocyte differentiation
antigens (MDAs) / melanoma-associated antigens
(MAAs) (IRIV) can enhance CTL
responses specific for synthetic peptides
simultaneously added to cultures in soluble form.
This effect was based on IRIV mediated activation
of CD4+ T cells. The "in vitro"
immunogenicity of a novel virosome formulation
coupling in a single reagent the adjuvant power of
IRIV to the capacity of liposomes to efficiently
encapsulate synthetic peptides was investigated.
L(27)Melan-A/Mart-1(26-35) HLA-A0201-restricted
peptide was used as a model antigen. The reagent
thus developed induced the proliferation of CD4+
T cells characterized by a Th1 cytokine
profile and CXCR3 expression. Most importantly, it
significantly enhanced the generation of
L(27)Melan-A/Mart-1(26-35) specific CTL, as
compared to soluble peptides, in particular at low
nominal epitope concentrations (<1 mg/ml). These effector
cells were able to efficiently kill HBL melanoma
cells expressing Melan-A/MART-1 and HLA-A0201. The
adjuvant effects observed were also detectable in
the absence of CD4+ T cellsref)
present on T-cells and dendritic cells is
important in activating and regulating immunity.
The effectiveness of CCL21 as an adjuvant with an
HVJ anionic liposomal TRP2 DNA (plasmid) vaccine
to enhance anti-TRP2 Ab, cytokines, delayed-type
hypersensitivity, T-cell responses, and tumor
protection against B16 melanoma cells was
investigated. Induction of anti-TRP2 immunity
depended mainly on cell-mediated immunity, which
was regulated by timing and route of CCL21
administration with DNA vaccine. The optimum
protocol was to administer CCL21 im 24 h before
DNA vaccine at the same vaccination site. 2
vaccinations (prime/boost) were essential for
induction of strong anti-TRP2 cell-mediated
immunity. CCL21 administration 3 days before or 24
h after DNA vaccine, simultaneous with DNA
vaccine, or at different sites (iv, opposite leg)
was not effective. This study demonstrated that
CCL21 was an effective adjuvant to enhance
TRP2-specific immunity induced by a plasmid DNA
cancer vaccineref
by using melanoma Ags common to normal melanocytes
(when vaccine is injected i.d. but not when injected
i.m.ref
: see danger theory) and risk
for subacute demyelinating polyneuropathy
using melanoma Ags common to normal Schwann cells. 26
patients with advanced melanoma were randomly assigned
to vaccination with a mixture of 4 gp100 and
tyrosinase peptides restricted by HLA-A1, HLA-A2, and
HLA-A3, plus a tetanus helper peptide, either in an
emulsion with GM-CSF
and Montanide ISA-51 adjuvant, or pulsed on MDDCs.
Systemic low-dose IL-2
was given to both groups. Anti-melanoma cell responses
are observed in the sentinel immunized lymph node
(SIN) that drain the vaccine site in 80% of patients
in the GM-CSF group, but only 13% of the patients that
are vaccinated with peptide-loaded DCs. Favourable
outcomes, including tumour shrinkage, are observed in
31% of patients who received the GM-CSF-based vaccine,
and 2 patients experienced stable disease for several
months. Favourable outcomes were only observed in 15%
of patients in the DC group, and only 1 patient
experienced stable disease. 2 patients who received te
GM-CSF-containing vaccine also developed vitiligoref
for prostate
adenocarcinoma. Side effect : autoimmune prostatitis. Low
doses of the pPSA vaccine were not capable of
inducing tumor protection, but when pIL-18 was
co-administered, complete tumor protection was
observed in all mice. Tumor protection was mediated
by both CD4+ and CD8+ T cells.
Detailed analysis of the immune response in mice
immunized with either pPSA or pPSA/pIL-18
demonstrated that pIL-18 skewed the PSA-specific
immune response toward Th1. More
importantly, stronger CD4+ and CD8+
T cell responses developed in the
pPSA/pIL-18-immunized mice, with faster kinetics.
These results suggest that IL-18 is a powerful
adjuvant molecule that can enhance the development
of antigen-specific immunity and vaccine efficacyref.
In a mouse model, the CD8+ T lymphocyte
response to a prostate cancer DNA vaccine encoding
PSA were evaluated after intradermal
electroporation. A significantly increased gene
expression (100- to 1000-fold) and higher levels of
PSA-specific T cells, compared to DNA delivery
without electroporation, was demonstrated.
Interestingly, investigation of a panel of different
electroporation conditions showed that only some
conditions that induce high levels of gene
expression additionally induced cellular immunity.
This suggests that electroporation parameters should
be carefully optimized, not only to enhance
transfection efficiency, but also to enhance the
immune response to the vaccineref.
Several DNA fragments encoding multiple CTL and T
helper cell epitopes were selected from human
prostate-specific membrane antigen (hPSM), mouse
prostatic acid phosphatase (mPAP), and human
prostate-specific antigen (hPSA). These DNA
fragments were ligated together to form a novel
fusion gene, termed the 3P gene. The secondary
lymphoid tissue chemokine (SLC), 3P and human IgG Fc
genes were inserted into pcDNA3.1 to construct a DNA
vaccine, designated pSLC-3P-Fc. After vaccination,
the DNA is taken up by cells that produce and
secrete the SLC-3P-Fc fusion proteins, termed
chemotactic antigen (chemo-antigen). The
secreted chemo-antigens, in addition to promoting
the co-localization of naive, non-polarized
memory T cells and DCs, are efficiently captured and
processed by dendritic cells via receptor-mediated
endocytosis and then cross-presented to both major
histocompatibility complex class I and class II in a
cognate manner. The results of this study
demonstrate that vaccination with pSLC-3P-Fc by gene
gun inoculation induced a strong antitumour response
in a mouse tumour model, which significantly
inhibited tumour growth and prolonged the survival
time of the tumour-bearing mice. In vitro,
the secreted SLC-3P-Fc fusion protein can attract
lymphocytes from human peripheral blood mononuclear
cells (PBMC); when human lymphocytes were stimulated
by pSLC-3P-Fc-transfected autologous PBMC, CTLs were
induced which could specifically kill hPSM-, hPAP-,
or hPSA-expressing tumour cells. These observations
provide a new vaccine strategy for cancer therapy
through promoting the co-localization of lymphocytes
and the concomitant enhancement of antigen-specific
CD4+ helper and CD8+ cytotoxic
T-cell responses against tumourref.
several DNA fragments from human prostate-specific
membrane antigen (hPSM), mouse prostatic acid
phosphatase (mPAP), and human prostate-specific
antigen (hPSA) genes were selected and fused to
create a novel hPSM-mPAP-hPSA fusion gene (named 3P
gene), and human secondary lymphoid tissue chemokine
(SLC), 3P, and human IgG Fc genes were inserted into
pcDNA3.1 to construct a DNA vaccine, designated
pSLC-3P-Fc. In this report, to establish a more
efficient treatment for immunotherapy against
prostate cancer, the construct was transfected into
B16F10 to generate gene-modified tumor cell vaccine
(named B16F10-SLC-3P-Fc). In poorly immunogenic
B16F10 mouse melanoma model, the immunization with
B16F10-SLC-3P-Fc resulted in a strong antitumor
response and 50% of tumor-bearing mice achieved
long-term survival (>120 days). In vivo depletion
of lymphocytes indicated that CD8+ T
cells were involved in the direct tumor killing,
whereas CD4(+) T lymphocytes were required for the
induction of CD8+ CTL response in
B16F10-SLC-3P-Fc-immunized mice. Splenocytes from
B16F10-SLC-3P-Fc-immunized mice specifically
recognized and lysed PSM, PAP, PSA, and 3P
expressing tumor cells. The combined therapy of
B16F10-SLC-3P-Fc plus anti-B7-H1 MAbs further
enhanced the immune response. Rechallenge experiment
showed that a persistent memory response was
successfully induced by the combined therapy. These
observations suggest pSLC-3P-Fc-modified tumor cells
could serve as a vaccine against prostate cancer,
and the therapy combined with anti-B7-H1 MAbs
further enhanced the antitumor immune responserefref.
A phase I study of a DNA vaccine targeting PAP in
patients with stage d0 prostate cancerref.
Previous studies indicated that Copenhagen rats
immunized with a recombinant vaccinia virus
expressing human PAP (hPAP) developed PAP-specific
CTL with cross reactivity to rat PAP (rPAP) and
evidence of prostate inflammation. Viral delivery of
vaccine antigens is an active area of clinical
investigation. However, a potential difficulty with
viral-based immunizations is that immune responses
elicited to the viral vector might limit the
possibility of multiple immunizations. In another
study, Lewis rats were immunized with either a
plasmid DNA-based (pTVG-HP) or vaccinia-based
(VV-HP) vaccine each encoding hPAP. We determined
that rats immunized with a DNA vaccine encoding hPAP
developed a Th1-biased immune response as
indicated by proliferating PAP-specific CD4+
and CD8+ cells and IFN-g production. Rats immunized
with vaccinia virus encoding PAP did not develop a
PAP-specific response unless boosted with a
heterologous vaccination scheme. Most importantly,
multiple immunizations with a DNA vaccine encoding
the rat PAP homologue (pTVG-RP) could overcome
peripheral self-tolerance against rPAP and generate
a Th1-biased antigen-specific CD4+
and CD8+ T cell response. Overall, DNA
vaccines provide a safe and effective method of
generating prostate antigen-specific T cell
responses. These findings support the investigation
of PAP-specific DNA vaccines in human clinical
trialsref, with highest levels of
expression observed in high-grade cancers,
metastatic disease, and androgen-independent tumorsref1,
ref2,
ref3,
ref4.
The membrane-associated character and correlation
between expression levels and disease grade have
enabled PSMA to become an important biomarker for
prostate cancer, and antibodies to PSMA are
currently being developed for the diagnosis and
imaging of recurrent and metastatic prostate cancer
as well as for the therapeutic management of
malignant diseaseref1,
ref2,
ref3,
ref4.
The LNCaP prostate cancer cell line had an estimated
180,000 molecules of PSMA per cell
chain is a primary binding and internalization subunit
for a Th2-derived immune regulatory
cytokine, IL-13. Although extremely high levels of
IL-13Ra2 chain
are expressed on a variety of human tumor cells and
specimens. D5 melanoma cells were stably transfected
with the human IL-13Ra2gene
(D5a2) to assess
the effect of an IL-13Ra2
DNA vaccine in immunocompetent animals. Prophylactic
immunization of mice with the IL-13Ra2 DNA vaccine
resulted in protection against D5a2
tumor development. In vivo depletion
experiments in C57BL/6 and RAG-2-/- mice
indicated that both T and B cells, but not NK cells,
were required for the tumor protection. In addition,
antibody induced by the IL-13Ra2
DNA vaccine showed a modest but significant inhibitory
effect on D5a2
cells in vitro, suggesting that the antibody is
biologically functional. The IL-13Ra2
DNA vaccine also exhibited antitumor activity against
established D5a2
tumors in mice. Histologic analysis of regressing
tumors identified infiltration of CD4+ and
CD8+ T cells and the expression of CXCL9
chemokine in tumorsref
for 90% of primary prostate
adenocarcinoma
and lung
spindle
cell carcinomas
and 50% of primary breast
carcinomas
: presented on HLA-A*02.01
produced an immune response in 60% of patients tested
- or 20 of 33 evaluable patients - with active or
relapsed AML,
refractory CML
or high-risk MDS.
Patients only get the vaccine 3 times, yet an immune
response has been measured in patients 4 years after
treatment. Of those 20 patients who mounted an immune
response against their cancer, 14 had an overall
survival of 4 years, and none of the 13 patients who
did not have such a response lived that long. 3
patients are in "molecular remission" in which there
is no evidence of the disease remaining.
with mutations in APC,
axin 1
and b1-catenin
genes.ref.
It is a member of the secretoglobin protein familyref
successfully used for DNA immunization in murine
models by researchers at Washington University School
of Medicine and the Siteman Cancer Center in St. Louis is
expressed at high levels in leukemic blast cells in
most acute
myeloid
leukemias
and acute
lymphoblastic leukemias.
In myelodysplastic
syndrome,
WT1 mRNA expression levels increase along with disease
progression; thus, WT1 mRNA is a tumor marker for
leukemic blast cells. WT mRNA is also expressed at
high levels in various types of solid cancers,
including cancers of the lung, breast, colon and
pancreas. Patients with WT1-expressing tumors produce
antibodies and cytotoxic T-lymphocytes against WT1
protein, indicating that WT1 protein is highly
immunogenic and a promising tumor antigen. MHC class
I-restricted CTL and class II-restricted helper
epitopes of WT1 protein were identified, and clinical
studies of cancer immunotherapy using these CTL
epitope peptides were performed without significant
adverse effect and with clinical results promising
enough to encourage further clinical trials. The
clinical efficacy of cancer immunotherapy targeting
the WT1 protein should be clarified by a large-scale
clinical studyref.
The expression of WT1 was examined in 494 cases of
human cancers, including tumors of the
gastrointestinal and pancreatobiliary system, urinary
tract, male and female genital organs, breast, lung,
brain, skin, soft tissues and bone by
immunohistochemistry using polyclonal (C-19) and
monoclonal (6F-H2) antibodies against WT1 protein.
Staining for C-19 and 6F-H2 was found in 35-100 and
5-88% of the cases of each kind of tumor,
respectively. WT1-positive tumors included tumor of
the stomach, prostate, and biliary and urinary
systems, and malignant melanomas. A majority of the
positive cases showed diffuse or granular staining in
the cytoplasm, whereas ovarian tumors and desmoplastic
small round cell tumors frequently showed nuclear
staining. Glioblastomas, some of soft tissue sarcomas,
osteosarcomas, and malignant melanomas of the skin
showed extremely strong cytoplasmic staining as
compared with other tumors. Western blot analysis
showed that WT1 protein was predominantly expressed in
the cytoplasm of the tumor cells in 2 cases of lung
adenocarcinoma, supporting the intracytoplasmic
staining for WT1 using immunohistochemistryref.
Mice immunized with the WT1 peptide or WT1 cDNA
rejected challenges by WT1-expressing tumor cells and
survived with no signs of auto-aggression to normal
organs which physiologically expressed WT1 in
prophylactic and therapeutic models. Furthermore, IgM
and IgG WT1 antibodies were detected in the patients
with hematopoietic malignancies, indicating that WT1
protein was highly immunogenic, and that
immunoglobulin class-switch-inducing WT1-specific
cellular immune responses were elicited in the
patients. CD8+ WT1-specific CTLs were also
detected in peripheral blood or tumor-draining lymph
nodes of cancer patients. These results provided us
with the rationale for elicitation of CTL responses
targeting the WT1 product for cancer immunotherapy. On
the basis of the findings mentioned above, a phase I
clinical trial of WT1 peptide cancer vaccine for the
patients with malignant neoplasms. These results
strongly suggested that WT1 peptide cancer vaccine had
efficacy in the clinical setting, because clinical
responses, including reduction of leukemic blast cells
or regression of tumor masses, were observed after the
WT1 vaccination in patients with hematopoietic
malignancies or solid cancersref
is a pivotal regulator of apoptotic cell death and it
is overexpressed in many cancers. Consequently, the
Bcl-2 protein is an attractive target for drug design,
and Bcl-2-specific antisense oligonucleotides or
small-molecule Bcl-2 inhibitors have shown broad
anticancer activities in preclinical models and are
currently in several clinical trials. The clinical
application of immunotherapy against cancer is rapidly
moving forward in multiple areas, including the
adoptive transfer of anti-tumor-reactive T cells and
the use of "therapeutic" vaccines. The overexpression
of Bcl-2 in cancer and the fact that immune escape by
down-regulation or loss of expression of this protein
would impair sustained tumor growth makes Bcl-2 a very
attractive target for anticancer immunotherapy.
Spontaneous T-cell reactivity against Bcl-2 in
peripheral blood from patients suffering from
unrelated tumor types (ie, pancreatic
cancer,
breast
cancer,
acute
myeloid
leukemia (AML),
and B-cell
chronic lymphocytic leukemia) has been described :
these Bcl-2-reactive T cells are indeed
peptide-specific, cytotoxic effector cells. Thus,
Bcl-2 may serve as an important and widely applicable
target for anticancer immunotherapeutic strategies
(eg, in the combination with conventional radiotherapy
and chemotherapy)ref.
and melanomaref.
The recombinant HSP105 did not induce DC maturation,
and the mice vaccinated with HSP105-pulsed BM-DCs were
markedly prevented from the growth of subcutaneous
tumors, accompanied with a massive infiltration of
both CD4+ T cells and CD8+ T
cells into the tumors. In depletion experiments, we
proved that both CD4+ T cells and CD8+
T cells play a crucial role in anti-tumor immunity.
Both CD4+ T cells and CD8+ T
cells specific to HSP105 were induced by stimulation
with HSP105-pulsed DCs. As a result, vaccination of
mice with BM-DCs pulsed with HSP105 itself could
elicit a stronger tumor rejection in comparison to DNA
vaccinationref.
: despite the ubiquitous nature of histones, T cell
recognition of histone H4 peptide was specifically
associated with the presence of prostate cancer in a
primary mouse model of prostatic adenocarcinomaref : WT1124-138
and WT1247-261 were shown to induce
peptide-specific anti-tumor CD4+ helper T
lymphocytes (HTL), which were restricted by frequently
expressed HLA class II alleles. Both peptides-reactive
HTL lines were capable of recognizing naturally
processed antigens presented by DCs pulsed with tumor
lysates or directly by WT1+ tumor cells
that express MHC class II molecules. Interestingly,
the 2 WT1 HTL epitopes described here are closely
situated to known MHC class I-restricted CTL epitopes,
raising the possibility of stimulating CTL and HTL
responses using a relatively small synthetic peptide
vaccineref-melanocyte
differentiation
antigens,
a 16.5kDa TAA, is the smallest member of the inhibitor
of apoptosis family that is abundantly expressed during
development but essentially absent in normal adult
tissues. Interestingly, survivin expression is
up-regulated in virtually all types of cancers studied,
as well as in vascular endothelial cells during tumor
associated angiogenesis. Survivin links apoptosis to
cell cycle progression and plays a pivotal role in
regulation of cell proliferationref.
These characteristics make survivin a potentially
promising generic target for cancer immunotherapy.
Overexpression of survivin in tumors is linked to
decreased patient survival, increased tumor recurrence,
and resistance to therapyref.
Spontaneous immune responses against survivin were
recently demonstrated in a variety of patients with
cancerref.
Survivin-based DNA vaccines were also shown to induce T
cell-mediated antitumor responses without severe
toxicities in preclinicalref
and clinical trialsref.
Hence, a genetic immunization strategy to induce
tumor-specific immune responses against human survivin
in a pre-clinical animal model was developed. In initial
studies, BALB/c mice were immunized by intramuscular
injection with DNA coding for human survivin
(pcDNA3.1/hSurv). In addition, a construct encoding a
secreted version of survivin (pSecTag2B/hSurv) was
designed. A plasmid coding for murine GM-CSF was
co-injected in both cases as a molecular adjuvant.
Expression of survivin following transfection in mouse
cells was corroborated. Humoral responses against human
survivin were detected in mice sera using 2 immunization
protocols (injections at 2- or 3-week intervals). The
humoral response was markedly improved by secretion of
survivin and co-expression of GM-CSF. The predominant
antibody subclass detected in responsive mice was IgG2a,
suggesting that a Th1-CD4+
cellular response had been induced. Furthermore, DNA
immunization with survivin encoding vectors generated an
effective CD8+ T cell response measured as an
increase of cytotoxic IFN-g
secreting CD8+ T cells. In conclusion,
intramuscular genetic immunization of mice with human
survivin encoding plasmids induced a survivin-specific
humoral as well as cellular immune response in recipient
mice. Secretion of survivin and co-injection of GM-CSF
as a genetic adjuvant appear to be more important in
generating an humoral than a cellular immune responseref
on recipient cells in allogeneic
HSCT
recipients mediate the GvL effect. For minor
histocompatibility antigens HA-1 and HA-2, normal cell
expression is restricted to hemopoietic cells, and
boosting the immune response to these antigens may
potentiate GvL effect without accompanying GvHD. To
increase efficacy, expansion of HA-1- or HA-2-specific
CTL before transplantation is desirable. However,
primary HA-1- or HA-2-specific CTL expanded in vitro are
often of low avidity. An alternative approach is to
prime specific CTL responses in vivo by vaccination.
Clearly, donor vaccination must be safe and specific.
DNA fusion vaccines able to induce high levels of
epitope-specific CTL using linked CD4+ T-cell
help have been developed. The vaccines incorporate a
domain of tetanus toxin (DOM) fused to a sequence
encoding a candidate MHC class I binding peptide. This
design generates antitumor CD8+ T-cell
responses and protective immunity in preclinical models.
For clinical application, vaccines encoding
HLA-A*0201-restricted peptides from human HA-1 and HA-2,
which were fused to DOM, were constructed and tested
performance in HLA-A*0201-transgenic mice. Priming
induced epitope-specific, IFN-g-producing
CD8+ T cells with cytotoxic function boosted
to high levels with electroporation. Strikingly, these
mouse T cells efficiently killed human lymphoblastoid
cell lines expressing endogenous HA-1 or HA-2. High
avidity is indicated by the independence of cytolysis
from CD8/MHC class I interaction. These safe
epitope-specific vaccines offer a potential strategy to
prime HA-1- or HA-2-specific CTL in transplant donors
before adoptive transferref
on recipient cells in the setting of allogeneic
HSCT
mediate graft-versus-leukemia effect by T-cells. For
minor histocompatibility antigens HA-1 and HA-2, normal
cell expression is restricted to hemopoietic cells, and
boosting the immune response to these antigens may
potentiate graft-versus-leukemia effect without
accompanying graft-versus-host disease. To increase
efficacy, expansion of HA-1- or HA-2-specific CTL before
transplantation is desirable. However, primary HA-1- or
HA-2-specific CTL expanded in vitro are often of
low avidity. An alternative approach is to prime
specific CTL responses in vivo by vaccination. Clearly,
donor vaccination must be safe and specific. DNA fusion
vaccines able to induce high levels of epitope-specific
CTL using linked CD4+ T-cell help have been
developed. The vaccines incorporate a domain of tetanus
toxin (DOM) fused to a sequence encoding a candidate MHC
class I binding peptide. This design generates antitumor
CD8(+) T-cell responses and protective immunity in
preclinical models. For clinical application, vaccines
encoding HLA-A*0201-restricted peptides from human HA-1
and HA-2 fused to DOM were constructed and their
performance was tested in HLA-A*0201-transgenic mice.
Priming induced epitope-specific, IFN-g-producing CD8+ T
cells with cytotoxic function boosted to high levels
with electroporation. Strikingly, these mouse T cells
efficiently killed human lymphoblastoid cell lines
expressing endogenous HA-1 or HA-2. High avidity is
indicated by the independence of cytolysis from CD8/MHC
class I interaction. These safe epitope-specific
vaccines offer a potential strategy to prime HA-1- or
HA-2-specific CTL in transplant donors before adoptive
transferref. for B
cell lymphomas
: Idiotypic (Id) vaccination has shown promising
results in patients with follicular
lymphoma.
With 2 ongoing, phase-III clinical trials enrolling
newly-diagnosed FL patients, idiotypic vaccinationref
is approaching the final stage of its clinical
development, that of demonstrating a possible benefit
to patients. However, even in the event that either or
both ongoing clinical trials succeed, a number of
relevant questions would still remain unanswered, in
particular whether idiotype (Id) vaccines may be
feasible for most if not all relapsed FL and for some
other B-cell malignancies. All Id vaccine production
attempts were carried out by the same personnel and
always using the same fusion partner (K6H6/B5, i.e.
ATCC number: CRL-1823), according to the standard
tumor/hetero-hybridoma fusion-based method previously
describedref1,
ref2,
ref3,
ref4.
Patients with FL, mantle cell lymphoma and small
lymphocytic lymphoma were supposed to receive Id
vaccine treatment if they achieved either complete
(CR) or partial (PR) response, while patients with
either diffuse large cell or Burkitt's lymphoma were
supposed to receive Id vaccine treatment only if they
achieved a CR. The feasibility of Id vaccination in
relation to induction treatment (if the salvage
therapy did not induce a response sufficient to
proceed with Id vaccination) was markedly different,
being 80-100% in indolent NHL subtypes and 40-50% in
aggressive ones. This difference was not, however, due
to an overall lack of efficacy of the respective
chemotherapy regimens, but rather to the different
eligibility criteria for Id vaccination following
chemotherapy. In fact, the overall response to
induction treatment for aggressive NHL was 75-80%
(CR+PR), but in this group only patients achieving CR
were considered eligible to receive Id vaccination. A
far more important factor that halted treatment was
Id-secreting hybridoma production. Fusion experiments
were successful in most FL cases at the very first
attempt, whereas in other NHL cases, irrespective of
the ultimate Id production outcome, as many as 5
attempts had to be carried out most of the time.
Similarly, in most FL cases, the average number of
successful fusion wells per 96-well plate was >>
15, whereas that of most of the other NHL cases was
typically < 5. Statistically significant
differences in fusion-related and overall feasibility
were found between cases of FL and those of all other
NHL, indolent and aggressive lymphoma, respectively.
Both fusion-related and overall feasibility of the Id
vaccine treatment for FL in first relapse (87%) were
comparable with those already described for both
newly-diagnosed and relapsed patients with the same
diseaseref1,
ref2,
ref3.
Interestingly, the Id vaccine production success rate
was substantially low in cases of mantle cell
lymphoma,
as opposed to what has been preliminarily described
with the very same method in newly-diagnosed MCL
patients (Neelapu SS, Wilson WH, Baskar S, White T,
Frye R, Pennington R, et al. Induction of T-cell
responses by tumor antigen vaccination in mantle cell
lymphoma following rituximab-based treatment. J Clin
Oncol 2003; 22 Suppl 1:663a). This apparent
discrepancy could be due, at least in theory, to MCL
cells at first relapse biologically resembling those
of aggressive NHL rather than FL clones, with obvious
possible repercussions on the fusion process. The main
cause for Id vaccine production failure was poor
hybridoma survival (83% of cases), which accounted for
100% of failures in aggressive lymphoma (9/9
cases). Loss of Id secretion by growing
hybridomas accounted for only 17% of cases of
Id vaccine production failure. In the vast majority of
cases, the feasibility of idiotypic vaccination for
patients with first-relapse B-cell malignancies
strictly depends on the ultimate ability to produce a
viable Id vaccine rather than on the probability of
inducing a clinical response suitable for subsequent
idiotypic vaccination. In this respect, first-relapse
FL clearly appears more suitable for hybridoma-based
Id vaccine production than any other first-relapse NHL
subtype tested. As a major consequence of these data,
clinical trials on Id vaccination for B-cell lymphomas
other than FL in first relapse have been closed.
However, it is possible that alternative methods to
produce the Id protein, particularly those based on
molecular techniquesref1,
ref2,
ref3,
ref4,
may prove far more efficient than the traditional
approachref.
Anti-idiotypic antibodies that bind to the BcR complex
appear to induce apoptosis. In a fraction of patients
treated with anti-idiotypic antibodies, recurrence is
associated with loss of the relevant idiotypic
determinants as genes encoding the cell surface BcR
continue to undergo somatic
hypermutation.
Vaccination with Id-pulsed dendritic cells (DCs) or
with soluble Id-KLH in adjuvant induced immune
responses that eliminated both B-cell lymphoma and
myeloma in tumor-bearing mice; however, the 2
vaccination regimens resulted in distinct immune
responses. Whereas soluble Id plus adjuvant induced
high levels of anti-Id antibodies, the Id-pulsed DCs
did not induce anti-Id or any antitumor antibodies.
Immunization with Id-pulsed DCs induced a significant
increase in the frequency of Id-reactive T cells.
Depletion studies in DC-vaccinated mice showed that
the predominant effector cells responsible for tumor
rejection were of the CD8 subset. The finding that
DC-based Id vaccines elicit tumor protection, which is
entirely based on cell-mediated effector mechanisms,
is of particular importance for plasma cell tumors
because these tumors do not express Id on the surface
and hence do not bind anti-Id antibodiesref. : for follicular
lymphoma is in phase 3 trial : 8 cycles
of CVP => if
at least partial response, => 7 s.c. vaccinations
given over 24 weeks (wk 0, 4, 8, 12, 16, 20 and 24)
+ 4 injections of GM-CSF each cycle; for CLL : with a median follow-up of 9.2
years, 45% of patients remained in continuous
remission, OS = 95%, DFS = 96.5 monthsref|
|
epitopes presentable by MHC class I molecules of |
|
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|
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|
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|
|
|
|
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|
plasmid priming + AAV boosting | ||||
| BCL1 | anti-Id antibodies-dependent (30-80%) |
anti-Id antibodies-dependent (30-100%) | ||||||||
| 38C13 (Href, Lref) | anti-Id antibodies-independent (30%) |
no anti-idiotype antibodies induced (exception : ) (40%) |
no anti-Id antibodies induced (80%) |
|||||||
| JLms (sIg-) | CD8+ T cell-dependent (20%) |
CD8+ T cell-dependent (50%) |
no anti-Id antibodies induced CD8+ T cell-dependent |
|||||||
cell lines expressing the epithelial marker, epithelial
cell adhesion molecule (EpCAM). VH gene
transcripts were identifiable by nested reverse
transcription-PCR either as single or dual VDJ
rearrangements in 4 of 6 lines, most being potentially
functional. V(D)J transcripts were observed in
sequential cultures, indicating stable expression. To
exclude coexisting lymphocytes, each cell line was shown
to be EBV negative, with CD19/CD20 and
cytoplasmic/surface immunoglobulin also absent by flow
cytometry. Identified VH transcripts were
then sought in individual tumor cells, isolated as EpCAM+
single cells by flow cytometry. Importantly, in 3 of 3
selected cell lines, VH genes were
identifiable in a significant fraction (approximately
32%) of single cells. In 5 of 6 identified VH
genes, somatic mutations were apparent with no
intraclonal variation, indicating cessation of
mutational activity. VH transcripts were pre-
and post-isotype switch, with activation of switch
events evident from expressed germ-line switch
transcripts in two of six lines. Strikingly, 6 of 6 cell
lines expressed activation-induced cytidine deaminase
(AID) essential for mutational and switch activity.
These data suggest either a de novo rearrangement
and modification of VH genes in epithelial
tumor cells or assimilation of lymphocyte-derived
chromatin. Constitutive AID activation in malignant
epithelial cells further raises a potential for inducing
aberrant mutational activityref
158 Valine/Valine (V/V) genotype both correlate with
better outcome in a group of 136 FL patients receiving
idiotype vaccination after induction chemotherapy. In CR
patients, the 5-year progression free survival (PFS) was
69% for patients with antibody response and/or V/V
genotype while only 40% for patients with neither,
median time to progression (TTP) 10.47 vs 3.46 years
(p=0.012). In PR patients, the 5-year PFS was 57% for
patients with antibody response and/or V/V genotype and
17% for patients with neither, median TTP not reached vs
1.31 years (p=0.0001). This study further confirms the
strong association of clinical outcome with antibody
response and with the functionally more active form of
the FcR in patients receiving idiotype vaccination
regardless their response to induction chemotherapyref.
are short amino acid sequences that are capable of
increasing transport through cellular membranes. The
murine B cell 38C13 lymphoma idiotype DNA vaccines was
tested with HIV Tat-derived transduction sequences for
efficacy against 38C13 challenge. The rate of tumor
onset was similar for the idiotype and transduction
domain-conjugated idiotype vaccine groups. At days 22-23
postchallenge, the number of surviving mice was
significantly higher in the group that had received a
DNA vaccine consisting of the 38C13 idiotype sequence
plus modified Tat transduction sequence, in comparison
with the group that received idiotype-only vaccines.
Although the overall survival difference was not
statistically significant following day 24, a trend
toward an increased survival rate for mice receiving
idiotype plus Tat-derived transduction domains was
maintained through day 106 postchallenge. Thus, the
addition to idiotype vaccines of specific sequences that
facilitate intracellular transport may have potential to
improve the effectiveness of such vaccinesref.
ref
for T
cell lymphomas
:. Favrille uses a similar
approach with its lead product candidate FavId for the treatment of
B-cell NHL. In January the Company completed
enrollment in a pivotal Phase 3 clinical trial of
FavId following induction therapy with rituximab,
the current standard of care. Favrille has received
Fast Track designation from the FDA for FavId. gene in the 5' end fused to a GDP-L-fucose:b-D-galactoside-2-a-L-fucosyltransferase
(FUT1) in an antisense orientation in the 3'
end. in acute
promyelocytic leukemia (APL) : lipo ATRA was highly
effective in inducing durable molecular remissions
in immunocompetent mice [C57BL/6 x C3H F(1)
(B6C3HF1)]; arsenic therapy was much less
effective, and did not clearly synergize with Lipo
ATRA to increase the remission rate in
immunocompetent mice. The survival of Lipo
ATRA-treated severe combined immunodeficient
(SCID) animals (lacking functional T and B cells)
was inferior to that of immunocompetent B6C3HF1
recipients (40% vs. 88% survival at 1 y, P <
0.001). These data suggest that adaptive immunity
cooperates with pharmacologic therapy to induce or
maintain remissions in murine APL. It also implies
that immunosuppressive anti-leukemia therapies
could paradoxically blunt effective anti-leukemia
immune responses that are important for clearing
small numbers of residual tumor cells after
chemotherapy-mediated cytoreductionref. oncogene to tetanus
fragment C (FrC) sequences can have a pronounced
effect on survival in mice with an animal model
of APL, both alone and when combined with all-trans
retinoic acid (ATRA). The survival advantage is
concomitant with time-dependent antibody
production and an increase in IFN-g. ATRA therapy on its
own triggers an immune response in this model.
When DNA vaccination and conventional ATRA
therapy are combined, they induce protective
immune responses against leukemia progression in
mice and may provide a new approach to improve
clinical outcome in human leukemiaref./c-Abl
in CML : the junctional sequences
represent potentially immunogenic TSAs.. No significant toxic
effects were observed. In 14 of 14 patients,
delayed-type hypersensitivity (DTH) and/or CD4
proliferative responses developed after
beginning vaccinations, and 11 of 14 patients
showed IFN-g release
by CD4 ELISPOT at one or more time points. These
responses were CD4+CD45RO+.
A peptide-specific CD8+ IFN-g ELISPOT was found in 4
patients. 4 patients in hematologic remission
had a decrease in Philadelphia chromosome (Ph)
percentage (3 concurrently receiving IFN-a and 1 on imatinib
mesylate), and 3 patients in molecular relapse
after allogenic transplantation became
transiently PCR- after vaccination; 2
of these patients received concurrent donor
lymphocyte
infusion (DLI). All 5 patients on IFN-a ultimately reached a
complete cytogenetic remission. A relationship
between the clinical responses and vaccination
cannot be determined from this trialref as an immunological
adjuvant. Delayed-type hypersensitivity (DTH),
humoral responses, and unprimed ex vivo autologous
proliferation (3H-thymidine
incorporation) and cytotoxicity (chromium-51
release) responses were measured. All 68
vaccinations were well tolerated without
significant adverse effects. In 3 of the 6
patients treated at the 2 highest dose levels of
vaccine, peptide-specific, T-cell proliferative
responses (n = 3) and/or DTH responses (n = 2)
were generated that lasted up to 5 months after
vaccination. CTLs have not been identifiedref, and no further
reduction of residual disease for at least 6
months preceding enrolment were recruited and
given 6 vaccinations with a peptide vaccine
derived from the sequence p210-b3a2 + molgramostim and QS-21 as adjuvants (CMLVAX100)
before assessment of immunological and disease
response, which included detecting amounts of
b3a2 transcripts by standardised quantitative
real-time RT-PCR. Of 10 patients on imatinib, 9
started CMLVAX100 having had a median of 10
months' stable cytogenetic disease (median 10%
Ph+ metaphases), whereas one started
in stable CCR. All patients' cytogenetic
responses improved after 6 vaccinations, with 5
reaching CCR. Notably, 3 of these 5 patients
also had undetectable amounts of b3a2 transcript
(BCR-ABL:ß2 microglobulin ratio
<0.00001). 6 patients on IFN-a treatment with a median
of 17 months' stable residual disease (median
13% Ph+ cells) were also vaccinated.
All but one had improved cytogenetic responses,
and 2 reached CCR. Overall, peptide-specific
delayed-type hypersensitivity was recorded in 11
of 16 patients, CD4 cell proliferation in 13 of
14 assessed, and interferon production in 5 of 5
assessed. Addition of CMLVAX100 to conventional
treatment in patients with CML might favour
further reduction of residual disease and
increase the number of patients reaching a
molecular responseref.
Imatinib does not impair specific
antitumor T-cell immunity in patients with CMLref or chemotherapy :ref and breast
cancer cells that can serve as targets
for immune recognition and treatment strategies.
Globo H has been synthesized, conjugated to KLH, and
administered with the immunologic adjuvant QS-21 as
a vaccine for patients with prostate cancer who have
relapsed after primary therapies such as radiation
or surgery. The vaccine, given as 5 subcutaneous
vaccinations over 26 weeks, has been shown to be
safe and capable of inducing specific high-titer IgM
antibodies against globo H. Its immunogenicity was
confirmed in prostate cancer patients with a broad
range of stages and tumor burdens. Observations of
several patients who had evidence of disease relapse
restricted to a rising biochemical marker, PSA,
indicated that a treatment effect could occur within
3 months after completion of the vaccine therapy.
This effect was manifested as a decline of the slope
of the log of PSA concentration vs. time plot after
treatment compared with values before treatment. 5
patients continue to have stable PSA slope profiles
in the absence of any radiographic evidence of
disease for > 2 yearsref.
An efficient route to complex synthetic
oligosaccharides attached to an affinity matrix for
identifying and isolating antibodies elicited
against such a carbohydrate-based vaccine in humans
has been reported. Pre- and postvaccination profiles
from serum samples of patients immunized with globo
H-KLH were compared. All anti-globo H antibody
activity was efficiently separated from other serum
constituents. The isolated antibodies were readily
quantified, and their specificities were analyzed.
Since no comparable data were available on
antibodies resulting from the vaccination of other
cancer patients, we compared the observed levels
with those quoted in studies with bacterial
polysaccharide vaccines that had been quantified.
Remarkably, cancer patients immunized with globo
H-KLH produce anti-globo H antibody levels often
exceeding those formed by immunization with
bacterial polysaccharides. In addition, substantial
quantities of both IgG and IgM antibodies were
elicited, clearly indicating a class switch to IgGref. and neuroblastoma.
Active immunization against the glycolipid GD2
using DNA vaccines encoding for cyclic GD2-mimicking
decapeptides (i.e., GD2 mimotopes) is effective
against neuroblastoma. For this purpose, 2 GD2
peptide mimotopes (MA and MD) were selected based on
docking experiments to anti-GD2 antibody
ch14.18 (binding free energy: -41.23 kJ/mol for MA
and -48.06 kJ/mol for MD) and Biacore analysis (Kd
= 12.3 x 10-5 mol/L for MA and 5.3 x 10-5
mol/L for MD), showing a higher affinity of MD over
MA. These sequences were selected for DNA vaccine
design based on pSecTag2-A (pSA) also including a
T-cell helper epitope. GD2 mimicry was
shown following transfection of CHO-1 cells with
pSA-MA and pSA-MD DNA vaccines, with twice-higher
signal intensity for cells expressing MD over MA.
Finally, these DNA vaccines were tested for
induction of tumor protective immunity in a
syngeneic neuroblastoma model following oral DNA
vaccine delivery with attenuated Salmonella
typhimurium (SL 7207). Only mice receiving the
DNA vaccines revealed a reduction of spontaneous
liver metastases. The highest anti-GD2
humoral immune response and natural killer cell
activation was observed in mice immunized with the
pSA-MD, a finding consistent with superior
calculated binding free energy, dissociation
constant, and GD2 mimicry potential for
GD2 mimotope MD over MAref, mammary, sebaceous and
some ovarian
carcinomas. Cancer cells frequently undergo
incomplete glycosylation resulting in the appearance
of precursor structures that normally would be
absent like the case with the T antigen. T antigen
can be detected by several different reagents
including monoclonal antibodies and several plant
lectins-e.g., Arachis hypogea (peanut
agglutinin)(particle-mediated
immunotherapeutic delivery (PMID)-peptide complex-based
vaccines-peptide complex-based
vaccinesref
(produced only for 90% of patients) (in
all stage IV patients, median survival
improved by more than 50%; median
survival in the Oncophage-treated arm
was 20.9 months, versus 12.8 months in
the physician's choice armref1,
ref2);
in
phase I/II trials at the Istituto dei
Tumouri, Milan (in association with
Sigma-Tau), Italy. Preliminary data from
the phase II trial for melanoma was
presented at the AACR-NCI-EORTC
International Conference in Florida,
USA, in October 2001. In phase I/II
trial in the USA (36 patients)ref
(produced for 100% of patients) : in
phase I/II trials (30 patients) at the
Istituto dei Tumouri, Milan (in
association with Sigma-Tau), Italy
(produced for 98% of patients) : in a
pivotal phase III clinical trial for
renal cancer at 80 clinical sites
worldwide. The trial is enrolling at
least 500 patients who are randomised to
receive surgical removal of the primary
tumour followed by out-patient treatment
with Oncophage((R)) or surgery only.
This study was initiated on the basis of
results from a pilot phase I/II study
and preliminary results from a phase II
study in patients with renal cell
cancer. In October 2001, Oncophage was
designated as a fast-track product by
the Food and Drug Administration in the
US for the treatment of renal cell
carcinoma. Oncophage is also in a phase
I/II (42 patients) and a phase II trial
(84 patients) in the US for renal cell
cancer : phase
II trial in the USA (35 patients) :
specific CTL lines were obtained after
repeatedly stimulating T cells with
autologous, HLA-A*0201+ DCs
pulsed with gp96 derived from HLA-A*0201+
human myeloma cell line (HMCL) U266 or
primary myeloma cells. hese T cells
lysed not only gp96-pulsed DCs, U266,
and other HLA-A*0201+ HMCLs IM-9 and XG1
but also effectively killed HLA-A*0201+
primary myeloma cells from patients. No
killing was observed against unpulsed
dendritic cells, dendritic cells pulsed
with control gp96, HLA-A*0201- HMCLs,
and primary myeloma cells, or HLA-A*0201+
nonmyeloma cells. Cytotoxicity was
mainly MHC class I/HLA-A*0201
restricted, suggesting that the CTLs
recognized gp96-chaperoned peptides on
HLA-A*0201 that were derived from shared
myeloma antigens and that myeloma cells
naturally present these peptides in the
context of their surface MHC molecules.
Upon antigen stimulation, these T cells
secreted IFN-g
and TNF-a,
indicating that they belong to Th1
subsetsref. (produced
only for 71% of patients) : phase I/II
trials at Johannes Gutenberg-University
Hospital, Mainz, Germany (30 patients) (produced
only for 30% of patients because of the
high concentration of proteases in that
tissue type) : phase I/II trials at
Johannes Gutenberg-University Hospital,
Mainz, Germany. A pilot phase I trial in
patients with pancreatic cancer began in
the US in 1997 with 5 patients enrolled.
In November 2000, Antigenics announced
that this trial had been expanded to a
phase I/II study which would now include
survival as an endpoint and would enroll
5 additional patients.
fusion protein fusion protein (such an
approach is likely to be more effective in
targeting antigens to macrophages than to DCs,
because macrophages have higher levels of
FcR's) mAb) fusion protein of APCs with tumour DNA encoding
the full ORF of the tumor antigen by using genetically modified can be exploited for clinical
use in cancer immunotherapy by targeting
autologous tumour vaccines to APC, thereby
increasing their immunogenicity. Autologous intact
tumour cells from haematological malignancies, or
autologous tumour cell membranes from solid
tumours are processed to express a-gal epitopes by
incubation with neuraminidase, recombinant a1,3GT and with
UDP-galactose. Subsequent immunization with such
autologous tumour vaccines results in in vivo opsonization
by anti-Gal IgG binding to these a-gal epitopes. The
interaction of the Fc portion of the vaccine-bound
anti-Gal with FcgRs of
APC induces effective uptake of the vaccinating
tumour cell membranes by the APC, followed by
effective transport of the vaccinating tumour
membranes to the regional lymph nodes, and
processing and presentation of the TAA peptides.
Activation of tumour-specific T cells within the
lymph nodes by autologous TAA peptides may elicit
an immune response that in some patients will be
potent enough to eradicate the residual tumour
cells that remain after completion of standard
therapy. A similar expression of a-gal epitopes can be
achieved by transduction of tumour cells with an
adenovirus vector (or other vectors) containing
the a1,3GT gene, thus
enabling anti-Gal-mediated targeting of the
vaccinating transduced cells to APC. Intratumoral
delivery of the a1,3GT
gene by various vectors results in the expression
of a-gal epitopes.
Such expression of the xenograft carbohydrate
phenotype is likely to induce anti-Gal-mediated
destruction of the tumour lesion, similar to
rejection of xenografts by this antibody.
Opsonization of the destroyed tumour cell
membranes by anti-Gal IgG further targets them to
APC, thus converting the tumour lesion, treated by
the a1,3GT gene, into
an in situ autologous tumour vaccineref.) under the control of
tissue-specific promoters : their apoptosis enhances
antigen uptake and presentation by professional
APCs.
transfected with tumour antigen-encoding gene(s) => autologous
bone
marrow transplantation (BMT) + Flt3 ligands +
anti-CD40 mAb to
overcome induction of central tolerance, which could
occur following repopulation of the thymus with
antigen-expressing DCs differentiated in vivo.
This strategy is more successful than administration
of ex vivo generated transduced DCs,
resulting in the long-term survival of 50% of
treated mice mobilization into
peripheral blood :, which binds to
CCR1 and CCR5ref-mobilized
peripheral
blood monocytes as a cell source of DCs :
previous investigations have suggested that
G-CSF-mobilized peripheral blood monocytes
produce reduced levels of proinflammatory
cytokines such as IL-12 and TNF-a. These Th1-type
cytokines are thought to promote antitumor
immune response. The functional abilities of
DCs generated from G-CSF-mobilized monocytes
obtained from 13 patients with CEA-positive
advanced solid cancers were assessed.
Peripheral blood mononuclear cells were
obtained from leukapheresis products collected
before and after systemic administration of
G-CSF (subcutaneous administration of
high-dose [5-10 mg/kg]
human recombinant G-CSF for 5 consecutive
days). In vitro cytokine production
profiles after stimulation with
lipopolysaccharide (LPS) were compared between
monocytes with and without G-CSF mobilization.
DCs generated from monocytes were also
examined with respect to cytokine production
and the capacity to induce peptide-specific T
cell responses. Administration of G-CSF was
found to efficiently mobilize peripheral blood
monocytes. Although G-CSF-mobilized monocytes
(G/Mo) less effectively produced Th1-type
cytokines than control monocytes (C/Mo), DCs
generated from G/Mo restored the same level of
IL-12 production as that seen in DCs generated
from C/Mo. T cell induction assay using recall
antigen peptide and phenotypic analyses also
demonstrated that DCs generated from G/Mo
retained characteristics identical to those
generated from C/Mo. G-CSF mobilization can be
used to collect monocytes as a cell source for
the generation of DCs for cancer
immunotherapy. DCs generated in this fashion
were pulsed with HLA-A24-restricted CEA
epitope peptide and administered to patients
safely; immunological responses were induced
in some patientsref starting from hematopoietic
progenitor
stem cells (HPSC) using different long
and short term culture systems : starting from, because monocytes
represent a readily accessible source of DC
precursors and can easily be obtained in
relatively large numbers from patients.
Methods for enrichment of monocytes collected
in a leukapheresis procedureref
: target was induced. Specific
cytotoxicity was augmented when cultures were
boosted continuously with IL-2 (20 U/mL
biological response modifier program) plus tumor
lysate stimulation. These results suggest that
nonadherent PBMCs may participate in enhancing
DC maturation. Besides the simplicity of this
culture technique, bulk DC cultures potentially
may be used with the same efficiency as
conventional purified DCs. Furthermore, bulk
culture-derived DCs may be used directly in
vivo as a tumor vaccine, or for furtherex
vivo expansion of co-cultured CTLs to be
used for adoptive immunotherapyref.
The number of DC obtained with 2% human serum
albumin (HSA)-supplemented cultures are
consistently higher than in cultures with
various concentrations of autologous plasma or
serum. DC prepared with HSA acquire the ability
to uptake dextran, and express high levels of
MHC and co-stimulatory molecules similar to DC
cultured with autologous plasma or serum.
Although DC cultured with autologous plasma or
serum consist of CD1a+ and CD1a-
populations, DC differentiated in the presence
of HSA express CD1aref.
: CB and APB
monocyte-derived ex vivo expanded DC
express similar DC markers CD83 (31.27+ 11.7%
versus 34.0+ 5.2%, CB versus APB), CD1a (23.4+
4.2% versus 27.6+ 6.3%), and CD80 (21.97+
12.01% versus 27.7+ 5.95). IHC shows that
cells with DC morphology express CD1a but not
CD14. Neither FLT-3L nor TGF-b enhance DC
expansion. Addition of 10% autologous plasma
to CB cultures promotes greater cell survival
and a 150% increase in CD1a+/CD80+
cell recovery. CB DC were 62% as effective
stimulators of adult allogeneic T-cells as APB
DC (p < .05) in allogeneic MLRref.
Cord blood mononuclear cells were cultured in
serum-free medium in the presence of GM-CSF,
SCF and Flt-3 ligand (FL), to generate DC from
their precursors, and thereafter transfected
with the eGFP reporter gene by
electroporation. Maturation of DC was induced
by stimulation with GM-CSF, SCF, FL and
phorbol myristate acetate (PMA). Transfected
DC strongly expressed EGFP, but transfection
efficiency of DC, defined as HLA-DR+
cells lacking lineage-specific markers, did
not exceed 2.5%. Expression of the reporter
gene was also demonstrated in the DC generated
from transfected, purified CD34+
cord blood cells, by stimulation with GM-CSF,
SCF, FL, and TNF-a.
Transfection of CD34+ cells was
very efficient, but proliferation of the
transfected cells was much reduced as
compared to the untransfected cells.
Therefore, the yield of transgene-expressing
DC was relatively low. In conclusion, nonviral
transfection of cord blood DC proved feasible,
but considering the requirements for
immunotherapy in cancer patients, transfection
of differentiated DC or generation of DC from
transfected hematopoietic stem cells provide
only a limited number of DC expressing the
transgeneref is
based on culture of adherent cells in medium
containingref
:
... at 37°C, 5% CO2. The addition of
GM-CSF + IL-4 at the onset of culture proved
disadvantageous and is, therefore, delayed for 24
h. DC development from apheresis cells occurs
faster than from fresh blood or buffy coat, and is
complete after 6-7 daysref.
and IL-3 for differentiation and the TLR3 ligand poly I:C for activation
before pulsing with the TSA. Such a NA17.A2-loaded
DC vaccine was well-tolerated in 8 stage III or IV
melanoma with only minor and transient flu-like
symptoms and inflammatory reactions at the
injection sites. In most patients, isotopic
imaging documented DC migration from the
intradermal injection site to the draining lymph
nodes. Finally, mixed lymphocyte-peptide culture
under limiting dilution conditions followed by
tetramer labeling indicated that 3 out of 8
patients mounted a CD8 T cell response against the
NA17.A2 antigenic peptideref
patients were functionally
abnormal. However, the phenotype and function of
patients' monocyte-derived DCs (MoDCs), which are
commonly used for immunotherapy, were poorly
defined. This study was undertaken to examine the
quality of MoDCs from myeloma patients compared
with cells from healthy donors. Patient-derived
MoDCs are phenotypically and functionally
defective. Compared with their normal
counterparts, patient-derived, mature MoDCs
expressed significantly lower levels of CD1a,
CD40, CD80, and HLA-DR and were poor at activating
alloreactive T cells, presenting recall antigen,
and activating autologous antigen- and
myeloma-specific T cells. These abnormalities may
be attributed to elevated production of autocrine
cytokines such as IL-6, activated p38 and STAT3,
and inhibited MEK/ERK signaling pathways in the
progenitor cells. Treatment with neutralizing
IL-6-specific antibody and, more importantly, p38
inhibitor, or both, could correct these
abnormalities. Treating patient-derived cells with
these agents not only significantly increased cell
yield but also produced MoDCs that were as
functional as their normal counterpartsref
cells are poorly
immunogenic and inhibit T-cell function. AML-derived
dendritic cells (AML-DCs) have better
antigen-presentation capacity than
undifferentiated leukemic blasts, but may not be
fully competent to stimulate T cells previously
inhibited by leukemic cells. IL-12
produced by AML-DCs plays a critical role in
counteracting the inhibitory activity of LCs on
T-cell function. IL-12 gene can be successfully
expressed into AML-DCs defective in endogenous
IL-12 production by using a novel nonviral
method that does not modify their phenotypical,
cytogenetic, and functional features.
Genetically modified AML-DCs restore a near
normal T-cell functionref and ovarian carcinoma were
reportedref1,
ref2,
ref3,
ref4,
ref5,
ref6,
ref7,ref8,
ref9,
ref10,
ref11,
ref12,
ref13,
ref14.
At present, DCs, for experimental and clinical
fusion trial, were obtained mainly from in
vitro standard 7-d protocol culture. In
fact, the kinetics of DCs differentiation from
blood monocytes under physiologic conditions
might not be reflected by current standard
protocols for the in vitro development of DCsref.
One research showed that a subpopulation of
blood monocytes differentiated into DCs within
48 h in a model simulating transendothelial
migration into lymphatic vesselsref.
Dauer et al.ref
reported that they cultured blood monocytes
within 48 h in vitro with the same combination
of proinflammatory mediators as standard 7-d
protocol and obtained mature DCs with their
ability similar to standard protocol DCs in
stimulating primary antigen-specific Th1-type
immune responses, and inducing the production of
IFN-g and activating
autologous naïve T cells, and the DCs were
referred to as FastDCs. Compared with common
standard protocol methods, this new strategy not
only simplified the process and reduced labor,
cost and time for the whole experiment
procedure, but also may be less disrupted by
microbial contamination. Fusion hybrids vaccines
between DCs and engineered J558/IL-12 myeloma
cells secreting Th1 cytokine IL-12
may be an attractive strategy for cancer
imlmunotherapyref.
22 patients were treated with DC vaccine of
fusion cells composed of autologous DCs and
tumour cells (DC/tumour-fusion vaccine), which
was generated by treating each cell type with
PEG. 9 of the 22 patients were treated with both
the DC/tumour-fusion vaccine and systemic
administration of rhIL-12. Serum levels of ANA were examined with an ELISA
kit. One patient with gastric carcinoma (patient
1, DC/tumour-fusion vaccine alone), one patient
with breast cancer (patient 2, DC/tumour-fusion
vaccine alone) and one patient with ovarian
cancer (patient 3, DC/tumour-fusion vaccine +
rhIL-12) showed significant elevations of serum
ANA levels during treatment. In patient 1
malignant ascitic effusion resolved and serum
levels of tumour markers decreased. Patients 2
and 3 remained in good physical condition during
treatment for 24 and 9 months, respectively.
Immunoblot analysis indicated antibody responses
to autologous tumour cells after vaccination in
patient 2. None of the treated patients showed
clinical symptoms suggesting autoimmune disease.
Patients with elevated serum levels of ANA had
significantly longer treatment periods than
those without it. Elevated serum levels of ANA
after DC/tumour-fusion cell vaccine might be
associated with anti-tumour immune response
induced by the vaccinationref.
The anti-tumor effects of vaccinating Balb/c
mice (H-2d) with CT26CL25 colon
carcinoma cells that had been fused with either
syngeneic DCs from Balb/c mice, allogeneic DCs
from C57BL/6 mice (H-2b) or
semiallogeneic DCs from B6D2F1 mice (H-2b/d).
Preimmunization with either semiallogeneic or
allogeneic DC/TC hybrids induced complete
protection from tumor challenge, whereas mice
preimmunized with syngeneic DC/TC hybrids were
only partially protected (75% tumor rejection).
The average number of pulmonary metastases after
intravenous tumor injection decreased
significantly following immunization with
semiallogeneic or allogeneic DC/TC hybrids (8.3
+/- 7.9 or 16.3 +/- 3.5, mean +/- SD) relative
to syngeneic DC/TC hybrids (67.8 +/- 6.3). These
data demonstrate that vaccination with
semiallogeneic DC/TC hybrids resulted in the
greatest anti-tumor efficacy. Anti-tumor effects
showed by in vivo studies were virtually
accomplished by the frequency of induced CTLs
specific to both gp70 and b-galactosidase
assessed
by using pentameric assay. Among the fusion
vaccines tested, semiallogeneic DC/TC hybrids
induced the highest ratio of Th1 cytokine IFN-g to Th2
cytokine IL-10. In addition, allogeneic or
semiallogeneic DC/TC hybrids elicited a
significantly stronger NK activity than
syngeneic DC/TC hybrids. These findings suggest
that in clinical settings, DCs derived from a
healthy donor (which are generally characterized
as more semiallogeneic than allogeneic) may be
more capable than autologous DCs of inducing
promising anti-tumor effects in vaccinations
with DC/TC hybridsref.alone significantly
suppresses the growth of primary 4T1 murine
mammary tumors and the incidence of lung
metastases in the prophylactic setting but is
less effective against pre-established tumors.
However, celecoxib administered after primary
tumor establishment synergizes with tumor
lysate-pulsed DC and the adjuvant, GM-CSF, to improve the antitumor
immune response by suppressing primary tumor
growth and markedly reducing the occurrence of
lung metastases. This triple combination therapy
elicits a tumor-specific immune response
evidenced by elevated IFN-g
and IL-4 secretion by CD4+ T cells
and results in increased infiltration of CD4+
and CD8+ T cells to the tumor site.
In addition, dietary celecoxib inhibits
angiogenesis evidenced by decreased vascular
proliferation within the tumor and serum
vascular endothelial growth factor levelsref freely traverse membrane
barriers. NY-ESO-1, an immunogenic antigen by
itself highly expressed in 60% of high-risk
myeloma patients, was purified to near homogeneity
both alone and as a recombinant fusion protein
with a PTD, derived from HIV-Tat. Efficient entry
of PTD-NY-ESO-1 into DCs, confirmed by microscopy,
Western blotting, and intracellular flow
cytometry, was achieved without affecting
dendritic cell phenotype. Experiments with
amiloride, which inhibits endocytosis, and N-acetyl-l-leucinyl-l-norleucinal,
a
proteasome inhibitor, confirmed that PTD-NY-ESO-1
entered dendritic cells by protein transduction
and was degraded by the proteasome. Tetramer
analysis indicated superior generation of
HLA-A2.1, CD8+ T lymphocytes specific
for NY-ESO-1157-165 with PTD-NY-ESO-1
compared with NY-ESO-1 control protein (44% versus
2%, respectively). NY-ESO-1-specific T lymphocytes
generated with PTD-NY-ESO-1 secreted IFN-g indicative of a Tc1-type
cytokine response. Thus, PTD-NY-ESO-1 accesses the
cytoplasm by protein transduction, is processed by
the proteasome, and NY-ESO-1 peptides presented by
HLA class I elicit NY-ESO-1-specific T lymphocytesref (obtainable even when only tiny
amounts of tumour tissue are available) by DOTAPref
patients and 7 HLA-A1+ or HLA-A2+
healthy donors transfected with mRNA from four
NB cell lines and cocultured with autologous
CD8+ lymphocytes. Expanded CTL
expressed an effector/memory phenotype and a Tc1-like
profile of cytokine secretion. CTL specificity
was demonstrated by IFN-g
release on incubation with HLA-matched NB cell
lines. The latter cell lines, but not
autologous T-cell blasts, were lysed by CTL in
an HLA-restricted manner. Cytotoxicity was
found to involve the release of granzyme B.
When tested for reactivity against
NB-associated antigens, CTL from normal
individuals recognized ALK and PRAME peptides
only, whereas patients' CTL reacted also to
survivin, telomerase, and tyrosine hydroxylase
peptides. This study demonstrates that DC
transfected with NB mRNA induce the generation
of patients' CTL specific for different
NB-associated antigens, supporting the
feasibility of NB T-cell immunotherapyref is superior to lipofection
and passive pulsing of mRNA and to
electroporation of plasmid cDNA. 0.5 mL of the
cell suspension is mixed with 20 mg of IVT mRNA, and
electroporated in a 0.4-cm cuvette using an
Easyject Plus device (EquiBio, Kent, United
Kingdom)ref1,
ref2.
Alternatively cells are electroporated in 2-mm
cuvettes (200 ml
of DC (5 x 106 cells) at 300 V for
500 ms using an
Electro Square Porator ECM 830 (BTX, San
Diego, CA). The amount of in vitro transcribed
RNA used was 2 mg/106
cellsref. : Adenovirus (Ad) type 5
(Ad5)-mediated gene transfer to mouse bone
marrow DCs and human monocyte-derived DCs is
inefficient because neither express the
cognate high-affinity Ads receptor.
Co-precipitating adenoviral vectors with
calcium phosphate (CaPi) increased
gene expression (2000-fold) and transduction
efficiency (50-fold) in mouse DC, primarily
owing to receptor-independent viral uptake.
Moreover, Ad5:CaPi-treated DCs were activated
to express the maturation surface molecules
CD40 and CD86, and to secrete proinflammatory
cytokines TNF-a and
IL-6. However, neither DC transduction nor
maturation was dependent on viral protein
interactions with cell surface integrin.
Ad5:CaPi also transduced human DC
more efficiently than Ad5 alone, similar to a
genetically modified vector (Ad5f35) targeted
to the CD46 receptor. As such, this approach
combines the efficiency of adenoviral-mediated
endosomal escape and nuclear trafficking with
the receptor independence of nonviral gene
delivery. Importantly, CaPi
co-precipitation could be used to functionally
modify DC to activate and expand CMV-specific
memory CTLsrefref
: still a challenge because of low
transduction efficiency and gene silencing in
DCs. An efficient method to prepare such DCs
by in vitro differentiation of
hematopoietic progenitor cells transduced with
chicken ovalbumin (OVA) cDNA via the
gene-silencing-resistant retroviral vector GCDNsap
packaged in vesicular stomatitis virus G
(VSV-G) protein has been established. When
c-KIT+/lineage- cells
were transduced with OVA followed by expansion
and differentiation, more than 90% of mature
DCs expressed the transgene. Mice inoculated
with those cells completely rejected the
OVA-expressing tumor E.G7-OVA, and the
anti-tumor effects were stronger than those
observed in mice inoculated with the same
number of OVA peptide-pulsed DCs. The mice
harbored more cytotoxic T lymphocytes (CTLs)
against E.G7-OVA and produced antibody against
OVA, suggesting the generation of multiple
CTLs recognizing different OVA epitopes and
OVA-specific CD4+ T cells.
Successive inoculations of the transduced DCs
in a therapeutic setting eradicated
preexisting E.G7-OVA and prevented the
progression of retransplanted tumors. Thus,
this vaccine therapy may represent a potent
immunotherapeutic approach for various
malignant tumors that express suitable TAAsref. : lentivirally
transduced DC have been shown to present
antigenic peptides, prime transgene-specific
T cells in vitro and elicit a
protective cytotoxic T-lymphocyte (CTL)
response in animal modelsref : one of the major
advantages of these vectors was reported to be
the absence of deleterious immune responses
following gene transfer. However, recent
studies have shown that AAV vectors elicit
humoral and cellular responses against the
transgene productsref., previously reported to
enhance AAV gene expression. rAAV
transduction of freshly purified MO followed
by 7 days of culture with cytokines to
generate DCs, and subsequent sorting for
coexpression of DC markers CD1a and CD40,
shows robust transgene expression as well as
evidence of nuclear localization of the rAAV
genome in the DC population. Phenotypic
analyses using multiple markers and
functional assays of one-way allogeneic MLRs
indicates that rAAV-transduced MO-derived
DCs are as equivalent to nontransduced
DCsref.) (this is required for
presentation by CD40-B cells as they do not appear to
efficiently process and present exogenous Ags through
the class I MHC pathway). The poly-epitope response is
usually more effective than the response against a
single epitope, as peptides may represent mutant as
well as wild-type portions of the protein. APC
transduced with viral vectors have been reported to
have impaired functions in terms of stimulatory
capacity.
: this would result in autocrine DC stimulation in
vivo after re-injecion.
in culture.
Co-culture of DCs with autologous T cells leads to an
increase in both the number of Tregs, as well as the
expression of FOXP3 protein per cell both in healthy
donors and myeloma patients. DC mediated expansion of
FOXP3high Tregs is enhanced by
endogenous but not exogenous IL-2, and DC-T cell
contact, including the CD80/86 membrane costimulatory
molecules. DCs also stimulate the formation of Tregs
from CD25- T cells. The efficacy of induction
of Tregs by DCs depends on the nature of the
DC maturation stimulus, with inflammatory cytokine
treated DCs (Cyt-DC) being the most effective Treginducers.
DCs induce Tregs using cells from both
healthy donors as well as myeloma patients, and the
induced Tregs effectively suppress T cell
responses in the mixed leukocyte reaction (MLR). A
single injection of Cyt-DCs led to rapid enhancement of
FOXP3+ Tregs in vivo in 3/3
myeloma patients. These data reveal a role for DCs in
increasing the number of functional FOXP3high
Tregs in humansref.
ref cells that generate prostatic
acid
phosphatase : is an immunotherapy
product consisting of APCs, loaded ex vivo
with a recombinant fusion protein consisting of PAP
linked to GM-CSF. As part of a Phase III trial for
FDA approval they gave the vaccine treatment to 82
men with metastatic prostate cancer unresponsive to
hormone therapy, while 45 got a placebo treatment.
On average the men who got the vaccine lived 26
months, while those who got a placebo lived 22
months (18% longer). 3 years later, 34% of vaccine
patients were still alive, compared 11% of
unvaccinated patients. A phase III study was
undertaken to evaluate the safety and efficacy of
sipuleucel-T in a placebo-controlled study. A total
of 127 patients with asymptomatic metastatic hormone
refractory prostate cancer (HRPC) were randomly
assigned in a 2:1 ratio to receive three infusions
of sipuleucel-T (n = 82) or placebo (n = 45) every 2
weeks. On disease progression, placebo patients
could receive APC8015F, a product made with frozen
leukapheresis cells. Of the 127 patients, 115
patients had progressive disease at the time of data
analysis, and all patients were followed for
survival for 36 months. The median for time to
disease progression (TTP) for sipuleucel-T was 11.7
weeks compared with 10.0 weeks for placebo (P =
.052, log-rank; hazard ratio [HR], 1.45; 95%CI, 0.99
to 2.11). Median survival was 25.9 months for
sipuleucel-T and 21.4 months for placebo (P = .01,
log-rank; HR, 1.70; 95%CI, 1.13 to 2.56). Treatment
remained a strong independent predictor of overall
survival after adjusting for prognostic factors
using a Cox multivariable regression model (P =
.002, Wald test; HR, 2.12; 95%CI, 1.31 to 3.44). The
median ratio of T-cell stimulation at 8 weeks to
pretreatment was eight-fold higher in
sipuleucel-T-treated patients (16.9 v 1.99; P <
.001). Sipuleucel-T therapy was well tolerated.
While the improvement in the primary end point TTP
did not achieve statistical significance, this study
suggests that sipuleucel-T may provide a survival
advantage to asymptomatic HRPC patients. Supportive
studies are underwayref1,
ref2 : and colorectal
cancer : Eladem© :
autologous dendritic cells pulsed with recombinant
human PSAref)))) : phase II trial is starting
in November 2004. The patients will receive 4
initial immunizations over a 6 week period with
1.25 mg HER-2 protein AutoVac formulated in
Alhydrogel adjuvant followed by booster
immunizations every 4 weeks for up to 26 weeks.
The trial will be carried out at 5-10 cancer
centers in Poland and Hungary. Pharmexa is also
currently preparing an application for another
phase II trial with the vaccine formulated in a
stronger adjuvant (an immunostimulatory substance
contained in all vaccines). This trial is also
planned to take place in Poland and Hungary and to
include up to 50 patients. The first trial, which
includes up to 50 breast cancer patients, will
start in Hungary and is expected to conclude by
mid 2006.)
to mature
DCs
: when large numbers of DCs were used, tumor growth was
suppressed up to 41% when compared to control mice.
Survival of the animals was prolonged to 54 days
compared to the 33-day survival the control mice. The
delayed-type hypersensitivity (DTH) response induced was
26-fold higher than in the controls. Larger numbers of
DCs also led to higher expansion of IFN-g-secreting-CD8+ T
cells. Furthermore, the secretion of IL-12p70 and IFN-g by spleen cells were enhanced
in proportion to the dosage. However, the level of IL-4
secreted from spleen cells was negligible compared to
the level of IFN-g that was
released. These results indicate that DCs induce Th1-dominant
immune response and that more DCs could lead to better
immunological results, a finding which was consistent
with our therapeutic resultsref.
Anyway the standard approach is use of mature DCs
achieved by DC-poietins
increase protection in mice on immunizationrefref gene
either linked to an Ag-encoding generef
as a bicistronic plasmidref
or as a separate plasmid mixed with the Ag encoding
plasmidref1,
ref2,
ref3,
ref4,
ref5,
ref6.
It has a paracrine effect at the injection site that
recruits a mixed cellular infiltrate including
neutrophils, macrophages, immature DCs, eosinophils
(and T ad B cellsref1,
ref2,
ref3)
capable of recognizing tumour Ags at metastatic
sites : separation of injections with pGM-CSF and
Ag-expressing plasmid plasmid into different sites
does not enhance immune responseref. injected locally or systemicallyref1,
ref2,
ref3,
ref4,
ref5
: systemic toxicityref and flaring of
autoimmune diseases have been reportedref1,
ref2.) mobilizes
DCs into blood, allowing generation in vivo
of large numbers of DCs for immunotherapy. These
immature DCs can be rapidly activated by soluble
CD40 ligand (CD40L). Flt3 ligand-mobilized DCs
(FLDCs) were isolated, activated with CD40L, loaded
with antigenic peptides from influenza M protein,
HBcAg, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected
into patients with resected melanoma. 3 injections
were given at 4-week intervals. No patient had a
measurable tumor. 6 patients were entered. FLDCs
were obtained, enriched, and cultured under Good
Manufacturing Practice grade conditions. Overnight
culture with soluble CD40L caused marked
up-regulation of activation markers (CD83 and
HLA-DR). These FLDCs were functional and able to
stimulate antigen-specific T cells in vitro.
No significant adverse events were attributable to
FLDCs. Peptide-pulsed FLDCs caused strong local skin
reactions up to 60 mm diameter with intense
perivascular infiltration of T cells, exceeding
those seen in previous peptide-based protocols.
Antigen-specific blood T-cell responses were
induced, including responses to an antigen for which
the patients were naive (HBcAg) and
MAGE-A10. MAGE-A10-specific T cells with a skewed
TcR repertoire were detected in 1 patient in blood
ex vivo and from tumor biopsies. Vaccination
with FLDCs pulsed with peptides is safe and primes
immune responses to cancer antigensref and G-CSFR, was
shown to be highly effective in expanding DC in
vivo. In an HLA-A2.1 transgenic mouse system,
ProGP-4-generated DC were found to be approximately
equivalent in presenting a CTL peptide to a CTL line
in vitro compared with bone marrow
(BM)-derived DC and >20-fold more efficient than
macrophages or B cells, and >100-fold better than
BM-DC, macrophages, or B cells at presenting PADRE,
a universal helper T cell epitope, to a T cell
clone. The heightened epitope presentation by
ProGP-4 DC was paralleled in vivo inasmuch
as a >6-fold increase in CTL induction was
observed compared with other APC populations
following ex vivo loading with peptide. The
in vitro and in vivo CTL responses
stimulated by ProGP-4 DC could be further augmented
by either culturing with TNF-a
or co-loading with PADRE. Peptide-loaded
ProGP-4-generated DC demonstrate potent antigenicity
and immunogenicity for CTL, making them an
attractive component of epitope-based vaccinesref.
: ligand(s)
to the cytokine cocktail further enhances the yield,
maturation, migratory and immunostimulatory capacity of
the DC generated. More importantly, culture conditions
also influence the outcome of the T cell response
induced.
/IL-1b/IL-6 or MCM alone induce CD4+
T cells that release intermediate levels of IFN-g and no IL-4 or IL-10.
Production of IFN-g is significantly
induced by addition of PGE2, while no effect on production
of IL-4 or IL-10 is
observed.low, IL-4neg cells, TNF-a/IL-1b/IL-6promoted growth of IFN-gintermediate,
IL-44neg
CD8+ T cells. Addition of PGE2 again only further polarized
this pattern enhancing IFN-g production by
alloreactive CD8+ T cells in both
cultures without inducing type 2 cytokines./IL-1b/IL-6
can substitute for MCM and that addition of PGE2further
enhances
the yield and quality of DC generated. TNF-a/IL-1b/IL-6
+ PGE2-cultured
DC
seem to be optimal for generation of IFN-gintermediate-producing
CD4/CD8+ T cellsref.
Yet, despite rigorous criteria used for their
characterization in vitro, it is not clear
whether the chosen conditions for maturation result in
DC with optimal in vivo functions, including
migration to lymph nodes and activation of Th1
CD4+ and CD8+ CTL. The
above-reported ex vivo maturation protocol is
suboptimal and leads to a partially matured DC, which is
refractory to optimal maturation stimuli encountered in
situ, such as imiquimod or Adjuprimeref.
In addition, reagents used for DC maturation using this
or other protocols can be expensive and not always
readily available for clinical use.
co-culture of day 7 monocyte-derived DC generated with
GM-CSF and IL-4 induces increased numbers of CD40+54+80+83-.
Also, IFN-a maturation leads
to an increase in IP-10 and MCP-1 chemokine secretion,
but only a minor increase in IL-12p40 secretion. In line
with this, maturation with IFN-a
has only a small effect on induction of autologous
T-cell stimulatory capacity of the DC. However, an
increase in DC allogeneic T-cell stimulatory capacity
was observed. These data suggest that IFN-a has a potential as a maturation
agent used in DC-based cancer vaccine trials, but not as
a single reagentref
ligand(s) cream can act as an
adjuvant for gene
gun DNA vaccination as good as rGM-CSFref.
When administered by subcutaneous injection at
the vaccination site immediately after
particle-mediated immunotherapeutic delivery
(PMID) of plasmid DNA using a gene gun,
resiquimod produces a similar Th1 biased immune response with
a 10-fold reduced dose compared with imiquimodref:
resiquimod 50 nM moderately enhances IFN-g production per cell (as
measured by a peptide-based ELISpot assay), and
produces a several-fold increase in the
stimulation index of antigen-specific T-cell
proliferation, increased antibody titer, and
strong Th1 cytokine biasrefmarkedly
enhances
the targeting of antigen to DEC-205+ DCs but
fails to produce sustained antigen-specific immunity
because of the failure to activate DCs in vivo.
The addition of an activating CD40-specific antibody to
anti-DEC-205-antigen conjugate did result in sustained
immunity, showing the importance of combining
MHC-targeting and APC-activation strategies.
and CD70 / CD27L along with an allogenic
MHC class I (H-2Kd) molecule expression on
melanoma cells (B16F10, H-2b) to favor the
antitumor immune response. B16F10 tumor growth slows
significantly when CD40L and CD70 are coexpressed by
tumor cells and the association with the allogenic
molecule (H-2Kd) enhances this effect. Growth kinetics
of mock and CD40L-CD70-H-2Kd-expressing
B16F10 tumors in immunocompetent versus nu/nu and beige
mice suggested that CD8+ T lymphocytes and NK
cells were involved in this antitumor immunity. A delay
in mock tumor growth was observed when CD40L-CD70-H-2Kd-expressing
B16F10 cells and mock tumor cells were injected
simultaneously and contralaterally. It was also shown
that in vivo immunization of immunocompetent mice with
CD40L-CD70-H-2Kd B16F10 tumor cells improved
the generation of cytotoxic lymphocytes against the
wild-type melanoma cells expressing the syngenic MHC
Class I molecule H-2Kb (B16K1). These
observations lay a path for new immunotherapeutic trials
using semiallogenic fibroblasts expressing costimulatory
molecules and tumor-associated antigensref. secreted by
DCs and pulsed with synthetic peptides subserve the
DC function in MHC class I-restricted, peptide-specific
CTL priming in vitro and in vivoref
with E1-, E3-deleted adenoviral vector
would enhance the T cell-mediated cellular immune
response with a consequent induction of anti-tumor
immunity to suppress tumor growth. To prove this
hypothesis, established tumors of different mouse
cancer cells (B16-F10 melanoma, H-2b, and
Colon-26 colon adenocarcinoma, H-2d) were
treated with intratumoral injection of bone
marrow-derived DC that had been modified in vitro
with an RGD fiber-mutant adenovirus vector expressing
mouse fractalkine (Ad-FKN). In both tumor models
tested, treatment of tumor-bearing mice with
Ad-FKN-transduced DC gave rise to a significant
suppression of tumor growth along with survival
advantages in the treated mice. Immunohistochemical
analysis of tumors treated with direct injection of
Ad-FKN-transduced DC demonstrated that the treatment
prompted CD8+ T cells and CD4+ T
cells to accumulate in the tumor milieu,
leading to activation of immune-relevant processes.
Consistent with the finding, the intratumoral
administration of Ad-FKN-transduced DC evoked
tumor-specific CTLs, which ensued from in vivo
priming of Th1 immune responses in the
treated host. In addition, the anti-tumor effect
provided by intratumoral injection of
Ad-FKN-transduced DC was completely abrogated in CD4+
T cell-deficient mice as well as in CD8+ T
cell-deficient mice. These results support the concept
that genetic modification of DC with a recombinant
fractalkine adenovirus vector may be a useful strategy
for cancer immunotherapy protocolsref.
, and head
and
neck cancersref molecules into recombinant DNA
and viral vaccine vectors for antigen-specific
vaccination that directly transduce or infect
APCs. Even though DCs naturally express B7
molecules, the increased level of expression of B7
as well as altered patterns or ratios of
expression of the different B7 family members
could significantly modify the outcome of T-cell
priming in vivo (iCD40)
receptor permits temporally controlled,
lymphoid-localized, DC-specific activation. iCD40
is comprised of a membrane-localized cytoplasmic
domain of CD40 fused to drug-binding domains. This
allows it to respond to a lipid-permeable,
high-affinity dimerizer drug while circumventing
ectodomain-dependent negative-feedback mechanisms.
These modifications permit prolonged activation of
iCD40-expressing DCs in vivo, resulting in
more potent CD8+ T-cell effector
responses, including the eradication of previously
established solid tumors, relative to activation
of DCs ex vivo (P < 0.01),
typical of most clinical DC protocols. In
addition, iCD40-mediated DC activation exceeded
that achieved by stimulating the full-length,
endogenous CD40 receptor both in vitro and
in vivo. Because iCD40 is insulated from
the extracellular environment and can be activated
within the context of an immunological synapse,
iCD40-expressing DCs have a prolonged lifespan and
should lead to more potent vaccines, perhaps even
in immune-compromised patientsref, TNF-a,
and IL-12
between mice and humans, with mice tolerating
50-300-fold higher serum concentrations. So, doses
of these cytokines that induce tumour regression in
mice are lethal in humans. Indeed the primary
motivation is to maximize the expression of the
cytokine at the site of antigen delivery.
Considering that the cytokine gene-transduced tumour
vaccines are typically administered intradermally,
subcutaneously or intramuscularly, whereas T-cell
priming generally occurs in the draining lymph node,
it is not surprising that tumour-cell vaccines
engineered to produce APC-targeted (particularly
DC-targeted) cytokines might ultimately be more
effective in priming immune responses. Targeting of
cytokines to areas of tumour metastasis, to which T
lymphocytes would subsequently traffic and carry out
their effector functions, is achieved by chimeric
antibody-cytokine fusion molecules, in which
cytokines such as IL-2 are linked to the Fc regions of
antitumour antibodies. Incorporation of genes
encoding T lymphocyte costimulatory or proliferative
cytokines into recombinant DNA or viral vaccines
quantitatively enhance T-cell responses and alters
the differentiation pattern of antigen-specific T
lymphocytes. ligands
: in syngeneic mice, ectopic expression of NKG2D
ligands causes NK cell-mediated rejection of
transfected tumor cellsref1,
ref2.
It also primes cytotoxic T cells (CTLs), which are
responsible for rejecting subsequent challenges with
tumor cells lacking NKG2D ligand expressionref.
Engagement of the murine NKG2D receptor (by using a
DNA vaccine encoding one of its ligands, H60)
enhanced innate and adaptive immune responses
induced by a survivin-based DNA vaccineref.
This, in turn, augmented the vaccine's antitumor
efficacy in prophylactic and therapeutic settings
against tumors of different origin and NKG2D
expression levelsref.
Engaging the NKG2D receptor markedly improved the
efficacy of a survivin-based
DNA
vaccine, delivered by attenuated S typhimurium.
The combination vaccine encoding both the NKG2D
ligand H60 and survivin activates both innate and
adaptive antitumor immunity, resulting in better
protection against tumors of different origin and
NKG2D expression levels. The enhanced vaccine
efficacy is in part attributable to increased
crosstalk between lymphocytes. Depletion of CD8 T
cells during priming reduces the vaccine-induced
activation of DCs and NK activity. Depletion of NK
cells during priming leads to reduced DC activation
and CTL activity. However, depletion of CD4+
T cells results in activation of DCs, NK and CD8+
T cells and enhances NK cell activity. The
pH60/survivin vaccine also increases DC and NK
cells, but decreases CD4+ T cells homing
to Peyer's patches, presumably as a result of
changes in the their homing receptor profile. Thus,
by preferentially activating and attracting positive
regulators, and reducing negative regulators in
Peyer's patches, this dual function DNA vaccine
induces a microenvironment more suitable for NK cell
activation and T cell primingref.
inhibitors prevent Trp starvation.
Inevitably, this approach will restrict DC traffic and,
therefore, confine the vaccine effect to one or a few
regional LNs. Unexpectedly, the state of maturation at
the time of injection had no influence on the proportion
of DC that localized to draining lymph nodes, as labeled
immature and mature DC were detected in equal numbers.
Immature DC that trafficked to lymph nodes underwent a
significant up-regulation of CD86 expression indicative
of spontaneous maturation. Moreover, immature DC exited
completely from the dermis within 36 h of injection,
whereas mature DC persisted in large numbers associated
with a marked inflammatory infiltrate. In vitro
maturation is not a requirement for effective migration
of DC in vivo and administration of Ag-loaded
immature DC that undergo natural maturation following
injection may be preferred for DC-based immunotherapyref.
Intradermal immunization with MART-1 peptide-pulsed DCs
results in an increase in circulating IFN-g-producing, antigen-specific T
cells, but the frequency of these cells did not
correlate with responseref.
Intradermal administration results in about a 3-fold
higher migration to lymph nodes than subcutaneous
administration, while mDC showed, on average, a 6-to
8-old higher migration than iDC. The first DC are
detected in lymph nodes 20-60 min after inoculation and
the maximum concentration was reached after 48-72 href.)
that respond by cytokine secretion to peptide-pulsed
targets and de novo DTH compared with other
routes in melanomaref1,
ref2,
ref3
and cutaneous
T-cell
lymphoma (CTCL)ref
patients, indicating that IN may be the preferred ROA
for mature DC vaccinesref.
Inevitably, this approach will restrict DC traffic and,
therefore, confine the vaccine effect to one or a few
regional LNs. Injections of mature peptide
NA17-A-pulsed, keyhole limpet hemocyanin–pulsed, and
Melan-A-pulsed DCs with potential Th1
polarization capacities at monthly intervals, first into
an afferent lymphatic and then twice intranodally into
the lymphatic system did not result in marked clinical
results, despite immunologic responses in some patients.
This highlights the need to improve our understanding of
dendritic cell physiologyrefref.
The inability of mature DCs to home to LNs from the
blood is an important obstacle to the use of
antigen-pulsed DCs as vaccines. . DC vaccination might
be improved if DCs could be manipulated to home to LNs
throughout the body after intravenous injection. For
this, DCs were retrovirally transformed with a chimeric
E-selectin-L-selectin molecule - a substitute for
L-selectin - that recognizes PNADref.
This molecule was chosen because expression of
L-selectin does not change when DCs are retrovirally
transfected with the full-length molecule, presumably
because the ectodomain is proteolytically removedref.
Importantly, L-selectin is clustered on the tips of
microvilli on leukocytes, which greatly enhances its
ability to form tethers under high shear flowref.
Because E-selectin-L-selectin fusion protein contains
the transmembrane and intracellular domain of
L-selectin, it is also targeted to tips of microvilli.
The ectodomain derived from E-selectin is not cleaved,
mediates slow rolling of DCs through PNAD interactions,
and enables DCs to extravasate directly from blood
through LN HEVsref.
So by reconstituting a missing component of the
LN-homing cascade, it is possible to target circulating
leukocytes to the LNsblockade blockade
cells. In HL cells grzB-induced mitochondrial release of
pro- apoptotic Smac is blocked, which results in
complete abrogation of cytotoxicity mediated by CTLs.
Cytosolic expression of recombinant mature Smac enhanced
caspase activity induced by grzB and restored the
apoptotic response of HL cells. Similarly,
down-regulation of XIAP by RNA interference markedly
enhanced the susceptibility of HL cells for CTL-mediated
cytotoxicity. XIAP gene knock-down sensitized HL cells
for killing by antigen- specific CTLs redirected by
grafting with a chimeric anti-CD30scFv-CD3z immunoreceptor. The results
suggest that XIAP targeting by Smac agonists or XIAP-
siRNA can be used as a synergistic strategy for cellular
immunotherapy of HLref
responses, but these may measure CD4 T cells instead of
CD8 T cells
assays allow identification and quantification of
numbers of cells producing cytokines such as IFN-g when encountering target
antigens, but cytokine production may not correlate with
tumor cell killingmay
not
correlate directly with tumor burden
to estimate the number of circulating tumor cells in
peripheral blood : patients with cancer in
whom cancer vaccines are presently being tested are,
almost without exception, of advanced age (65-80 years),
many decades after the thymus has stopped producingnaive T
lymphocytes. Therefore, the generation of an effector-cell
population in response to a vaccine depends on the
recognition of the vaccine antigen by one or more memory
cells in the T-cell repertoire of the patient. In mouse
models, it can be clearly shown that young mice make
stronger primary responses than old mice. There is an
important discrepancy between preclinical studies in mouse
models and clinical trials of cancer vaccines : few
studies, if any, use old mice. Those that do, report an
age-related increase in susceptibility to cancer due to
changing patterns of T-cell subsets, as well as difficulty
in the induction of effective antitumour immune responses.
Hence elderlies could need more adjuvant(s).
in first remission. Humoral antigen- and tumor-specific
responses were detectable but delayed, and they
correlated with peripheral blood B-cell recovery. In
contrast, vigorous CD4+ and CD8+
antitumor Th1 cytokine responses were induced
in most individuals in the absence of circulating B
cells. Analysis of relapsing tumors showed no mutations
or change in expression of target antigen to explain
escape from therapy. These results show that severe
B-cell depletion does not impair T-cell priming in
humans. Based on these results, it is justifiable to
administer vaccines in the setting of B-cell depletion;
however, vaccine boosts after B-cell recovery may be
necessary for optimal humoral responsesref.
is a Th1-type cytokine that
has demonstrated potential as a biological adjuvant in murine
tumor models. The anti-tumor potential and mechanism of action
of simultaneous Apoptin and IL-18 gene transfer in C57BL/6 mice
bearing Lewis lung carcinoma (LLC). The growth of established
tumors in mice immunized with pAPOPTIN in conjunction with
pIL-18 was significantly inhibited compared with the growth of
tumors in mice immunized with the empty vector (EV) or pAPOPTIN
alone. Furthermore, the immunization of mice with pAPOPTIN in
conjunction with pIL-18 elicited strong NK activity and LLC
tumor-specific CTL responses in vitro. In addition, T
cells from lymph nodes of mice vaccinated with pIL-18 or
pAPOPTIN + pIL-18 secreted high levels of the Th1
cytokine IL-2 and IFN-g, indicating
that the regression of tumor cells is related to a Th1-type
dominant immune response. These results demonstrate that
vaccination with Apoptin together with IL-18 may be a novel and
powerful strategy for cancer immunotherapyref.
, mucin-1 and a triad
of costimulatory molecules (TRICOM), which include
B7.1, ICAM-1 and LFA-3. Vaccination is administered by
subcutaneous injection followed by 4 days of local
recombinant adjuvant GM-CSF at the vaccination site. The
vaccine has been developed for patients with advanced
pancreatic cancer and has now entered a randomized Phase III
clinical trial. Currently in phase III studyref
19 peptide sequences with HLA-A*0201 motifs were selected
by computer-based algorithms. 5 peptides (KDR82-90,
KDR288-297, KDR766-774, KDR1093-1101, or KDR1035-1044)
stimulated specific cytotoxic T lymphocytes (CTLs) from
PBMCs of three HLA-A*0201 donors. The decapeptide
KDR288-297 was efficient in sensitizing target cells for
recognition by a CTL clone at a concentration of 10 nM.
More importantly, KDR288-297-specific CTLs lyzed target
cells transfected with HLA-A2/KDR cDNAs and a range of
HLA-matched KDR+ angiogenic endothelial cells
(aECs) and recognized CD34+ endothelial
progenitor cells as well. The specificity of CTLs was
further confirmed by tetramer assay and cold-target
inhibition assay. In addition, ex vivo exposure of aECs to
the inflammatory cytokines enhanced CTL reactivity, which
is in keeping with upregulated KDR and HLA class I
expression. In Matrigel assays, recognition of aECs by
specific CTLs triggered an anti-vascular effect. These
findings provide the first proof of the antigenic property
of KDR protein, and may be useful for devising new
immunotherapeutic approaches to human cancersref.
DNA vaccination against Flk-1 resulted in T cell
activation, suppression of neoangiogenesis, and a marked
reduction in atherosclerosis which was independent of
hypercholesterolemia in both male and female LDL-R-/-
mice. Immunohistochemical characterization of aortic sinus
lesions showed that the decreased lesion area was not
associated with reduced plaque stability and had a lower
density of microvesselsref(stabilises
pericyte-endothelial interactions during angiogenesis and
is highly expressed on endothelium during several
diseases, including arthritis, age-related macular
degeneration and cancer) : a region unique to Tie-2 (amino
acids 1-196) that is homologous in humans and mice. Using
computer algorithms, several HLA-A*0201 epitopes that are
identical in mice and humans were predicted within this
region; however, binding assays showed that the majority
of these epitopes were of low affinity. Modification of
the anchor residues of 4 epitopes enhanced HLA binding.
These epitopes linked via alanines to a known helper
epitope from HBVc128-140
(TPPAYRPPNAPILAAFLPATLTMV) were incorporated by
site-directed mutagenesis into a Tie-2 DNA construct. As
FcR has been shown to increase antigen uptake and delivery
to both class I and II a DNA Fc-fusion vaccine using the
amino acids 1-196 of Tie-2 was designed. Immunisation of
HLA*0201 transgenic mice with one of the modified Tie-2
constructs stimulated CTLs that recognised both wild-type
and modified peptide-pulsed target cells. In contrast, no
CTLs were generated in mice immunised with wild-type Tie-2
construct, demonstrating that the modified epitope was
necessary in the generation of CTLs. Moreover, CTLs from
mice immunised with the modified construct killed
HLA-A*0201 endothelial cells overexpressing Tie-2ref.
fusion vaccine to break the immune tolerance of self
antigens associated with angiogenesis. PCR amplification
of mature b-defensin 2 (Def)
and extracelluar segment of Vascular Endothelial
Cadherin (Cad) was conducted. (GGGS)3 was
used as linker peptide for fusion plasmid(Def-Cad). All
the 3 segments were inserted into pSecTag2B plasmid
(pSec), and CT26 tumor cells were transfected. Expression
was verified by RT-PCR and chemotaxis assay. The alginate
bead model was used to study the antiangiogenic effect of
fusion plasmid vaccination. All constructions were
verified by sequencing and were found expressed in
mammalian cell. The b-defensin
2 fusion protein had similar capacity to chemoattract DCs
as compared with nonfusion protein (P>0.05). The
alginate bead assay revealed that the tumor-induced
angiogenesis in fusion plasmid immunized mice was
significantly suppressed when compared with that in
nonfusion plasmid (P<0.01). An active immunization
strategy based on b-defensin 2
fused with angiogenesis related antigen of endothelial can
inhibit angiogenesis and may be a useful approach for
cancer therapyref.or MMP-14. To enhance the
immunogenicity of the mRNA-translated FAP product, a
lysosomal targeting signal derived from LAMP-1
was fused to the COOH terminus of FAP to redirect the
translated product into the class II presentation pathway.
DCs transfected with mRNA encoding the FAP-LAMP fusion
product stimulated enhanced CD4+ and CD8+
T-cell responses. The growth of solid tumors is
accompanied by tissue remodeling that appears to involve
interactions between the tumor cells and the stromaref.
The reactive stromal fibroblasts are not transformed, but
they are distinguished from normal fibroblasts by their
production of certain extracellular matrix proteins and
proteasesref1,
ref2.
Among the latter, FAP appears to be consistently expressed
as a cell-surface protein in the stroma of malignant solid
tumorsref.
The presence of FAP in 2 malignant melanoma cell linesref1,
ref2
also suggests that FAP expression can be induced in the
cancer cell itself as a result of malignant
transformation. FAP is a serine protease that is closely
related to another type II integral membrane protein, CD26/DPP-IVref1,
ref2.
The catalytic site in CD26/DPP-IV and FAP contains the
characteristic catalytic triad of Ser630/624,
Asp708/702, His740/734 (residues are
numbered according to human CD26/DPP-IV and FAP
respectively), and the active serine is situated in a
nucleophile elbow in the sequence Gly-Trp-Ser-Tyr-Glyref1,
ref2,
ref3.
Both enzymes cleave NH2-terminal dipeptides
from polypeptides with the sequence NH2-Xaa-Pro/Ala, where
Xaa can be any natural amino acidref1,
ref2,
ref3.
In addition, FAP was found to possess gelatinase activity
in vitro, and this observation gave rise to the
notion that the enzyme might play a role in tissue
remodeling by digesting type I collagen of the
extracellular matrixref1,
ref2.
Rat CD26/DPP-IV has also been reported to possess
gelatinase activityref,
and although this activity is seldom attributed to
CD26/DPP-IV, the ability of CD26/DPP-IV to form a complex
with FAP at invadopodia of migratory fibroblasts has
suggested a possible role for the coordinated activity of
both enzymesref
. The amino boronic dipeptide, Val-boro-Pro (PT-100),
appeared to be an interesting drug candidate in this
context. PT-100 competitively inhibits the dipeptidyl
peptidase (DPP) activity of FAP and CD26/DPP-IV, and there
is a high-affinity interaction with the catalytic site due
to the formation of a complex between Ser630/624
and the boron of PT-100ref1,ref2.
The potential of PT-100 as an antitumor agent was
intriguing in the light of our earlier observation that
PT-100, apparently through its interaction with FAP, could
stimulate hematopoiesis via the increased production of
cytokines [including G-CSF] both in vitro in
stromal cell supported human bone marrow cultures and in
vivo in miceref.
The cytokines involved in the stimulation of hematopoiesis
by PT-100 are also known to promote innate as well as
T-cell-mediated antitumor responses. In particular, G-CSF
gene transfection of the colon adenocarcinoma C-26 cell
line has been shown to inhibit tumor take in mice and
cause regression of an established tumor in sublethally
irradiated mice inoculated with the transfected cellsref.
The release of G-CSF by the tumor cells appeared to
promote tumor infiltration by polymorphonuclear leukocytes
expressing IL-1b and TNF-a mRNA followed, sequentially, by
macrophages and T cells. PT-100 abrogates tumor growth as
a single agent and augments antibody-dependent
cell-mediated cytotoxicity. Similarly to hematopoietic
stimulation, the antitumor activity did not require an
interaction with CD26. The data suggest that, via the
up-regulation of cytokine and chemokine expression in both
the tumor and the draining lymph nodes, PT-100 can
stimulate immune responses against tumors involving both
the innate and adaptive branches of the immune systemref.
Mice immunized against FAP using DCs transfected with FAP
mRNA inhibit growth of melanoma, carcinoma, and lymphoma
models and the magnitude of the antitumor response was
comparable to that of vaccination against tumor
cell-expressed antigens. Both s.c. implanted tumors and
lung metastases were susceptible to anti-FAP
immunotherapy. The antitumor response could be further
enhanced by augmenting the CD4+ T-cell arm of
the anti-FAP immune response, achieved by using a
lysosomal targeting sequence to redirect the translated
FAP product into the MHC class II presentation pathway, or
by covaccination against FAP and a tumor cell-expressed
antigen, tyrosinase-related protein 2 (TRP2). No morbidity
or mortality was associated with anti-FAP vaccination
except for a small delay in wound healingref.
Fibroblasts are also the primary source of collagen type
I, which contributes to decreased chemotherapeutic drug
uptake in tumors and plays a significant role in
regulating tumor sensitivity to a variety of
chemotherapies. To specifically kill tumor-associated
fibroblasts, an oral DNA vaccine targeting FAP, which is
specifically overexpressed by fibroblasts in the tumor
stroma, was constructed. Through CD8+ T
cell-mediated killing of tumor-associated fibroblasts, the
vaccine successfully suppressed primary tumor cell growth
and metastasis of multidrug-resistant murine colon and
breast carcinoma. Furthermore, tumor tissue of
FAP-vaccinated mice revealed markedly decreased collagen
type I expression and up to 70% greater uptake of
chemotherapeutic drugs. Most importantly, pFap-vaccinated
mice treated with chemotherapy showed a 3-fold
prolongation in lifespan and marked suppression of tumor
growth, with 50% of the animals completely rejecting a
tumor cell challenge. This strategy opens a new venue for
the combination of immuno- and chemotherapiesref.
: immunization induced cytotoxic lysis of PDGFRb-expressing target cells and
protected mice from the growth and dissemination of murine
colon, breast and lung carcinoma. Furthermore, this novel
vaccine suppresses angiogenesis in vivo and reduces the
numbers of tumor-associated, PDGFRb-expressing
pericytes
as suggested by a decrease in intra-tumoral expression of
PDGFRb and NG2ref
vaccination : depletion of CD4+CD25+
regulatory T cells (Treg) by treatment with CD25
antibody synergizes with vaccination protocols to engender
protective immunity in mice. The effectiveness of targeting
CD25 to eliminate Treg is limited by the fact that CD25, the
low-affinity IL-2R, is up-regulated on conventional T cells.
At present, foxp3 is the only product known to be
exclusively expressed in Treg of mice. However,
foxp3 is not expressed on the cell surface and hence cannot
be targeted with antibodies. In this study, we tested the
hypothesis that vaccination of mice against foxp3, a
self-antigen expressed also in the thymus, is capable of
stimulating foxp3-specific CTL that will cause the depletion
of Treg and enhanced antitumor immunity. Vaccination of mice
with foxp3 mRNA-transfected dendritic cells elicited a
robust foxp3-specific CTL response and potentiated
vaccine-induced protective immunity comparably with that of
CD25 antibody administration. In contrast to CD25 antibody
treatment, repeated foxp3 vaccination did not interfere with
vaccine-induced protective immunity. Importantly, foxp3
vaccination led to the preferential depletion of
foxp3-expressing Treg in the tumor but not in the
periphery, whereas CD25 antibody treatment led to depletion
of Treg in both the tumor and the periphery.
Targeting foxp3 by vaccination offers a specific and simpler
protocol for the prolonged control of Treg that
may be associated with reduced risk of autoimmunity,
introducing an approach whereby specific depletion of cells
is not limited to targeting products expressed on the cell
surfaceref. and
cancer immunotherapyref
vaccine (Norelin©;
source : YM
BioSciences, Inc.) for androgen-dependent prostate
adenocarcinoma :
a double-chain miniprotein with each chain containing 3
linear repeats of the self-peptide gonadotropin-releasing
hormone (GnRH3), the hinge region of human IgG1
(hinge), and a T-helper epitope from the measles virus
protein (MVP). The GnRH3-hinge-MVP miniprotein was
conjugated to purified recombinant Hsp65 of Mycobacterium
bovis and used to immunize rats primed with
subcutaneous injections of BCG in the absence of adjuvantsref
vaccine : CTP37 (Avicine©; source : AVI BioPharma, Inc./SuperGen Corporation)
contains 37 amino acids from the carboxyl terminus of hCG.
Vaccination result in 2 distinct antibody responses to
separate epitopes within the peptideref immunogen (G17DT)
(Gastrimmune©; source : Aphton Corp.)
neutralises the gastrin 17 hormone and the Gly-G17 hormone.
Gastrin 17 is a growth factor for pancreatic, stomach and colorectal
carcinomas,
and a potent stimulator of gastric acid secretion. The
anti-gastrin immunogen, G17DT, consists of a large carrier
protein, called diptheria toxoid (DT). A synthetic peptide,
which is similar to a portion of the gastrin 17 hormone
(GT), is attached to the carrier protein. When administered
to patients, G17DT induces an immune response producing
antibodies, which cross-react and neutralise the target
hormone thus preventing its interaction with disease-causing
or -participating cells : the antibodies both reduced G17
stimulated gastric acid secretion and inhibited gastrin from
interacting with the CCK-2 receptor. Therapeutic efficacy of
both passive and active immunisation with G17DT has been
established in a number of tumour systems including both
primary and metastatic disease. Furthermore, additive
effects with 5-fluorouracil (5-FU)/leucovorin have been
confirmed in both colon and gastric tumour models. Phase
I/II studies in advanced gastrointestinal (GI) malignancies
have shown no systemic or autoimmune reactions to active
immunisation with G17DT. Use of an optimised dose has
yielded a high proportion of responders (> 80%), with
minimal side effects and antibody titres measurable within
2-4 weeks. Taken together these results suggest that the
G17DT immunogen is a promising agent for the treatment of GI
cancer and Phase III trials, currently underway, will
definitively evaluate this early promiseref.
Aphton has entered into an agreement with Aventis Pasteur
for the marketing of G17DT in all human cancer indications
in North America and Europe. Under the terms of the
agreement, Aphton is responsible for product development,
clinical trials and regulatory agency approvals. The
agreement was originally with Pasteur Merieux Connaught, a
subsidiary of Rhone-Poulenc. However, in December 1999,
Rhone-Poulenc merged with Hoechst to form Aventis. As a
result of the merger, Pasteur Merieux Connaught underwent a
name change to Aventis Pasteur. In July 2002, Aphton
announced that it would negotiate with companies wanting to
licence the vaccine in territories other than North America
and Europe. In February 2003, Aphton announced it had
received fast track designation for G17DT in combination
with cisplatin and fluorouracil
for use in stage IV gastric cancer to improve overall
survival. In July 2002, the US FDA granted G17DT orphan drug
status for the treatment of gastric
cancerref,
an indication that was broader than the indication Aphton
originally sought. The vaccine was also granted orphan drug
status in Australia for gastric cancer in December 2002. In
July 2002, Aphton announced that the US FDA had granted
G17DT orphan drug status for the treatment of adenocarcinoma
of the pancreas. In September 2002, the US FDA also granted
G17DT, used in combination with gemcitabine, fast track
status for the treatment of pancreatic
cancer
patientsref.
In addition, the vaccine was also granted orphan drug status
in Australia for pancreatic cancer in December 2002. In
March 2003, Aphton announced that the Committee for Orphan
Medicinal Products had recommended to the European
Commission that G17DT be granted orphan drug status for both
pancreatic and gastric cancer in the EU. Aphton expects this
will be approved and ratified within a few weeks. In August
2002, Aphton filed an IND with the US FDA, as well as the
European equivalent, to conduct clinical trials for patients
suffering from gastro-esophophageal
reflux
disease (GERD).
The trials will assess, among other endpoints, the efficacy
of G17DT in providing symptomatic relief to patients with
GERD. In November 2002, Aphton announced that it had
received approval to initiate a clinical trial in Europe.
Additionally, Aphton has agreed with the FDA to conduct
toxicology and other preclinical studies prior to initiation
of a trial in the US. Patient recruitment for the European
study was scheduled to begin in January 2003ref
vaccines currently are tested on men whose cancers are growing
and no longer responsive to hormone therapy, so that < 20% of
men respond to vaccines aloneref.
To date, the cancer vaccines that have been tested definitively
in randomized phase 3 clinical trials did not show statistically
significant disease-free or overall survival, the ultimate test
of clinical efficacyref.
Clinical studies using adoptive transfer of T cells activated
and expanded ex vivo have unequivocally demonstrated
proof of principle that cancer patients possess tumor-specific T
cells capable of mediating anticancer activity in vivo,
as measured by regression of metastatic tumorsref1,
ref2.
Translation of these findings into a standardized, reproducible
and exportable therapy is still many years away. First and
foremost, the natural state of endogenous tumor-reactive T cells
appears to be characterized by general hyporesponsiveness or
anergy. This is likely to result from a number of mechanisms
that tumors utilize to induce antigen-specific immune toleranceref1,
ref2.
Although a number of the newer-generation vaccines can
effectively transfer antigen to and activate dendritic cells in
vivo, T cell tolerance remains a major barrier that is
difficult to overcome by therapeutic vaccination. Preclinical
models demonstrate that for poorly immunogenic tumors,
therapeutic vaccines alone are ineffective at curing animals
with a significant tumor burden, particularly once tolerance has
been established. Combinations of cancer vaccines administered
in conjunction with inhibitors of immunologic checkpoints and
agonists for TLRs or T cell costimulatory pathways can overcome
tolerance and generate significant antitumor responses even in
cases of metastatic cancerref1,
ref2,
ref3.
Treatment of patients with an antagonist antibody to a powerful
immunologic checkpoint, CTLA-4, resulted in antitumor responses
in a number of melanoma patients, but was associated with a high
incidence of severe autoimmune responsesref.
These and other studies emphasize that antigen-driven responses
such as vaccines will need to be combined with manipulation of
costimulatory and checkpoint pathways to achieve sufficient
therapeutic potency with acceptable toxicity. Because
various of these costimulatory pathways and immunologic
checkpoints work at distinct points in the evolution of the
effector immune response, it is likely that the most potent
antitumor immune responses will be generated by combination
strategies that affect multiple regulatory points. Complex whole
cell-derived vaccines have given clinically superior responses
compared to vaccines containing well-defined antigens, such as
peptides or gangliosides; however, well-defined vaccines are
theoretically more desirable because of their reproducibility.
Therapeutic cancer vaccines are expected to boost an already
existing, albeit weak, immune response rather than prime new
responses : if the existing response was primed against a tumour
that originated at a mucosal site, this response might be more
effectively boosted by a mucosal rather than systemic route of
immunization. Barriers to cancer vaccine development :
,
which has a 2-year mortality rate of >97%. This lack of
division based on disease severity can lead to an imbalance
in risk-benefit judgments in regulating the development of
novel approaches to treat lethal cancers.This effort should clearly be integrated with RAID's mission
of developing academically held immunotherapeutics using the
BRB as a resource for coordination of the production of these
materials. Such an enterprise cannot be successful unless its
budget is at least doubled and its physical plant
significantly upgraded. These increases represent a tiny
fraction of the total NCI budget and would go a long way to
fulfilling the stated mission: significant reduction in cancer
morbidity and mortality over the next decade. Resources might
also come through the NIH Roadmap for Medical Research, as
this endeavor is clearly aligned with the NIH's vision of
creating translationally relevant core technologies and making
their outputs broadly available to the community. The FDA must
take responsibility for better educating itself and the
oncology community regarding the cutting-edge
immunotherapeutics currently being developed. This would be
most efficiently accomplished by significantly expanding the
scope of BRMAC's composition and advisory role. In addition,
the FDA could develop an online database of immunotherapeutics
that covers mechanisms of action, potential and actual
toxicities, contacts (within academia, companies and the FDA)
with expertise in a particular area and regulatory
points-to-consider for investigational new drug applications.
This would alleviate much of the confusion and misperceptions
about regulatory issues regarding specific classes of agents.
As suggested above, the FDA has great power to facilitate the
development of combinations of experimental biologic agents by
promoting the message that combinatorial experimental
therapies with a sound scientific basis are encouraged rather
than discouraged. This message can only come with the
confidence of a well-educated FDA that communicates more
effectively with both academia and industry. In the case of
cancer therapeutics, creation of separate centers for the
evaluation of therapeutics for acutely lethal diseases may
facilitate a more accommodating atmosphere for novel
combinatorial immunotherapeutics by reorientation of
risk-benefit judgments. Only with concerted measures such as
these will the tremendous opportunities that immunologic
science has provided over the past decade be effectively
tapped for the sake of the most effective therapy for patients
with cancer.
Thus far, the emphasis has been on the activation of one arm
of the immune system, in the majority of cases T-cell
immunity, since T cells are believed to be the major players
in tumor control. The available experimental evidence argues
that activation of multiple arms of the immune system is a
must to enhance the ability of immunotherapy to control tumor
growthref1,
ref2
Companies
manufacturing
cancer immunotherapeutics
Web resources :
dependence, drug overdose, prevention of brain or
cardiac toxicity and protection of a fetus during pregnancy
in a drug abuser must induce high concentrations of
circulating drug-specific antibodies which can bind and
sequester nicotine in serum and extracellular fluid : as
antibodies themselves do not cross the blood-brain
barrier
(BBB),
they reduce nicotine distribution to brain and hence many of
nicotine's physiologic and behavioral effects (drug-seeking
and drug-taking behaviour) when the drug is repeatedly
available, especially in high doses. Vaccines should be
compatible with other treatment medications that may be
simultaneously administered vaccine : vaccinevaccine vaccine :
conformationally constrained haptensref;
vaccination during concurrent nicotine administration is
feasibleref;
vaccination reduces nicotine distribution to brain not
only by sequestering nicotine in serum but also by
redirecting tissue distribution disproportionately away
from brain, such that nicotine concentrations are reduced
to a greater extent in brain than in other tissuesref.
Female rats immunized with a nicotine conjugate vaccine
received a single dose of nicotine 0.03 mg/kg i.v. on
gestational day 16 to 22. 5 minutes later, vaccinated rats
had substantially higher bound and lower unbound serum
nicotine concentration and lower brain nicotine
concentration than controls. Fetal brain nicotine
concentration was reduced by 43% in vaccinated rats,
comparable to the reduction in the maternal brain nicotine
concentration. The whole-fetus nicotine concentration was
not altered by vaccination. A similar experiment was
performed in which pregnant rats were passively immunized
with rabbit nicotine-specific IgG 7 or 21 mg/kg just
before nicotine dosing. The effects of passive
immunization on nicotine distribution in the mother were
IgG dose-related and the higher dose reduced nicotine
distribution to fetal brain by 60%. These data suggest
that vaccine effects on nicotine distribution to serum and
brain are similar in pregnant female rats to those
previously reported in adult males. Vaccination of female
rats before pregnancy, or passive immunization during
pregnancy, can reduce the exposure of fetal brain to a
single dose of maternally administered nicotineref.
with Th1
polarization
: vaccine :
IL-18 is an inducer of IFN-g in T
lymphocytes and NK cells, and may contribute to
lupus-like autoimmune disease because cells from lpr mice
are hypersensitive to IL-18 and express high levels of
IL-18. To assess the contribution of IL-18 to the
pathogenesis in the animal model, in vivo
inhibition of IL-18 was attempted. Young lpr
mice were vaccinated against autologous IL-18 by
repeated administration of a cDNA coding for the murine
IL-18 precursor. Vaccinated mice produced autoantibodies
to murine IL-18 and exhibited a significant reduction in
spontaneous lymphoproliferation and IFN-g production as well as less
glomerulonephritis and renal damage. Moreover, mortality
was significantly delayed in anti-IL-18-vaccinated miceref vaccine : 4 i.m.
injections monthly, followed by booster injections every
3 months, of adjuvanted formol-inactivated IFN-a2a
in high risk HIV-positive patientsref
vaccine : MIF/TTX DNA vaccination may be useful
for ameliorating the symptoms of rheumatoid
arthritisref
with Th2
polarization
: vaccine : a modified
endogenous cytokine (AutoVac TNF106, a recombinant
murine TNF-a molecule
containing a foreign immunogenic Th epitope)ref.
Anti-TNF-a autoantibodies are
present in the serum of normal individuals as well as in
certain autoimmune disorders, and may modulate disease
progression in the setting of HIV-1
infection. Anti-TNF-a
autoantibody levels are significantly higher in GRIV
seropositive slow/non-progressors, compared to
seronegative controls. Anti-TNF-a
autoantibodies correlated positively with viral load,
and inversely with CD4+ cell count, %CD4+
cells, and CD4:CD8 ratio. Anti-TNF-a
autoantibodies also correlate positively with antibodies
to peptides corresponding to the CD4 binding site of
gp160, the CD4 identity region, the V3 loop, and the
amino terminus of Tat; anti-TNF-a
autoantibodies also correlated positively with
antibodies to Nef protein. The production of anti-TNF-a autoantibodies appears to be an
adaptive response to HIV infection and suggests the
potential utility of modified cytokine vaccines in the
treatment of HIV infections as well as AIDS-related and
unrelated autoimmune and CNS disordersref.: (Ang I) can be
achieved in hypertensive patients. The secondary objective
was to determine whether the antibodies block the renin
system. Patients (n=27) with essential hypertension
responsive to an ACEi (angiotensin-converting enzyme
inhibitor) or ARB (angiotensin blocker) were randomly
assigned to receive 3-4 injections of the Ang I vaccine PMD3117
or aluminium hydroxide (Alhydrogel) over a 6 week period.
Antibody titre was measured prior to each injection and
every 30 days until disappearance. Indices of renin
blockade were changes in renin and aldosterone (blood and
urine) and a within-patient comparison of the pre- and
post-vaccination rise in 24 h ambulatory blood pressure
after 2 weeks of withdrawal of ACEi or ARB. The anti-(Ang
I) antibody titre rose from the second injection in both
regimes and peaked on day 64. Median half-life was 85 (95%
CI, 44 and 153) days (where CI is confidence interval).
Vaccination did not influence blood pressure, but
significantly blunted the fall in plasma renin following
withdrawal of ACEi or ARB. At 42 days after the first
injection, aldosterone excretion was decreased by PMD3117
to 6 (95% CI, 1 and 31)% of values in patients receiving
Alhydrogel trade mark (P=0.012). In patients with
essential hypertension, PMD3117 generated a prolonged
antibody response to Ang I. Biochemical measurements show
evidence of blockade of the renin system, but higher
titres will be required to achieve a decrease in blood
pressureref.
Immunization studies in rat and human subjects compare the
effectiveness of tetanus toxoid (TT) and keyhole limpet
haemocyanin (KLH) vaccines for immunotherapy following
conjugation with an AI peptide analogue (AI).
Cardiovascular responses were assessed in immunized rats
and human subjects (2-dose trial only), following
increasing i.v. infusions of either AI or angiotensin II
hormone (AII). The AI-TT and AI-KLH conjugate vaccines
induced an equivalent immune response, and inhibition of
the pressor effects to exogenous AI in rats. Single-dose
clinical trials with both conjugate vaccines only resulted
in an immune response to the KLH carrier protein. A 2-dose
clinical trial of AI-KLH conjugate vaccine resulted in a
significant immune response to AI. A shift in diastolic
blood pressure (DBP) dose-response was demonstrated
following challenge with AI and AII for the study
volunteer showing the largest anti-AI IgG induction. KLH
was shown to be a suitable alternative to TT as a carrier
protein for AI, thus supporting continued evaluation of
our AI-KLH conjugate vaccine for treatment of hypertension
in manref : ref
containing 0.5 or 5 mg/ml Mycobacterium tuberculosis.
It appears to preferentially affect T cells specific for
CNS autoantigens, altering their Ag/MHC recognition not
unlike altered peptide ligands (APL). Cop-1 vaccination
boosts the local immune response needed to combat
destructive self-compounds associated with motor neuron
death.GA also induces populations of GA-reactive Th2regulatory cells
that may provide bystander suppression in the CNS. Daily
administration is approved for multiple
sclerosis
(MS)therapy
and
is suggested in therapy of acute and chronic motor neuron
diseasesref.
CD28-CD57+ T cells predominate in
CD8 responses to glatiramer acetateref,
can be recapitulated in
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-intoxicated mice. Adoptive transfer of
copolymer-1 immune cells to MPTP recipient mice leads to
T cell accumulation within the substantia nigra pars
compacta, suppression of microglial activation, and
increased local expression of astrocyte-associated glial cell line-derived
neurotrophic factor (GDNF).
This immunization strategy resulted in significant
protection of nigrostriatal neurons against MPTP-induced
neurodegeneration that was abrogated by depletion of
donor T cellsref.
1H MRSI performed in MPTP-treated mice demonstrated that
N-acetyl aspartate (NAA) was significantly
diminished in the substantia nigra pars compacta (SNpc)
and striatum, regions most affected in human disease.
When the same regions were coregistered with
immunohistochemical stains for tyrosine hydroxylase,
numbers of neuronal bodies and termini were similarly
diminished. MPTP-intoxicated animals that received Cop-1
immune cells showed NAA levels, in the SNpc and
striatum, nearly equivalent to PBS-treated animals.
Moreover, adoptive transfer of immune cells from
ovalbumin-immunized to MPTP-treated mice failed to alter
NAA levels or protect dopaminergic neurons and their
projections. These results demonstrate that 1H MRSI can
evaluate dopaminergic degeneration and its protection by
Cop-1 immunization strategies. Most importantly, the
results provide a monitoring system to assess
therapeutic outcomes for PDref.,
which were remediable by T cell restoration. T
cell-based vaccination, using glatiramer acetate
(copolymer-1, a weak agonist of numerous self-reactive T
cells), can overcome the behavioral and cognitive
abnormalities that accompany neurotransmitter imbalance
induced by (+)dizocilpine maleate (MK-801) or
amphetamine. The results, by suggesting that peripheral
T cell deficit can lead to cognitive and behavioral
impairment, highlight the importance of properly
functioning adaptive immunity in the maintenance of
mental activity and in coping with conditions leading to
cognitive deficits. These findings point to critical
factors likely to contribute to age- and AIDS-related
dementias and might herald the development of a
therapeutic vaccination for fighting off cognitive
dysfunction and psychiatric conditionsref.
over the 24 weeks of phase I/II clinical trials
(injection of MBP-reactive T cells, mucosal immunization
with MBP, MOG, GA in a
mouse model of optic nerve and spinal cord injury
induces clearance of toxic molecules, regulatory
cytokines, and neurotrophins or neuronal metabolic rest)ref1,
ref2,
ref3,
ref4,
ref5,
ref6,
ref7
(oral and nasal administration of MBP and MOG in a mouse
model of cerebral ischemia regulates immune responses)ref1,
ref2,
ref3.
: NMDRA-Radmistered
orally with an AAV in a mouse model of kainte-induced
seziures to block glutamate toxicityref:
the formation of abnormal protein aggregates in the brain,
known as Lewy bodies, has been linked to several
neurological disorders in adults. These include
Parkinson's disease, a condition that can involve slow
movements, tremors and impaired coordination. Genes may
predispose someone to the disease, say researchers. Others
point to environmental toxins as a potential
trigger. Whatever the cause, doctors currently lack
a cure for Parkinson's disease and related Lewy-body
illnesses. Many think that getting the immune system to
attack the protein aggregates is a good step towards
finding a treatment. So several research teams have been
pursuing therapeutic vaccines. Biologists have already
succeeded in giving mice specially designed immune cells
to save them from neurological damage. Now they have gone
a step further by getting mice to produce their own immune
protection through a series of injections. The vaccine,
developed by Leslie Crews of the University of California,
San Diego, and colleagues, is based on the protein in Lewy
bodies, known as a-synuclein.
An overdose of this protein, which acts at the tips of
nerve cells, apparently creates these aggregates in mice.
Animals genetically engineered to overproduce the protein
also exhibit Parkinsonian symptoms. The team gave such
mice monthly injections of their vaccine, and monitored
the response. About half of the animals produced high
levels of antibodies that fought the creation of Lewy
bodies. The vaccine helped to clear the a-synuclein from the brain. After 8
months of treatment, the older mice that received the
vaccine showed a 47% decrease in a-synuclein
protein
compared with their control counterparts. But the vaccine
is far from a sure bet to battle Parkinson's diseaseref.
The mice do not develop exactly the same condition that
appears in humans, so it is hard to extrapolate to people.
The biggest challenges in developing vaccines for
conditions like Parkinson's disease include the
availability of animal models that best reflect human
disease. The Lewy bodies' role in the disorder remains
unclear; preventing them from forming may not prevent
illness. It's not known whether they are a cause or an
effect. Another group working on PD vaccines is that of
Howard Gendelman of the University of Nebraska Medical
Center in Omaha which is using glatiramer
acetate
: amyloid b precursor
protein (APP). Antibodies to Ab
could exert a beneficial action by directly neutralizing
potentially synaptotoxic soluble Ab
species in the brain. Intracerebroventricular injection of
naturally secreted human Ab
inhibited long-term potentiation (LTP), a correlate of
learning and memory, in rat hippocampus in vivo
but a monoclonal antibody to Ab
completely prevented the inhibition of LTP when injected
after Ab. Size fractionation
showed that Ab oligomers, not
monomers or fibrils, were responsible for inhibiting LTP,
and an Ab antibody again
prevented such inhibition. Active immunization against Ab was partially effective, and the
effects correlated positively with levels of antibodies to
Ab oligomers. The ability of
exogenous and endogenous antibodies to rapidly neutralize
soluble A oligomers that disrupt synaptic plasticity in
vivo suggests that treatment with such antibodies
might show reversible cognitive deficits in early ADref. adjuvant
: parenteral immunization of APP Tg mice with synthetic
Ab in CFA
and then boosting with Ab in
IFAcan
markedly
decrease the number and density of Ab
deposits in the brain, with concomitant improvements in
neuritic dystrophy and gliosisref.
Neither T cell infiltrates nor brain inflammation were
observed in these animal studies. It appears that the
induction of antibodies to Ab
plays the primary role in the vaccine-mediated clearance
of Ab from the brain, as
passive transfer of Ab
antibodies has shown similar beneficial
neuropathological effectsref.
Remarkably, a single parenteral administration of a mAb
to Ab has recently been shown
to produce rapid (within hours) benefits on behavioural
measures of cognitive function in a mouse model,
apparently by interfering with diffusible, putatively
synaptotoxic forms of Ab
(e.g., Ab oligomers) without
lowering the overall amount of Ab
deposits in the brainref.
2 broad, not mutually exclusive theories about the
mechanism by which An antibodies work in mice have
emerged. First, evidence for an Fc-mediated uptake and
clearance of Ab-antibody
complexes by local activated microglia has been obtainedref.
Second, evidence of a net movement of Ab peptide out of the brain as a
result of its binding and mobilization by Ab antibodies, in the serum and in
the CSF, has been providedref.
Passive immunization using mAb to Ab
results in brain hemorrhageref.
to patients with mild to moderate AD at weeks 0, 4,
12, 24, and 36. Although a phase I safety study with a
small number of patients failed to reveal significant
side effects, a subsequent phase IIa trial was
discontinued shortly after its initiation when
approximately 5% of the treated patients developed
what appeared to be a CNS inflammatory reaction. A
recent postmortem case report has shown some
macrophages and T cell infiltrates in the CNS after Ab immunizationref.
Such cellular reactions were not detected in mice and
other mammals exposed to the vaccine during
preclinical safety and efficacy testing. According to
recent findings that some elderly subjects and
patients with AD have remarkably increased T cell
reactivity to Abref,
it is possible that AD patients with high Ab T cell reactivity develop
severe T cell reactions in the CNS when immunized and
boosted with Ab emulsified
in the Th1 adjuvant QS21. Nonetheless,
titers of Ab antibodies did
not correlte with the occurrence of severity of
symptoms or relapsesref,
suggesting that the meningoencephalitis was not
necessarily directly linked to Ab-reactive
T cells. Ab-reactive T
cells presumably contributed to effective production
of A antibodies, but it is yet not clear whether they
contributed to severe brain inflammation. Therefore,
it is possible that the Ab-reactive
T cells induced in the context of Th1
adjuvant mediated the first wave of T cell infiltrate
to the CNS, which was then followed by T cell
responses to myelin antigens caused by the
inflammatoryy response in brain. It is also possible
that the prolonged exposure to the adjuvant QS21
induced the stimulation of pathogenic myelin-specific
T cells, independent of Ab-reactive
T cells. The possibility that pathogenic myelin
reactive T cells were involved is supported by MRI
analysis which showed lesions in white matter of the
brain where Abis not
normally expressed. There are several reasons why Ab immunization of APP-Tg mice
did not cause severe inflammatory responses in the
CNS: type T cell responses similar to unimmunized AD, but
lacked plaque-associated dystrophic neurites and
astrocyte clusters and cerebral hemorrhages in 6%
of recipientsin
the
CNSref.
It was recently shown that overexpression of TGF-b in the CNS of APP-Tg mice
results in a significant reduction of Abplaque burden by promoting
microglial clearance of the peptideref.
Adult mouse astrocytes also showed profound capabilities
of degrading Abin vitro and
in situref.
Thus immune approaches targeted to induce Ab antibodies and Th2
immune responses may result in activation of microglia
and astrocytes with a beneficial effect on AD pathology.
: in rodents, after spinal lesion, neutralizing the
neurite growth inhibitor reticulon
4
(RTN4) / Nogo-A promotes axonal sprouting and
functional recovery. To evaluate this treatment in
primates, 12 monkeys were subjected to cervical lesion.
Recovery of manual dexterity and sprouting of
corticospinal axons were enhanced in monkeys treated with
Nogo-A–specific antibody as compared to monkeys treated
with control antibodyref
vaccines : active vaccination of mature rats with ghrelin immunoconjugates decreases
feed efficiency, relative adiposity, and body weight gain in
relation to the immune response elicited against ghrelin in
its active, acylated form. 3 active vaccines based on the
28-aa residue sequence of ghrelin, a gastric endocrine
hormone, were used to immunize adult male Wistar rats (n =
17). Synthetic ghrelin analogs were prepared that spanned
residues 1-10 [ghrelin (1-10) Ser-3(butanoyl) hapten, Ghr1],
13-28 [ghrelin (13-28) hapten, Ghr2], and 1-28
[ghrelin(1-28) Ser-3(butanoyl) hapten, Ghr3], and included
n-butanoyl esters at Ser-3. Groups immunized with Ghr1 or
Ghr3 showed greater and more selective plasma binding
capacity for the active, Ser-3-(n-octanoyl) form of ghrelin
as compared with Ghr2 or KLH vaccinated controls.
Accordingly, they gained less body weight, with sparing of
lean mass and preferential reduction of body fat, consistent
with reduced circulating leptin levels. The ratio of
brain/serum ghrelin levels was lower in rats with strong
anti-ghrelin immune responses. Effects were not attributable
to nonspecific inflammatory responses. Vaccination against
the endogenous hormone ghrelin can slow weight gain in rats
by decreasing feed efficiencyref extractions from the
human placenta after a live full-term delivery. This therapy was
first introduced in the 1970s by Valentin I. Govallo, M.D., Ph.D.ref,
who noted the immunological similarities between pregnancy and
cancer. The goal of cancer immunotherapy, according to Govallo, is
to view the fetal allograft as an "impregnating tumor" and create
an immunological state in the oncological patient analogous to a
spontaneous abortion in a pregnant women. The placenta shares
identical growth mechanisms, antigenic determinants, and
immune-escape properties with cancer cells; this includes numerous
tumor-associated antigens, angiogenic growth factors, complement
regulatory proteins, and defective apoptotic mechanisms which aid
in their survival. Placental vaccination may function as a
multi-epitope vaccine; the body recognizes the placental antigens
of this vaccine as foreign, and thus stimulates a cross reactive
humoral and cell-mediated immune response targeting cancer
tumor-associated antigens as well as proteins that aid in cancer
angiogenesis, complement regulation, and apoptotic resistance.
With recent advancements in molecular and cellular cancer
immunology, the model introduced by Govallo may provide an
important strategic approach to cancer immunotherapyrefref1,
or HIV-1ref1,
ref2and
ref3)
or cancerref.
that
enhances the survival of CD8+ T cells, and the FcgR / CD32, onto which they loaded
anti-CD3 and anti-CD28. Within a week of exposing these
cells to a mixed T cell population, flow cytometry showed
that the K562 cells had died, leaving behind an essentially
pure population of T cellsref.
This method was the first to stimulate large numbers of
expanded T cells and to maintain long-term expansion of the
CD8+ population. But these K562 cells activate
all T cells in a population, not specific subpopulations.
Antigen-specific T cells can be expanded in 2 ways using
this technique: either purify the antigen-specific T cells
before expanding them, or engineer K562 cells that express
HLA molecules. By selecting a cell line expressing the
appropriate HLA (i.e., MHC class I) type, the researchers
could then attach the antigen and expand antigen-specific T
cells from a mixed population,
multiple myeloma, and prostate
cancer.
The company has since initiated a Phase II trial for
patients with multiple
myeloma
and non-Hodgkin's
lymphoma
(NHL).
Expand T cells ex vivo up to 1,000-fold within 14
days while maintaining immune function with Dynabeads©
ClinExVivoTM
CD3/CD28 T-cell expansion technology)
will develop qualitatively different sets of DC
characteristics in response to differing stimuli, although no
stimuli consistently induced all of the characteristic DC
features. There were no clear differences in the responses
relative to specific oncogene expression or stage of
maturation arrest (AML vs CML). Signal transduction agonists
that bypassed membrane receptors/proximal signaling (in
particular, the combination of PMA and A23187) consistently
induced the greatest capability to activate T cells.
Interestingly, this ability did not clearly correlate with
expression of MHC/costimulatory ligandsref. and IL-4 for 7 days. After
confirming the phenotype and function, 100,000 DCs were
injected directly into the cancer nodules of 4 patients with hepatocellular
carcinoma
(HCC)
under ultrasonography guidance 48 h after the administration
of 100% ethanol. All patients were monitored for any
alteration in generalized condition, signs of inflammation,
and liver and kidney function for the next 14 days. In
addition, the final assessment of the safety of the
administration of DCs into cancer nodules was performed 6
months after therapy commencement. The injection of 100%
ethanol disrupted the HCC nodules in all 4 patients. DCs were
distributed uniformly in the cancer nodules as assessed by
ultrasonography. The administration of DCs into cancer nodules
was well tolerated by all patients and there were no immediate
or delayed side effects. The tumor marker decreased in one
patient after the direct administration of DCs. Direct
administration of DCs into the cancer nodules of patients with
HCC was saferef
at 2-week intervals in human cancer patients results in therapeutic
activation
of Va24+Vb11+
NKT cells,
which in turn cause a highly coordinated secondary activation
of acquired and innate immunity, resulting in modulation of
NK, T-, and B-cell numbers and increased serum IFN-g. Most patients experienced temporary
inflammatory symptoms, such as enlargement of palpable tumour
deposits and lymph nodes in all 5 patients with nodal
metastases, as well as respiratory symptoms in individuals
with pulmonary metastases. The therapy resulted in sustained
4-12-month-long decreases in serum tumour markers in 2
patients with adenocarcinoma. One patients with renal-cell
carcinoma developed extensive tumour necrosis, and 2 patients
with hepatic metastases had reduced serum hepatocellular
enzyme levels, indicating an antitumour effect. That is the
first clinical evidence that Va24+Vb11+ NKT cell memory
produces faster, more vigorous secondary immune responses by
innate and acquired immunity upon restimulationref.ref
: immunotherapy with NK cells has been limited by the
inability to obtain sufficient numbers of pure NK cells
suitable for manipulation and expansioncan
mediate Ab-dependent cellular cytotoxicity, and can contribute
to enhanced efficacy of MAb treatment. Under the given culture
conditions, only moderate expansion of CD56+/CD3-/CD16+
cells occurs, with the majority of cells being CD56+/CD3+/CD16+
cells.ref
,
and 2.5% human serum/plasma to achieve concentrations
sufficient to treat patients with >52 x 1010
cells. The protocol involves cultures initiated at 2.52 x
105 cells/mL in 25 mL in 1 L Vuelife culture
bags, with addition of fresh media plus IL-2 every 3 days
to maintain an optimal density of NK-92 cells for
expansion. Daily disruption of cell aggregates enhances
NK-92 cells expansion and viability during the culture
period. Final yields of approximately 1.1-1.32 x 106
cells/mL in a 1.2 L volume (1.36-1.562 x 109
cells; 218-250 fold expansion) over 15-17 days is
achievable under cGMP-compliant conditions with >85%
viabilityref (n = 7) and
non-Hodgkin lymphoma (n = 2), all of whom had relapsed
after an autologous transplantation, were treated with
escalating doses of CIK cells (3 patients at each dose
level of 1 x 109 , 5 x 109 , or 1 x
1010 cells). The CIK cells were produced by
culturing unselected cells from steady-state apheresis
products with IFN-g,
OKT3, and IL-2.
After 21 days in culture, with the addition of fresh media
and IL-2 every 3 to 4 days, the median culture was 97%
viable (range, 61%-100%), 98% CD3+ (range,
66%-99%), 76% CD8+ (range, 27%-96%), 23% CD4 +
(range, 6%-78%), 20% CD3+56+ (range,
8%-58%), and <1% CD16+56+ (range,
0.2%-7.7%). The CD3+56+ cells have
previously been shown to exhibit the most cytotoxic
activity. The absolute number of CD3+56+
cells typically expanded 290-fold (range, 3- to 4000-fold)
under these culture conditions. In vitro cytotoxic
activity was measured against a human B-cell tumor line
(OCI-Ly8). At a 40:1 effector-target cell ratio, CIK cells
killed 32% (range, 2%-69%) of the target cells. A total of
21 infusions were administered to 9 patients. The number
of CIK cells infused ranged from 1.0 x 109 to
1.0 x 1010 per treatment. Toxicity was minimal,
and there were no immediate adverse reactions to the
infusions. 2 patients had partial responses, and 2
patients had stabilization of disease: 1 for > 18
months. Considering that these were heavily pretreated
patients with advanced hematologic malignancies, CIK cells
expanded in this fashion may have utility for the
treatment of high-risk patients with evidence of MRD after
autologous transplantationref.
CD4+25+ regulatory T cells inhibit
the anti-tumor activity of CIK cells. The interaction
between CIK cells and DCs is sufficient for the blockage
of the properties of regulatory T cells. CIK cells have
the desirable properties for immunotherapy approaches,
especially after co-culture with DCsref.
were expanded from normal PBMC by culturing with zoledronate and a low
concentration of IL-2 for 2 weeks. For
the activation of gd T cells, gd T cells were cultured with type I
IFN (HLBI, IFN-a2b and
IFN-b) for 1-3 days. The
cytotoxicity of HLBI-activated gd
T cells against leukemia cell lines and fresh leukemia cells
was evaluated by 51Cr-release assay. gd T cells, which were expanded and
purified with magnetic beads using an anti-gd TCR MAb, were demonstrated to be
cytotoxic against leukemia cell lines of both lymphoid and
myeloid origin and fresh myeloid leukemia cells. By
culturing expanded gd T cells
with type I IFN, the expression of the activation marker
CD69 was increased and the cytometric bead array showed an
elevated production of IFN-g by gd T cells. In addition, the
cytotoxicity of gd T cells
against leukemia cells was definitely enhanced by culturing
gd T cells with HLBIref :
immunotherapy of B-cell malignancies with genetically
engineered human NKTref
recipients, they are termed vaccine-enhanced donor-lymphocyte
infusions (ve-DLI)
expanded by ...
autologous CTLs by DC/melanoma cell hybridomaref,
and nonspecific adhesion molecules including ICAM-1
and LFA-3 (both of which are involved in CD28
signaling)
to the intracellular domains of ... :Approach to manufacturing and characterizing bulk populations of gene-modified autologous T cells for use in treating follicular lymphoma PBMC from healthy donors, obtained after informed consent, were stimulated in vitro with Ab to CD3e (OKT3) and rhuIL-2 and then electroporated with plasmid DNA containing a human CD19-specific chimeric Ag receptor (CAR) gene and HSV-1 thymidine kinase (TK) gene. Stably transfected cells were selected in cytocidal concentrations of hygromycin B over multiple 14-day stimulation culture cycles and then cryopreserved. Vials of cryopreserved/selected T cells were used to initiate T-cell expansion cultures to produce cell products for clinical infusion. These cultures were characterized for phenotype, function and suitability for use in adoptive immunotherapy studies. Bulk populations of gene-modified T cells derived from peripheral blood of healthy donors express CD19+ chimeric Ag receptor at low levels and can specifically lyse CD19+ target cells in vitro. These cells display a differentiated T-effector phenotype, are sensitive to ganciclovir-mediated killing and display a non-transformed phenotype. TCR Vb usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5x109 and 1x1010 cells/run. One of three transductions resulted in a population of cells that was not suitable for infusion but was identified during release testing. No populations displayed any evidence of bacterial, fungal or mycoplasma contamination. Genetically modified T cells have been characterized by cell-surface marker phenotype, functional activity against CD19+ targets and requisite safety testing. These pre-clinical data confirm the feasibility of this approach to manufacturing T-cell productsref. Characterization of an artificial antigen-presenting cell to propagate cytolytic CD19-specific T cellsref. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cellsref
ref1,
ref2-specific
effector
cells : because no functional differences have
been found between antibodies containing the k or l
chainsref
and because k light chain
deficiency has been described in animalsref
and humansref1,
ref2
without increased susceptibility to infection, sparing
the normal population of B lymphocytes expressing the
nontargeted light chain should have minimal adverse
effects on patient humoral immunity. The scFv sequence
was cloned in frame with the human IgG1-CH2CH3
domain and with the z chain
of the TCR/CD3 complex in the SFG retroviral backbone.
T-lymphocytes expressing the anti-k-light
chain CAR showed cytotoxic activity against Igk+ tumor cell lines
and B-CLL cells both in vitro and in vivo.
Incorporation of the CD28 endodomain within the CAR
enhanced the in vitro and in vivo
expansion of transgenic T cells after tumor associated
antigen stimulation. Free Igk+
did not compromise the ability of redirected
T-lymphocytes to eliminate Igk+
tumors, since these free Igs served to sustain
proliferation of CAR-CD28 transgenic T cells, while
inhibit proliferation of CAR-only transfenic T cellsref
: the CE7R chimeric immunoreceptor was constructed by
PCR splice overlap extension and is composed of a
single-chain antibody extracellular domain (scFv)
derived from the L1-CAM-specific murine CE7 hybridoma
fused to human IgG1 hinge-Fc, the
transmembrane portion of human CD4, and the cytoplasmic
tail of huCD3-zeta chain (scFvFc:z)ref-specific
effector
cellsref1,
ref2.
Such proliferation abrogated tolerance and the rescued cells
became effective in treating leukemia. Therefore,
high-affinity CD8+ T cells are not necessarily
deleted by encounter with self-antigen in the periphery, and
can potentially be rescued and expanded for use in tumor
immunotherapyref
to the CD3z chain, allows activation of
murine T cells on engagement with NKG2D ligand+
tumor cells leading to antitumor responses in mice. A
human version of the chNKG2D receptor was expressed on
primary human T cells, and antitumor responses were
determined. Human peripheral blood mononuclear
cell-derived T cells were retrovirally transduced with a
human chNKG2D receptor gene. These chNKG2D-bearing human T
cells responded to NKG2D ligand-positive tumor cells by
producing Th1 cytokines, proinflammatory
chemokines, and significant cellular cytotoxicity. This
response could be blocked by anti-NKG2D antibodies, and it
was dependent on NKG2D ligand expression on the target
cells but not on expression of MHC molecules. In addition,
the activity of chNKG2D-bearing T cells remained
unimpaired after exposure to a soluble NKG2D ligand,
soluble MICA, at concentrations as high as 1.5 mg/mL. These data indicate the
feasibility of using chNKG2D receptors in primary human T
cells and suggest that this approach may be a promising
means for cancer immunotherapyref.-specific
effector
Th1 lymphocytes
and Tc1 CTLs
for prevention or treatment of posttransplant
lymphoproliferative
disorders (PTLDs)ref1,
ref2.
Extension of a similar strategy to Hodgkin's lymphomaref1,
ref2
and nasopharyngeal
carcinoma
(NPC)
is limited by the poor immunogenicity of the limited set
of EBV latency antigens expressed in these malignancies,
making T-cell expansion difficult
improves anti-viral T-cell responses and reduces the
functional differences observed between EBV+
and EBV- respondersref.
CD4+ T cells in the therapeutic LCLs-primed
T-cell culture are diverse in producing Th1 and Th2cytokines,
and may exert specific cytotoxicity via exocytosis of
granulysinref
: adoptive transfer of EBV-specific polyclonal T-cell
lines in 3 patients with extranodal NK/T-cell lymphoma,
nasal type, and evaluated the treatment for safety,
immunologic reconstitution, and clinical outcomes. The
tissue samples collected from the 3 patients were
confirmed by polymerase chain reaction analysis to be
EBV positive. In the cases of the first and second
patients, EBV-transformed B-lymphoblastoid cell lines
(LCLs) and T-cell lines were generated from peripheral
lymphocytes of HLA-matched sibling donors. The third
patient's T-cell lines were induced with autologous
lymphocytes. Polyclonal T-cell infusion was carried out
after high-dose radiotherapy because active relapsed
disease remained in all of the patients. The first
patient received 4 weekly infusions of 2 3 107
cells/m2, and the second and third patients
underwent treatment with 2 cycles of infusions of the
same dosage. All T-cell lines showed >60% NK
activity, CTL responses of >40% against autologous
LCLs, and no CTL activity against patient-derived
lym-phoblasts. The level of cytotoxicity increased
substantially in all patients after cell infusion. The 2
patients who received T-cell therapy twice had
stabilized disease for > 3 years. These safe
treatments exhibited no severe inflammatory response,
and no serious toxicity developed during T-cell therapy.
Adoptively transferred cells may provide reconstitution
of EBV-specific CTL responses in patients with active
relapsed angiocentric lymphoma. These results provide a
rationale for the immunotherapy of angiocentric lymphomaref-specific
effector
Th1 lymphocytesref1,
ref2
: Riddell et al enriched CD8+ CMV-specific CTLs
from donor lymphocytes in vitro prior to administration.
This method simultaneously addressed 2 issues. First,
alloreactive donor CTLs that could cause GvHD were
eliminated by culturing in the absence of their target
antigens. Second, by growing anti-CMV CTLs in the presence
of viral target antigens, the anti-viral effector cells
were stimulated to divide and rapidly expand to much
larger numbers than were initially isolated from the
donor. Following adoptive transfer, the CMV
antigen-specific CTLs did not produce GvHD or other
morbidity, and could persist and retain anti-CMV cytotoxic
activity for at least 12 weeks. The effectiveness of
antiviral adoptive immunotherapy for rapid reconstitution
of cellular immunity and definitive control of CMV and
other viral infections has now been demonstrated by a
number of other groups since these pioneering studiesref1,
ref2.
Unfortunately, despite the first demonstrated success of
these methods over 10 years ago, adoptive immunotherapy
has still not been widely applied clinically for a number
of reasons. In vitro expansion and/or selection of
antiviral CTLs is technically complex, labor intensive,
and expensive. Furthermore, because growing sufficient
numbers of cells for therapeutic efficacy is a lengthy
process (weeks or months), this approach is not applicable
to acute clinical situations that require rapid
availability of donor T-cells. Thus, because of these
restrictions, the therapeutic potential of adoptive
antiviral immunotherapy has not been fully realized. The
alternative approach of combining low-dose, unselected DLI
with vaccination (veDLI) will be a simpler and more
broadly applicable method for promoting rapid immune
reconstitution after transplantation than traditional
adoptive immunotherapy. A recently published clinical
trial supports this proposition. Rapoport et al randomized
multiple myeloma patients undergoing autologous HSCT to
receive DLI (composed of in vitro stimulated PBMC) with or
without combined vaccination (7-valent pneumovax)ref.2
The
investigators demonstrated that a combination of
pretransplant vaccination prior to PBMC collection, T cell
activation in vitro, and subsequent
post-transplant T-cell add-back along with vaccination was
highly effective at significantly increasing CD4+
T cell–dependent pneumococcal antibodies. Interestingly,
neither T-cell add-back nor post-HSCT vaccination alone
was effective at stimulating antigen-specific immunity,
suggesting the importance of combining these modalities
for optimal immune reconstitution in HSCT recipients.
While this is an important study, it unfortunately
utilized in vitro culture and activation of T cells
prior to infusion, which may place this approach outside
the capabilities of many centers. Future studies to
investigate whether in vitro T-cell stimulation is
necessary for the success of this protocol should be
informative. veDLI has a number of potential advantages
over classic adoptive immunotherapy. Adoptive
immunotherapy is a therapy that is difficult to apply
routinely, since a subset of CTLs must be enriched and
expanded in vitro from a specific donor for a selected
recipient. In contrast, a relatively small number of
CMV-vaccine constructs could be available off the shelf
for veDLI in the vast majority of transplant recipients.
For example, because the frequencies of HLA-A1, HLA-A2,
and HLA-A24 antigens are 30%, 45%, and 15% in Caucasians,
substantially more than half of all patients could be
vaccinated with a single construct expressing CMV HLA-A1–,
A2–, and A24–restricted immunodominant peptides. Thus,
anti-CMV vaccines could be used, like drug therapy, with
no on-site preparation required. In contrast to the
expense and technical complexity of in vitro CTL
expansion for adoptive immunotherapy, anti-CMV vaccination
takes advantage of normal immune mechanisms to drive in
vivo antiviral CTL expansion. Neither alloreactive
nor other CD8+ T-cell subsets would be
stimulated by an appropriately constructed vaccine.
Importantly, vaccination in lymphopenic HSCT recipients
should be extremely effective since the existing T-cell
"vacuum" leads to unusually rapid proliferation among a
broad array of T cells (homeostatic proliferation)ref.
In this setting, CD8+ T cells presented with
strongly reactive peptides (e.g., antiviral T cells
following vaccination) proliferate most rapidly, making up
a greater proportion of the final lymphocyte population at
the conclusion of homeostatic proliferationref.
And, because veDLI harnesses normal immune mechanisms for
activation, expansion, and subsequent physiological
contraction of the antiviral CTL responseref,
the resulting immunity may be more durable and
long-lasting than that produced by infusions of in
vitro expanded effector CTLs. Vaccination against
CMV antigens could also have a number of important
salutary effects on GvHD in addition to CMV disease. By
repressing CMV replication, vaccination would obviate the
need for ganciclovir drug therapy, eliminating
ganciclovir-associated neutropenia, immuno-suppression,
and associated opportunistic infectionsref.
Any spurious activation of alloreactive CTLs through
heterologous immunity should also be reducedref,
and initiation or exacerbation of GvHD by CMV infection
should be eliminated. By reducing the severity of GvHD,
steroids (if needed at all) could be administered at lower
doses, which should allow accelerated immune
reconstitutionref.
Furthermore, GvHD would not be expected to accentuate CMV
disease, because CMV replication should be suppressed
following vaccination. Given the potential advantages of
post-HSCT vaccination, it is surprising that with notable
exceptionsref
more effort has not been applied to optimize this therapy
for clinical use. Boeckh and colleagues have also recently
expressed this sentiment, and speculated that it may be
due in part to the minimal data currently available on
immune responsiveness following transplantationref.
The ubiquitous environmental bacterium Listeria
monocytogenes (Lm) has many characteristics of a
potent vaccination vector. It is avidly phagocytized by
host macrophages and other antigen-presenting cells
(APCs). Some bacteria are degraded within phagolysosomes
of APCs, and bacterial peptides are loaded onto MHC class
II proteins, stimulating a CD4+ T-cell
response. However, Lm can also induce phagolysosome
breakdown and escape into the cytoplasm of the APC. During
its intracellular phase, Lm expresses secreted proteins
that can be delivered to the APC’s endogenous
antigen-processing pathway and presented on MHC class I
proteins to elicit a potent CD8+ T-cell
responseref.
Lm can also activate strong innate immune responses and
humoral immunityref.
For use in vaccination, Lm can be rapidly modified to
express recombinant proteins using the pPL2 plasmid
integration vector. This plasmid is first engineered using
standard recombinant DNA techniques to express desired
antigens (such as ovalbumin or the murine CMV [MCMV]
immunodominant peptide HGIRNASFI24) as in-frame fusion
proteins with the N-terminal region of the bacterial LLO
gene. Following transformation, the plasmid becomes stably
integrated into an innocuous site of the bacterial genome
(adjacent to the tRNAArg gene), greatly reducing the
chances that recombinant protein expression will be lost
during bacterial passage. Engineered Lm strains can hold
large recombinant DNA segments (for example, multiple CMV
genes encoding several immunodominant epitopes), eliciting
a broad immune response following vaccination. Infection
with recombinant Lm expressing LCMV nucleoprotein antigenref,
papilloma virus antigenref,
or influenza NP antigenref
leads to potent CD8+ T-cell responses against
the recombinant peptides and protects experimental animals
from lethal LCMV infection, papilloma virus infection, or
tumors expressing NP, respectively. Additional desirable
vaccination characteristics of Lm include effectiveness in
recipients with preexisting antivector immunity, allowing
repetitive immunizations; compatibility with simple large
scale clinical-grade production; robust sensitivity to
ampicillin and other antibiotics, which enhances the
safety margin; and effectiveness following oral,
intravenous, intraperitoneal, intradermal, intramuscular,
and subcutaneous administrationref.
Notably, the absence of these characteristics in viral
vectors, such as vaccinia and adenovirus, limit their
clinical usefulness. In order to develop a clinically
useful Lm-based vaccine platform, investigators at Cerus
Corporation began by deleting actA (actA)ref,
a critical virulence factor involved in cell-to-cell
bacterial spread. Administration of similarly attenuated
Lm strains to normal human volunteers was well tolerated
with no untoward side effectsref.
Nonetheless, concern remained that these attenuated
strains may still pose a small but important risk to
immunocompromised patients such as HSCT recipients.
Unfortunately, reports in the literature showed that
extensively attenuated (i.e., heat killed) bacteria, which
should be safe for administration to immunosuppressed
patients, have limited efficacy and do not induce
functional IFN-g–expressing CD8+
effector T cells or stimulate a memory T-cell responseref.
To further improve the safety of the Lm platform, while
retaining its vaccination potential, a novel vaccination
technology was developed. First, the investigators took
advantage of proprietary DNA-crosslinking psoralens
developed by Cerus. The psoralen presently in clinical
use, S-59
{3-[(2-aminoethoxy)methyl]-2,5,9-trimethyl-7H-furo[3,2-g][1]
benzopyran-7-one hydrochloride}, is a highly-reactive
compound that in the presence of UVA light covalently
crosslinks nucleic acids in an irreversible reaction,
blocking DNA replication. S-59 is approved for commercial
sale in Europe for pathogen inactivation of platelets, and
to date S-59 photochemically treated platelet units or T
cells have been safely administered to > 1000
individuals in > 20 clinical trialsref.
Second, the investigators created Lm strains with an
additional defined deletion of the ultraviolet light
resistance (uvr) AB gene (uvrAB), which functions during
nucleotide excision repair. The resulting Listeria
actA/uvrAB mutants cannot repair DNA crosslinks resulting
from S-59 treatment, and very few (in fact, as little as
one) S-59 DNA crosslinks per bacteria render them
incapable of dividingref.
However, because the remainder of their genome is
unaltered by the low frequency of crosslinks, these
bacteria retain the capacity to infect APCs and express
antigens that are presented via both MHC class I and class
II pathwaysref.
This killed but metabolically active (KBMA) Listeria
platform thus defines a new class of vaccinesref.
Consistent with the theoretical advantages of this
technology, we have shown (below) that KBMA Lm-MCMV
(expressing the MCMV immunodominant antigen HGIRNASFI) is
safe enough to administer to highly immunocompromised BMT
mice on the day following transplantation without
lethality and effectively induces an anti-HGIRNASFI CD8+
T-cell response with accompanying long-term lytic
activity. A variety of alternative vaccination
methodologies are being developed for CMV, including
recombinant CMVgB (which is designed to elicit
neutralizing antibodies), whole killed virus, viral
peptides, and plasmid-based DNA vaccines (Zaia JA, Sissons
JG, Riddell S, et al. Status of cytomegalovirus prevention
and treatment in 2000. Hematology Am Soc Hematol Educ
Program. 2000;:339–355). Among these approaches, DNA
vaccination against MCMV has been extensively studied in a
murine systemref1,
ref2.
Thus, it is worth considering why KBMA Lm vaccination may
be a superior technology, based on safety and efficacy
concerns. With respect to safety, DNA vaccines represent a
well-defined immunogen contained on a cell-free plasmid
vector. However, when used alone, DNA vaccines have
limited efficacy and for this reason are typically
combined with attenuated microbial vectors in a
heterologous prime-boost strategyref.
With this approach, DNA vaccination is given first to
"prime" or "focus" the immune response on a defined
antigen(s). Days to weeks afterwards, virus expressing the
same antigen(s) is given to expand or "boost" the immune
response, through a mechanism that probably depends on
both higher antigenic expression from, and generation of a
pro-inflammatory immune response to, the virusref.
Notably, efforts to create HIV vaccines have affirmed the
conclusions that DNA vaccination must be combined with a
viral boost (such as from modified vaccinia virus Ankara)
to raise sufficiently high levels of protective cellular
immunity against HIV-like viruses in nonhuman primatesref.
Thus, even with DNA-based plasmid immunization, recipients
must be boosted with a microbial vector, which makes this
approach unsuitable for immunocompromised HSCT recipients
unless extremely safe microbial vectors like KBMA Lm are
used. In contrast, the use of KBMA Lm for both prime and
boost is not only safe but also rapidly elicits CTL
responses after transplantation. In initial studies, we
have used mouse models, including a murine BMT system, to
demonstrate proof of concept for veDLI (submitted for
publication). Vaccination was performed using Lm
engineered to secrete the MCMV H-2b
immunodominant peptide HGIRNASFI24 (Lm-MCMV) in-frame with
the bacterial LLO gene using the pPL2 integration vector.
In wild-type (non-transplanted) C57BL/6 mice, a single
inoculation of 107 colony-forming units (cfu)
Lm-MCMV (which is < 0.03 LD50) elicited a
strong HGIRNASFI-specific CD8+ T-cell response,
as quantified by intracellular cytokine and tetramer
staining. An average of 8% of splenic CD8+ T
cells were HGIRNASFI-specific 7 days after vaccination.
The long-term (> 200 day) persistence and function of
anti-HGIRNASFI CD8+ T cells was comparable
between vaccinated mice and those that had survived a
sublethal MCMV infection. Importantly, Lm-MCMV vaccination
of wild-type mice led to marked reductions in viral load
after challenging with experimental MCMV infection. In
order to assess the effects of DLI and/or Lm-MCMV
vaccination in reconstitution of anti-MCMV immunity after
transplantation, a syngeneic C57BL/6 C57BL/6 BMT
model was employed. Recipients were conditioned with a
lethal dose of total body irradiation (11 Gy) to eliminate
their T-cell function, and then transplanted with 5 x 106
donor bone marrow cells that were depleted of mature T
cells. These mice are immune deficient for several weeks
after transplantation and are acutely sensitive to CMV
lethality during this periodref.
Subgroups of BMT mice then received DLI (30 x 106
splenocytes harvested from congenic donors that were
either naïve or immunized with 107 cfu Lm-MCMV
7 days previously) and/or vaccinated with 107
cfu Lm-MCMV. When using the novel nonrepli-cating KBMA
Lm-MCMV vaccine, immunization could be performed
immediately after transplantation with no lethality over
the course of multiple studies. Consistently over the
course of these studies, BMT recipients who received only
DLI or vaccination (or neither) had few if any detectable
antiviral T-cells and minimal HGIRNASFI-specific cytolytic
activity. In marked contrast, the combination of DLI and
vaccination (veDLI) produced a significant expansion of
HGIRNASFI-reactive CD8+ T cells to levels of
4-25% of total CD8+ cells (depending on the
specific protocol). These results are very similar to the
results of the clinical trial reported by Rapoport et al,
where DLI and vaccination had to be combined to elicit
specific pneu-mococcal immunity following HSCTref.
Importantly, because KBMA-Lm-MCMV vaccination could safely
be administered immediately after transplantation,
antiviral T-cell expansion could be observed as early as 8
days after BMT, markedly shortening the post-transplant
period of defective immunity. As with vaccination of
nontransplanted mice, veDLI following BMT led to high
levels of persistent antigen-specific lytic activity for
> 200 days.-specific
effector
Th1 lymphocytesref-specific
effector
Th1 lymphocytesref1,
ref2,
ref3,
ref4.
Most of the approaches to generate antigen-specific T
cells using peptide-pulsed DCsref1,
ref2
and antigen-pulsed DCsref1,
ref2
or genetically modified APCsref
are labor intensive and require several weeks for the
generation of a clinical product. Animal models suggest
that the reconstitution of Aspergillus-specific
immune responses after allogeneic SCT is protective
against the development of IAref1,
ref2,
ref3.
Using the IFN-g secretion
assay, human activated T cells were isolated upon
stimulation with a cellular extract (EC SAB) of Aspergillus
fumigatus. Culturing this cell population for 14
days, an average of 1.1 x 107 cells were
obtained from a single 100-mL blood draw in 7 of 7 healthy
individuals. Within another 14 days, these cells were
expanded to an average number of 2.0 x 108 Th1
cells secreting IFN-g on
stimulation with Aspergillus antigens. Testing
various fungal antigen extracts, similar proportions of
IFN-g-producing CD3+/CD4+
cells were obtained upon activation with antigen extracts
of A fumigatus, A flavus, A niger,
and Penicillium chrysogenum, whereas no
significant IFN-g production
was observed upon activation with antigen extracts of Alternaria
alternata and Candida albicans. In addition,
generated T cells were able to induce damage to A
fumigatus hyphae, and significantly increased hyphal
damage induced by human neutrophils. CD4+
T-cell-mediated alloreactivity of generated anti-Aspergillus
T cells was clearly reduced compared with that of the
original cell populationref.
In a recent study, 35 haploidentical hematopoietic
transplant recipients with evidence of invasive
aspergillosis, indicated by the presence of pulmonary
infiltrates and positive galactomannan results, but not by
the isolation of Aspergillus species, received Aspergillus-specific
T-cell therapyref.
The pathogen-specific T cells, generated with extracts of
A fumigatus, controlled antigenemia and infectious
mortality, but unfortunately, no data were presented
showing whether these T cells were Aspergillus-specific
indeed.
Therefore, one cannot exclude that the generated cells
were functionally active against other filamentous fungi
than A fumigatusref.
insensitive to TGF-b, a powerful,
naturally occurring substance in the body that enables
cancer cells to evade surveillance by the body's immune
system. The immunosuppressive effect of TGF-b in cancer progression is well
established. After inserting a mutated form of the TGF-breceptor into CD8+ T
cells, tumor-specific immune cells were transplanted into
mice that had been given a particularly aggressive form of
prostate
cancer,
called TRAMP-C2. TRAMP-C2 prostate cancer cells produce
large amounts of TGF-b, and
possess such potent immunosuppressive power that regular
CD8+ T cells are unable to infiltrate tumor
tissues. The mice received a single injection of
tumor-reactive TGF-b-insensitive
CD8+ T cells, tumor-specific TGF-b-sensitive CD8+ T cells
or untreated CD8+ T cells at 3 (early cancer),
7 or 21 days (advanced cancer) after they had been
injected with the prostate cancer cells. Tumor-reactive
TGF-b-insensitive CD8+
T cells infiltrated prostate cancer tumors and effectively
destroyed the TRAMP-C2 cellsref.
She received unilateral nephrectomy and CTL therapy for
the treatment of retroperitoneal lymph node metastasis
of renal cell carcinoma. With CTL therapy, her
retroperitoneal lymph node mass decreased in size. 1
year after the third round of CTL therapy, her serum
creatinine was increased and massive proteinuria
occurred. Her renal biopsy specimen revealed necrotizing
and crescentic glomerulonephritis with immune complex
deposition. Her retroperitoneal lymph node mass
continued to decrease in size. Consequently, for the
purpose of avoiding interfering with the CTL therapy, we
performed double filtration plasmapheresis (DFPP)
monotherapy for removal of immune complexes without
using immunosuppressive drugs or prednisolone. After 24
sessions of DFPP, her serum IgG was reduced from 3942
mg/dL to 2400 mg/dL, and proteinuria (from 9.0 g/day to
0.9 g/day) and renal function (serum creatinine; from
5.6 mg/dL to 2.2 mg/dL) also improved. However, 3 months
after the final DFPP, she expired due to perforation of
the colon. The autopsy sample of the kidney showed that
most of the glomeruli were obsolescent, but
immunoglobulin depositions were reduced and necrotizing
lesions were diminished. In the patients with RPGN
associated with renal cell carcinoma, renal functional
recovery has not been observed upon immunosuppressive
treatment. Consequently, plasmapheresis is considered to
be one of the effective and safe methods for patients
with this associationref.
(TIL-B) (only in 24% of breast adenocarcinomas).
When present, TIL-B can comprise up to 40% of the TIL
population. Histologically, TIL-B in breast tumors are
arranged in aggregates, and are predominantly TSA- and
breast-associated Ags-specific IgG+, as opposed
to the low level predominantly IgA+ population
normally seen in healthy breast tissue, or the
predominantly IgM+ population of peripheral
blood.
injections to encourage the TILs to proliferate in vivo
: this increases survival time from 6.7 days to almost 17
days. Please note the expanded cultures include both helper
and cytolytic T cells and the non-myeloablative conditioning
regimen used might also have eliminated regulatory cells or
other mechanism that limit lymphocyte expansion. Reinfused
CTLs make up 37% of the total TIL population, whereas
these cells made up < 1% of the total CTLs in the
peripheral blood and high levels of CD27.
Incubation in vitro with high doses of IL-2 (3,000
IU/ml) or administration of IL-2 in vivo resulted in
substantial up-regulation of CD70 expression and the
concomitant loss of cell surface CD27 expression on CD8+
cells. Withdrawal of IL-2 from activated CD8+ T
cells that had been maintained in IL-2 resulted in a reversal
of the expression of these 2 markers, whereas reciprocal
changes were seen following treatment of PBMCs with IL-2. The
proliferation observed in cells stimulated with IL-2 primarily
occurred in a subset of the CD8+70+ T
cells that up-regulated IL-2R expression but did not occur in
CD8+70- T cells. Blocking CD70 resulted
in a significant reduction of T cell proliferation induced by
high-dose IL-2, indicating that the interaction of CD70 with
CD27 played a direct role in T cell activation mediated by
IL-2. Finally, studies conducted on tumor-infiltrating
lymphocyte (TIL) samples that were administered to melanoma
patients indicated that the size of the pool of CD8+27+
T cells in bulk TILs was highly associated (p = 0.004) with
the ability of these TILs to mediate tumor regression
following adoptive transferref.
:
human myeloid DCs are superior to other APCs for the
maintenance of FOXP3+ Treg's in culture.
Coculture of DCs with autologous T cells leads to an increase
in both the number of Treg's, as well as the
expression of FOXP3 protein per cell both in healthy donors
and myeloma patients. DC-mediated expansion of FOXP3high
Treg's is enhanced by endogenous but not exogenous
IL-2, and DC-T-cell contact, including the CD80/CD86 membrane
costimulatory molecules. DCs also stimulate the formation of Treg's
from CD25- T cells. The efficacy of induction of Treg's
by DCs depends on the nature of the DC maturation stimulus,
with inflammatory cytokine-treated DCs (Cyt-DCs) being the
most effective Treg inducers. DC-induced Treg's
from
both healthy donors and patients with MM are functional and
effectively suppress T-cell responses. A single injection of
cytokine-matured DCs led to rapid enhancement of FOXP3+
Treg's in vivo in 3 of 3 MM patients. These
data reveal a role for DCs in increasing the number of
functional FOXP3high Treg's in humansref.
Discrimination of human Treg from effector CD4+CD25+
non-nTreg cells and their selective survival and
proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4+CD25high
T cell subsets stimulated via TCR and CD28 or by IL-2 survived
and expanded up to 40-fold in the presence of 1 nM rapamycin,
while CD4+CD25low or CD4+CD25–
T cells did not. The expanding pure populations of CD4+CD25high
T cells were resistant to rapamycin-accelerated apoptosis. In
contrast, proliferation of CD4+CD25– T
cells was blocked by rapamycin, which induced their apoptosis.
The rapamycin-expanded CD4+CD25high T
cell populations retained a broad TCR repertoire and, like CD4+CD25+
T cells freshly obtained from the peripheral circulation,
constitutively expressed CD25, Foxp3, CD62L,
glucocorticoid-induced TNFR family related protein, CTLA-4,
and CCR-7. The rapamycin-expanded T cells suppressed
proliferation and effector functions of allogeneic or
autologous CD4+ and CD8+ T cells in
vitro. They equally suppressed Ag-specific and
nonspecific responsesref.
decreases the risk of GvHD
effects following induction of host-versus-graft tolerance by
T-cell depleted
(TCD) allogeneic HSCT
(=> no GvHD).
The patient must have a full donor or mixed hematopoietic
chimerism to receive a DLI. DLI treatment can also be used for
HHV-5
/
CMV
retinitisref.
In patients, the infusion of TK+
lymphocytes and the subsequent administration of GCV results
in a time-wise modulation of anti-host reactivity for a GvL
effect, while controlling GvHD. Whereas TcR triggering alone
led to effector memory (EM) TK+ lymphocytes, the
addition of CD28 co-stimulation through cell-sized beads
resulted in the generation of central memory (CM) TK+
lymphocytes. In a quantitative model for GvHD using NOD/SCID
mice, CM TK+ lymphocytes were more potent than EM
TK+ lymphocytes. GCV administration efficiently
controlled GvHD induced by CM TK+ lymphocytes.
These results warrant the clinical investigation of CM suicide
gene-modified human T lymphocytes for safe and effective
allo-HCTref.
Anyway the use of gancyclovir as a suicide
gene
therapy
strategy for DLI is not advisable since the drug is often
required to treate opportunistic herpetic infections at the
same time the DLI are wished. GVL activity following
administration of DLI to established mixed chimeras is primarily
dependent upon reactivity to allogeneic MHC antigens rather
than minor histocompatibility or tumor-associated antigens.
CD8+ T cell-dependent GVL responses against an MHC
class II- tumor following delayed DLI require CD4+
T cell help and are reduced significantly when host APCs lack
MHC class II expression. CD4+ T cells primed by
host APCs were required for maximal expansion of GvH-reactive
CD8+ T cells but not their synthesis of IFN-g. In contrast, the GVL requirement for
CD4+ T cell help was bypassed almost completely
when DLI was administered to freshly irradiated recipients,
indicating that the host environment is a major factor in
influencing the cellular mechanisms of GVLref.,
adoptive transfer of alloreactive or tumor-reactive
lymphocytes may be much more effective due to the
preponderance of anticancer effector cells on the one hand,
and elimination or depletion of the patient's regulatory cells
that may downregulate anticancer effector mechanismsref. expression by
IL-15-transduced lymphocytes may prolong lymphocyte
survivalin vivo and could potentially enhance
antitumor activity. A retroviral vector encoding a
codon-optimized IL-15 gene has been generated and alternate
codon usage significantly enhanced the translational
efficiency of this tightly regulated gene in retroviral
vector-transduced cells. Activated human CD4+ and
CD8+ human lymphocytes expressed IL-15Ra and produced high levels of cytokine
upon retroviral transduction with the IL-15 vector.
IL-15-transduced lymphocytes remained viable for up to 180
days in the absence of exogenous cytokine. IL-15
vector-transduced T cells showed continued proliferation after
cytokine withdrawal and resistance to apoptosis while
retaining specific Ag recognition. In the setting of adoptive
cell transfer, IL-15-transduced lymphocytes may prolong
lymphocyte survival in vivo and could potentially
enhance antitumor activityref
can abrogate immunological unresponsiveness to syngeneic tumors
in vivo and in vitro, leading to spontaneous
development of tumor-specific effector cells as well as
tumor-nonspecific ones, leading to autoimmunityref1,
ref2.
To evaluate the relative benefit and risk, a new test system to
measure simultaneously the immune reactivity to a TAA, neu, and
an unrelated self-antigen, thyroglobulin, was established.
BALB/c mice were inoculated with TUBO cells expressing an
activated rat neu and treated with anti-CD25 mAb to deplete TReg
cells. The tumors grew, then regressed, and neu-specific
antibodies and IFN-g-secreting T
cells were induced. The same mice were also exposed to mouse
thyroglobulin by chronic i.v. injections. These mice produced
thyroglobulin-specific antibody and IFN-g-secreting
T cells with inflammatory infiltration in the thyroids of some
mice. The immune responses to neu or thyroglobulin were greater
in mice undergoing TUBO tumor rejection and thyroglobulin
injection than in those experiencing either alone. These results
illustrate the importance of monitoring immune reactivity to
self-antigens during cancer immunotherapy that involves
immunomodulating agents, and the pressing need for novel
strategies to induce antitumor immunity while minimizing
autoimmunityref.
Elimination of CD4+/CD25+ Tregs
using the recombinant IL-2 diphtheria toxin conjugate DAB389IL-2 /
denileukin diftitox is capable of enhancing the
immunostimulatory efficacy of tumor RNA-transfected DC vaccines.
DAB389IL-2 is capable of selectively eliminating
CD25-expressing Tregs from the PBMCs of cancer
patients without inducing toxicity on other cellular subsets
with intermediate or low expression of CD25. DAB389IL-2-mediated
Treg depletion resulted in enhanced stimulation of
proliferative and cytotoxic T cell responses in vitro but
only when DAB389IL-2 was omitted during T cell
priming. DAB389IL-2 significantly reduced the number
of Tregs present in the peripheral blood of
metastatic renal
cell
carcinoma
patients and abrogated Treg-mediated
immunosuppressive activity in vivo. Moreover, DAB389IL-2-mediated
elimination of Tregs followed by vaccination with
RNA-transfected DCs significantly improved the stimulation of
tumor-specific T cell responses in RCC patients when compared
with vaccination alone. The design of immune-based strategies
may incorporate the Treg depletion strategy to
achieve potent antitumor immunity with therapeutic impactref.
Tumor antigen recognition by a subset of CD4+ T cells
led to their differentiation into cells capable of suppressing naive
and Th1 effector cells. Such tumor-induced
regulatory T cells (TMTregs) arose both from
precommitted "natural" regulatory T cells and CD4+CD25-GITRlow
precursors. Once induced, TMTregs were capable of
maintaining suppressor activity long after transfer into
antigen-free recipients. Suppression was mediated by GITRhigh
cells residing within both CD25+ and CD25-
subsets. Vaccination of the tumor-bearing host concomitantly
expanded TMTregs and effector cells, but suppression
was dominant, blunting the expansion of naive tumor-specific T
cells and blocking the execution of effector function in
vitro and in vivo. These studies illustrate the
possibility that therapeutic vaccination could actually worsen
host tolerance to tumor antigens and support treatment paradigms
that seek to not only increase the frequency of tumor-specific T
cells, but to do so in conjunction with strategies that
inactivate or remove regulatory T-cell populationsref
decreases the risk of GvHD and
EBV-associated posttransplant
lymphoproliferative
disorders (PTLDs):
: depleting the donor bone marrow or PBSCs of T lymphocytes
administered during the first 3 to 6 months following HSCT
have 40-85% effectiveness but can induce post-transplant
lymphoproliferative
disorder (PTLD)
of donor T lymphocytes : immunotherapy
of autoimmunity +
cyclophosphamide
for acquired
hemophilia
Aref to Th2 responses : after
anterior chamber inoculation of antigen, eye-derived APCs
arrive in the spleen, where they engage newly recruited NKT cells and
stimulate their production of immunosuppressive cytokine IL-10,
that contributes to the generation of Ag-specific CD8+
Tr cells that actively suppress Th1
cells (and so DTHs).. Once transplanted they can
induce tolerance in the recipient.
consists of injection of unmodified donor hematopoietic
cells (including tolerogenic immature
DC)
into immunocompromised adult recipients. The establishment
of mixed, host and donor, hematopoietic cellular chimerism
through BMT leads to specific tolerance in an otherwise
fully immunocompetent host. However, the use of allogeneic
BMT to induce tolerance has several potential drawbacks,
including the severity of the preparative regimen required
to achieve bone marrow engraftment, the potential of
inducing GvHD,
and the possibility of engraftment failure. Because the
timing of the availability of cadaver organs cannot be
predited or planned, pretransplant-conditioning regimens
cannot be used in the clinical setting. A
posttransplant-conditioning regimen consisting of total
lymphoid irradiation (TLI) and anti-thymocyte globulin
+ CsA
has been shown to induce mixed chimerism and tolerance in
completely MHC-mismatched individuals.
female NOD mice, but irradiated splenocytes require longer.
A significant proportion of the new islets in mice treated
with live cells were of donor origin. Fusion between host
and donor cells is not thought to have occurred as the
regenerated islet cells are of normal size, and do not
contain enlarged nuclei or multiple nucleoli. Also, there
are few, if any, islet cells with an XXY or XXXY genotype.
Therefore, live splenocytes can not only re-educate
peripheral T cells, but also reconstitute functional islets,
and further studies attributed this property to a subset of
CD45- non-lymphoid cells. However, in NOD mice
treated with irradiated splenocytes, none of the
regenerating islet cells contained a Y chromosome,
indicating that these cells are of host origin. Adult NOD
mice must also contain endogenous precursor cells that can
contribute to islet regeneration over a longer time scale,
once the autoimmunity has been corrected by donor
splenocytesref-based approach to modify
autologous bone marrow cells to express donor MHC class I Ags.
Expression of allogeneic MHC Ags on otherwise autologous bone
marrow-derived cells (BMC) eliminates many of the
complications associated with allogeneic BMT used to establish
mixed cellular chimerism. : immunotherapy
of hypersensitivities
(allergen-specific
immunotherapy
(SIT)) and transplant
rejection
: administration of whole antigen(s) as and TGF-b1that are produced by TReg cells potently suppress IgE
production, while simultaneously increasing production of the
non-inflammatory immunoglobulin isotypes IgG4 and IgA (blocking
antibodies) by B cells. The strategy is for the IgG or
IgA antibodies to interact with allergen before the allergen
can interact with IgE antibodies on the mast cells. This
isotype skewing has also been reported in individuals
naturally exposed to high allergen doses - for example,
multiply stung bee keepers and patients with chronic helminth
infections have IL-10-mediated tolerance and 100–1000-fold
increased levels of antigen-specific IgG4. In
addition, there is clear evidence that the effector cells of
allergic inflammation (mast cells, basophils and eosinophils)
require T-cell cytokines for priming, survival and activity
that are not provided by the TReg cells induced
during allergen-specific immunotherapy. In addition, the
controlled release of histamine from effector cells of allergy
has a suppressive effect on Th2 responses through H2 receptor, which also induces
the production of IL-10 by T cells and DCs. Setbacks are that
therapeutic benefits are not consistently evident. Also, a
rise in the titer of blocking antibodies does not mean that
the individual has been cured or aided. To achieve efficacy a
dose of approximately 5-20 mg
of the major allergen needs to be tolerated with each
maintenance injection that is then administered at regular
intervals for a period of 3-5 years : systemic anaphylaxis may
occur in up to 10% of patients receiving SIT. cytokine
production of IL-4, IL-5, and IL-13 and histamine
release in serum were significantly decreased. Moreover,
the development of pollen-induced clinical symptoms was
inhibited in our experimental sneezing mouse model.
These results indicate the potential of transgenic rice
seeds in production and mucosal delivery of
allergen-specific T cell epitope peptides for the
induction of oral tolerance to pollen allergensref and class II molecules.
After administration of 90 mg/kg prior to transplant,
rabbit IgG remains present for 4-5 weeks, while T
cell-reactive antibodies persist up to 3 weeks with
antibody levels equivalent to 0.2-4.1mg/ml
rabbit ATGref
cell lines, including those resistant to conventional
anti-myeloma agents. Importantly, the anti-myeloma
activity was preserved even when myeloma cells were
grown with different cytokines demonstrating the ability
to overcome microenvironment-mediated resistance.
Thymoglobulin induced apoptosis of freshly isolated
primary myeloma cells from patients. Using a competitive
flow cytometric analysis, we were able to identify the
potential antigen targets for Thymoglobulin preparation.
Finally, in a plasmacytoma mouse model of myeloma,
Thymoglobulin delayed the tumor growth in a
dose-dependent manner providing convincing evidence for
continued evaluation of this agent in the clinic in
patients with myeloma, either alone or in combination
with other agentsref.. Polyclonal
antithymocyte globulins (ATG) or monoclonal anti-CD52
(alemtuzumab) are included in conditioning regimens to enhance
engraftment and reduce risk of severe GvHD. Both agents have
been reported to induce depletion of B cells.
Complement-mediated and complement-independent activity of
ATG-Fresenius and Alemtuzumab was investigated on 4 MM cell
lines (RPMI-8226, U266, KMS-12-BM, and EJM) and bone marrow
samples from 6 MM patients. Cytotoxicity was determined by
staining with annexin V-fluorescein isothiocyanate and
7-amino-actinomycin D followed by flow cytometry. ATG at a
concentration of 500 mg/mL induced
up to 100% and 85% complement-dependent killing of MM cell
lines and primary MM samples respectively. In the absence of
complement ATG still could induce up to 50% and 80% apoptosis
in MM cell lines and primary MM samples, respectively.
Preincubation of MM cells with a general caspase inhibitor
abrogated ATG-induced complement-independent cell death.
Alemtuzumab-mediated MM cytotoxicity was only observed in
KMS-12-BM cells, and in none of the patient samples. ATG
induces marked cytotoxic activity both in MM cell lines and in
primary MM samples. Further elucidation of antibodies and
antigens involved may pave the way for antibody-based MM
therapyref
sera :heterologous
serum
: 50,000 IU (50-100 mL per die).
heterologous serum : 20-40 mL
heterologous serum : from 20,000-40,000 IU for
laryngo-pharyngeal forms begun from less than 48 hrs, up
to 80,000-120,000 IU for clinical forms begun from more
than 3 dd.
and related gangrene Bacteria heterologous serum :
75,000 IU heterologous serum : horses repeatedly
inoculated with a chicken vaccine against H5N1 bird flu to make them produce antibodies.
They then collected the horses’ blood, separated out the
antibodies and split them to make them less likely to
cause an allergic reaction when injected into a human.
When they injected mice with 0.1 mg of these antibodies 24
hours after they had been given an otherwise lethal dose
of H5N1,
all the mice lived (Jiahai Lu at Sun Yat-sen University in
Guangzhou)ref sera :
therapeutic antivenom against snakes was first produced by
Albert Calmette in 1894. Since then antivenoms have saved
the life of countless snakebite victims. However, there are
still many problems associated with antivenom production,
for example variable percentage of responder horses, low
neutralizing potency of antivenom, the large amount of snake
venom needed for immunization and the difficulties
encountered in producing potent polyvalent antivenoms. These
problems have led to shortage and high cost of antivenom
and, in some cases, failure of treatment.In 1997, a new
immunization protocol for antivenom production was reported.
It involves the injection of venom at low dose (approx.
2mg/horse) emulsified in Complete Freund's adjuvant in low
volume (0.1-0.2 ml/site) in a total of 10 sites around the
neck area of the horse. This immunization protocol has
minimized the local reaction at the injection site thus
allowing the use of the potent oil adjuvant. This, together
with the increase in total surface area of the droplets,
allow a more effective immune response to take place, e.g.
enhancing the migration and activation of more antigen
presenting cells and lymphocytes. The low dose, low volume
multi-site immunization has resulted in dramatic
improvements on the antivenom production in terms of amount
of venom used for immunization, the time required to reach
hyperimmune stage, the percent of responder horses and the
potency of the antivenom. Furthermore, this protocol has
made it possible to produce potent truly polyvalent
antivenoms against several elapid and viperid snakes. This
immunization protocol has alleviated various problems
associated with antivenom production and has implications
for immunization in generalref.
Currently there is a crisis in the supply of antivenom for
treatment of snake bite in sub-Saharan Africa. Commercial
pressures have resulted in the reduction or even cessation of
production of antivenom by European manufacturers while
continued production of antivenom in Africa has been
threatened by the privatisation of the only remaining company
based in Africa. As a consequence, there has been an increase
in snake bite morbidity and mortality in many African
countries. Two Latin American antivenom manufacturers have
agreed to produce antivenom suitable for Africa, using venoms
from the species which are of the greatest medical importance
in sub-Saharan Africa. Preclinical in vivo assays of
neutralising potency demonstrated that a new Pan African
antivenom produced in Colombia compared favourably with the
existing commercial monospecific and polyspecific antivenoms.
This new antivenom, and a similar product being manufactured
in Costa Rica, are now candidates for clinical testing at an
appropriate site in Africaref.
antivenom
antivenom
antivenom
antivenom
antivenomantivenomantivenom
antivenom : purity of the parent toxin influenced greatly
the immunogenicity in guinea pigs of HR1 component of
Habu-venom toxoid. The potency of HR1 toxoid in terms of
the immunizing unit (ImU) appeared to be related to the
antigen and aluminum contents. The immune response of the
animals differed depending on the purity of the toxoid
when the dose-response curve was examined over a wide
range of the antigen dosage. Anti-HR1 titers of the guinea
pigs immunized with crude toxoid (adsorbed) reached a
plateau at about 10-20 U/ml ane further rise was not
remarkable even when the amout of the antigen was
increased several times, while 200 U/ml (maximum > 400
U/ml) of anti-HR1 was produced with a large amount of
highly purified HR1 toxoid. Purified toxoid showed an
excellent immunogenicity also in monkeys. The amounts of
antigens necessary for the basic immunization to resist
the challenge with a certainly lethal dose of crude venom
were 18 and 0.5 mg as protein, for a crude and a highly
purified toxoid, respectively. The total amount of
aluminum was also reduced from 8.1 (crude toxoid) to 0.5
mg (purified toxoid). Multiple injections for the primary
immunization enhanced early production of anti-HR1 in
monkeys. However, at later stage of immunization,
significant correlation was observed between the amount of
HR1 toxoid (in ImU) for the primary immunization and the
circulating anti-HR1 titer irrespective of the schedule of
immunization. Anti-HR2 > 10 U/ml was produced
consistently in monkeys with HR2 toxoid of 1.0 ImU for the
basic immunization. However, excess HR2 toxoid of > 10
ImU seemed rather unfavorable for anti-HR2 productionref.
Effects of pH and lysine during detoxification with
formalin on the immunogenicity of the toxoidref.
antivenom
antivenomantivenom
antivenom : the antibody preparation elicited by dimeric
glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin)
-cobrotoxin enriches the content of antibodies recognizes
the C-terminal region of native cobrotoxin,
NK), the King cobra (Ophiophagus
hannah
OH) and the banded krait (Bungarus
fasciatus,
BF). 2 groups of horses were immunized. Group 1,
comprising 5 horses, was immunized twice with a mixture of
postsynaptic neurotoxins followed by an additional six
immunizations with a mixture of crude venoms of the three
elapids. Group 2, comprising four horses, was immunized
with a mixture of crude venoms throughout the course. For
the first immunization, the immunogens were emulsified in
complete Freund's adjuvant and injected using a low dose,
low volume multi-site immunization protocol previously
developed in this laboratoryref.
The second immunization was carried out with the
immunogens in Incomplete Freund's adjuvant. Blood was
drawn to assay the antibody titer by ELISA. Sera at the
peak of ELISA titers were pooled and assayed for the ED50.
The ED50's of antivenom from Group 1 horses
against NK, OH and BF venoms were 1.44, 0.22 and 0.23 ml
serum/mg venom, respectively, while those from Group 2
horse sera were 0.88, 0.20 and 0.49 ml serum/mg venom,
respectively. The potency of sera from Group 2 against BF
venom was significantly higher, while the potencies
against NK and OH venoms were comparable to those of the
corresponding monovalent antivenoms produced under the
same protocol. This potent, truly polyvalent antivenom
should be useful in saving lives of victims envenomed by
these elapids and the immunization protocol should be
useful in the production of potent polyvalent antivenoms
against other medically important elapidsref|
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Pentaglobin© | |
| manufacturer | Alpha Therapeutic Corp. | Alpha Therapeutic Corp. | Baxter Corp./Hyland Immune Division | Immuno-U.S. Inc. | Baxter Corp./Hyland Immune Division; distrib. by American Red Cross (ARC) | Bayer | Aventis Behring | ZLB Bioplasma | Kedrion S.p.a. | Baxter S.p.a. | American Red Cross | Baxter Corp. | Novartis Pharmaceuticals | Biotest |
| formulations | 5% (half the solvent can be used to create a 10% = 100
mg/ml solution) :
|
5% :
|
5% :
|
5%
|
||||||||||
| method of preparation (including viral inactivation) | cold alcohol fractionation, PEG/bentonite fractionation, ion-exchange chromatography, solvent detergent treatment | Cohn-Oncley ultrafiltration, ion-exchange chromatography, solvent detergent treatment | cold ethanol, PEG, trypsin | Cohn-Oncley ultrafiltration, ion-exchange chromatography, solvent detergent treatment | Cohn-Oncley pH 4.25, solvent detergent treatment | Cohn-Oncley pasteurization, ultrafiltration | KistlerNitschmann; pH 4.0 + trace pepsin | Cohn alcohol fractionation, New York Blood Center solvent-detergent treatment, pH 4.0 | ||||||
| form | liquid | liquid | lyophilized | lyophilized | lyophilized | liquid | lyophilized | lyophilized | liquid | |||||
| shelf-life | 24 months | 24 months | 24 months at T < 25°C (2 hrs after reconstitution) | 24 months | 27 months | 26 months | 24 months | 24 months | storage +2-+8°C | |||||
| reconstitution time | liquid solution | liquid solution | < 5' at RT; > 20' if cold | < 10' at RT | < 5' at RT; > 20' if cold | liquid solution | < 20' | several minutes | ||||||
| recommended concentration | 5% | 10% | 5% | 5% | 5% | 10% | 5% | 3% | ||||||
| recommended infusion rate | 3 mL/kg/hr | 3 mL/kg/hr | 0.5 ml/kg/h => 8 mL/kg/hr | 2 mL/min | 4 mL/kg/hr | 4.8 mL/kg/hr | 3.6 mL/kg/hr | 2.5 mL/min | 0.46-0.92 ml/kg/h for 20-30 minutes, then increase up to 1.85 ml/kg/h | 0.16 ml/kg/min | ||||
| time to infuse 70 g (1g/kg) | 7 hr | 3.5 hr | 5.3 hr | 12 hr | 5.3 hr | 2.3 hr | 5.8 hr | 16 hr | ||||||
| sugar content | 5% D-sorbitol | 5% D-sorbitol | 2% glucose | 5% glucose | 2% glucose | sugar-free (glycine) | 5% sucrose | 5% sucrose | maltose | glucose (1 g/g IgG), macrogol | ||||
| sodium content (determines osmolality) | 1.3 mEq/L | < 1 mEq/L | 0.85% at 5% concentration | 0.3% | 0.85% at 5% concentration | trace | 0.5% | up to 0.9%, depending on diluent | ||||||
| osmolality | 300 mOsm/L | 300 mOsm/L | 5% 636 mOsm/L, 10% 1,250 mOsm/L | > 240 | 5% 636 mOsm/L, 10% 1,250 mOsm/L | 274 mOsm/L | 5% 309 mOsm/L, 10% 600 mOsm/L | in sterile water (mOsm/L); 3% 192, 6% 384, 12% 768; in normal saline (mOsm/L) : 3% 498, 6% 690, 12% 1074 | ||||||
| pH | 5.2-5.8 | 5.2-5.8 | 6.8 | 6.4-7.2 | 6.8 | 4.25 | 6.8 | 6.6 | ||||||
| IgG content |
> 95% of 50 g/l | 38 mg/ml | ||||||||||||
| IgG subclasses | as in normal blood |
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| IgA content |
15.1 mg/mL | 20-50 mg/mL | < 3.7 mg/mL | < 10 mg/mL | < 3.7 mg/mL | 270 mg/mL | < 25 mg/mL | 720 mg/mL | < 0.05 mg/ml | < 0.05 mg/ml | 6 mg/ml | |||
| IgMcontent |
6 mg/ml |
transport
receptors : normally, IgG antibodies bind to a
protective receptor in endocytotic vesicles, called FcRn,
and then return intact to the circulation. Without this
protective mechanism, IgG would pass to the lysosomes of
the cells and be degraded. FcRn exists in many tissues,
including skin and muscle, but it is highly expressed on
vascular endothelial cells, which may be a major site of
IgG catabolism. The large amount of IgG in IVIg
preparations could, in theory, saturate the FcRn,
accelerate the catabolism of endogenous pathogenic IgG,
and reduce the level of pathogenic autoantibodies.
and TNF-a=>
downregulation
of the in situ expression of TGF-b
and TGF-b mRNA and reduction of
cytokine-induced MHC-I and ICAM-1 expression,
which may exert an additional immunomodulating or
immunosuppressive effect, CCR1, CCR2, CCR3, CCR4, and CCR5 and
downregulation of the expression of MIP at the
protein and mRNA levels and C4b limits the
number of C3 molecules available for incorporation into
the C5 convertase assembly and prevents the formation
and in situ deposition of MAC in targeted tissues and C5a
anaphylatoxinsref that inhibit
phagocytosis-blocking
antibodies that may help explain the action of IVIg
in diseases in which Fas-mediated tissue destruction plays
a pathogenic role.| Indications |
|
|
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| substitutive therapy | primary
immunodeficiencies |
initial dose : 0.4-0.8 g/kg following doses : 0.2-0.8 g/kg |
every 2-4 weeks to keep basal IgG levels > 4-6 g/l |
| secondary
immunedeficiencies |
0.2-0.4 g/kg | every 3-4 weeks to keep basal IgG levels > 4-6 g/l | |
| prophylaxis in CLL |
400 mg/kg | every 3 weeks | |
| prophylaxis in MM |
400 mg/kg | every 4 weeks | |
| prophylaxis after surgery |
400 mg/kg | weekly up to 4 infsions or dimission from ICU (+ASA) | |
| AIDS children |
0.2-0.4 g/kg | every 3-4 weeks | |
| immunomodulation | ITP |
0.8-1 g/kg OR 0.4 g/kg/day |
the first day, possibly repeating a second time
within 3 days OR for 2-5 days |
| Guillain
Barré
syndrome |
0.4 g/kg/day | for 3-7 days (5 consecutive days) | |
| multifocal
motor
neuropathy |
0.4 g/kg/day or 2 g/kg | for 5 consecutive days or 2-4 days | |
| Kawasaki
disease |
1.6-2 g/kg or 2 g/kg | in doses divided in 2-5 day, in association with
ASA. OR in single dose, in association with ASA |
|
| myasthenia
gravis |
0.4 g/kg/day | for 5 consecutive days in critical phases not well compensated by specific therapy; | |
| allogeneic
HSCT |
treatment of infections and prophylaxis of GvHD | 0.5 g/kg | every week since day 7 before HSCT to month 3 (every 2 days in the induction phase for a total of 10 doses; twice weekly in maintenance for a total of 8 doses; associate ganciclovir in both phases) |
| persistent deficiency in antibody production | 0.2-0.6 g/kg | every week and then every month until antibody levels return normal | |
: normal serum viscosity is 1.2 to 1.8 cp. IVIg therapy
causes an increase in serum viscosity of up to 0.5 cp in
most patients and even higher in patients with high-normal
or slightly elevated serum viscosity, such as those with
essential mixed cryoglobulinemia, hypercholesterolemia, or
hypergammaglobulinemia. Patients who develop a serum
viscosity greater than 2.5 cp have an increased risk for
thromboembolic events, which might account for the rare
cases of stroke,
pulmonary embolism, or AMI that can occur after IVIg
treatment. Patients with recent DVT and immobilized
patients face a higher risk for subclinical thrombosis and
may be more likely to develop a thromboembolic event after
IVIg. For these patients we recommend prudence in the use
of IVIg, screening for clots in the lower extremities with
an US examination, and a very slow infusion rate. It
remains uncertain whether low-dose heparin or antiplatelet
agents can prevent thromboembolic events in these
patients. IVIg therapy can also induce a hyperviscosity
syndrome in children with HIV infection who may have high
pretreatment levels of serum immunoglobulins. A reversible
cerebral vasospasm has been noted in patients treated with
IVIg
attack in patients with a history of migraine, which
can be prevented by pretreatment with propranolol. The
complication of aseptic meningitis is also high in
migraine patients. IVIg therapy has been associated with a
stroke
in a woman with a history of migraine
can occur in patients who have a severe deficiency of or
totally lack IgA because of IgA antibodies. Selective IgA
deficiency
is common, with a prevalence of about 1/1,000, but is
asymptomatic. About 29% of people with selective IgA
deficiency have anti-IgA antibodies, which may be a risk
factor for the development of adverse reactions. When
these patients receive IVIg, the small amount of IgA that
it contains may lead to an anaphylactic reaction due to
the formation of macromolecular complexes between the
infused IgA and anti-IgA antibodies. The reaction is rare
and occurs mostly in patients with CVID
occurs rarely and is mostly reversible when it does occur.
The greatest risk exists for patients with
preexisting kidney disease and volume depletion,
especially the elderly and those with diabetes or poor
hydration. Fatalities can occur as a consequence but are
rare. A patient's serum creatinine may increase within the
first 10 days after treatment with IVIg, but the level
usually returns to baseline within 2 to 60 days after the
infusion stops. This complication has almost always been
associated with the high concentration of sucrose in some
proprietary IVIg products. Osmotically induced tubule
injury and vacuolization are the common histopathologic
findings in the renal biopsy. Identical osmotic tubule
nephrosis is caused by intravenous solutions that contain
as much hypertonic sucrose as some preparations of IVIg.
Diluting the IVIg preparation and slowing the rate of
infusion minimizes the risk. Close monitoring of
creatinine and BUN is also essential for patients with
preexisting kidney diseases who must receive IVIg
treatment.:
IVIg contains complement-activating anti-A/B IgG blood
group antibodies that have the potential to induce
hemolysis. In our experience, hemolysis is only a
theoretical possibility, and we have not seen it occur in
any of our patients. The European Union Pharmacopeia
mandates that anti-A and anti-B titers should be less than
1:64 in IVIg preparations.
increases 6-fold or more, probably due to enhanced
rouleaux formation and reduced surface area on the
patient's erythrocytes caused by IVIg. The increase can
persist for 2 to 3 weeks and should not be considered a
sign of a developing vasculitis
(130 mg/L) because of the additional dilution of the
sample required owing to the high serum protein
concentration that occurs after IVIg infusion.
should be delayed for 3 months since administration of
IVIg (up to 1 year for measles vaccination)...
hyperimmune homologous serum from subjects infected with Escherichia
coli O111:H15.
hyperimmune homologous serum : 0.2 mL/weight Kg.
hyperimmune homologous serum (TIG) : 3,000-6,000 IU. It doesn't allow
remission of present symptoms but reduces mortality.
Human Botulism Immune Globulin Intravenous (Human) (BIG-IV)
homologous serum / vaccinia
immunoglobulins (VIG) : 0.6
mL/kg of body weight, i.m.or
i.v.. It is an isotonic sterile solution of the
immunoglobulin fraction of plasma from persons vaccinated
with vaccinia
vaccine).
Because therapeutic doses of VIG might be substantial (e.g.,
42 ml for a person weighing 70 kg), the product should be
administered in divided doses over a 24- to 36-hour period.
Doses can be repeated, usually at intervals of 2-3 days,
until recovery begins (e.g., no new lesions appear). They
are contraindicated for the treatment of vaccinial
keratitis. : -vir- : -bac- : -lim- : -les- :
-col-:
-mel- :
-mar- :
-got- :
-gov- :
-pr(o)-|
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| -cir- | -xi | -mab | abciximab |
| -lim- | -zu | -mab | daclizumab |
mAbs for pathogen
neutralization and antiviral therapy. Antibody binding can
directly and effectively block the activity of many pathogens,
often without requiring Fc-mediated cytotoxicity. Indeed, this
has always been the promise of antibody-mediated viral
neutralization.
3 r-mAbs : they are
effective if given up to 2 days after exposure, protecting
from 1-2 hours after administration up to 3-6 months. The
botulinum antitoxin, in uncontrolled studies, has been
associated with lower mortality rates and, if administered
early after onset of symptoms, a shorter course of
illness. A licensed trivalent antitoxin (includes
antitoxin against A, B, and E only) is available. Contrary
to the package insert directions, current recommendations
are to administer 1 10-mL vial of antitoxin per patient,
intravenously in a normal saline solution over 20 minutes.
Antitoxin need not be repeated, since the circulating
antibodies have a half-life of 5 to 8 days. The antitoxin
is of equine origin and requires skin testing for
hypersensitivity before administration of the antitoxin.
About 9-21% of patients will develop either acute or
delayed-type sensitivity reactions. Serum sickness
reactions appear to be dose-related and may be less likely
with the newer dosing recommendations. A human-derived
antitoxin for infant botulism is available from the
California Health Departmentref.
protective antigen (PA) mAbs : raxibacumab (ABthrax®;
source : Human Genome
Sciences) is a human IgG1
l mAb that prevents
the anthrax toxins from entering and killing cells by
blocking the binding of protective antigen to cell surfacesref.
In human subjects, intravenous raxibacumab at a dose of 40
mg/kg had a half-life of 20 to 22 days and provided a
maximum concentration of the drug in excess of levels that
are protective in animalsref mAb :
anti-lipid A mAbs for treatment of patients with severe sepsis, especially those
with shock, and probable gram-negative bacilli (GNB)
bacteremia
: sevirumab / EV2 7 / SDZ
MSL 109 (Protovir®) is specific to the CMV
glycoprotein H with high neutralizing capacity. It is
ineffective in clearing CMV-DNA and CMV antigen from the
plasmaref,
is not beneficial in CMV-seropositive HSCT recipientsref,
and not offer a significant advantage when added to
traditional anti-CMV therapy in cytomegalovirus retinitisref : :
XTL-002 / HepeX-C is a fully human mAb, also employed
to prevent HCV re-infection in HCV-associated liver
transplant patients (source : XTL Biopharmaceuticals Ltd)
: anti-CD4 mAb TNX-355 (source : Tanox,
Inc.): passive transfer of mouse immune serum
has been shown to reduce pulmonary viral titres in mice
infected with SARS coronavirusref mAb : MGAWN1
(Macrogenics)
mAb: once
thought impossible, have taken the lead in terms of survival
and delayed treatment. Passive immunization constitutes
another strategy to treat EBOV infections and was first
attempted during the initial outbreak in 1976, where an
infected laboratory employee was successfully treated with
interferon and convalescent serumref. During the 1995
outbreak, convalescent serum was administered to eight
infected individuals, seven of which survivedref. However, evaluations
of passive therapies in animal models have had mixed
success. The administration of immunoglobulin G (IgG)
purified from EBOV-hypervaccinated horses failed to protect
macaques against an EBOV challengeref1,ref2 and the neutralizing
human monoclonal antibody KZ52, isolated from a survivor of
EBOV infection, also failed to protect macaquesref. Purified IgG from
vaccinated and subsequently challenged nonhuman primate
survivors provided 100% protection from Ebola or Marburg
virus challenge when administered up to 48 h post-infectionref.
This advance was followed closely by a cocktail of 3 mouse
monoclonal antibodies (m1H3, m2G4, and m4G7 : ZMabref1, ref2)
that was 100% effective at 24 h and 50% effective at 48 h in
preventing death against Ebola virus challenge in cynomolgus
macaquesref.
Later, this same cocktail when combined with
Adenovirus-vectored interferon-α was 75% and 100% effective
in cynomolgus and rhesus macaques, respectively, when
administered 72 h post-infectionref.
Similarly, an alternate cocktail of 3 humanized monoclonal
antibodies (clones c13C6, h13F6 and c6D8 : MB-003;
Mapp Pharmaceuticals) provided 100% (1 h) and 67% protection
(24 h and 48 h post-infection)ref
in the Ebola virus rhesus macaque model and 43% survival
when treatment occurred at 120 h post-infection following
onset of viremia and feverref.
These two trials represent the first true therapeutic
interventions whereby in response to a diagnostic
indication, the onset of viremia and/or fever, treatment is
initiated. ZMapp (Mapp Biopharmaceutical Inc.
= Mapp Biopharmaceutical + LeafBio + Defyrus Inc. +
U.S. government + Public Health Agency of Canada) is
composed of 3 humanized mAbs (c13C6, c2G4, and c4G7))
manufactured by expression in tobacco plantst (Nicotiana
benthamiana), two coming from ZMab and one from
MB-003. The report that two seriously ill American
volunteers, Kent Brantly and Nancy Writebol, received an
experimental cocktail of three monoclonal antibodies, never
before administered to humans, has raised questions around
the globe. Dubbed “secret serum” by the media, the treatment
has generated hope, suspicion, accusations of inequity, and
requests for additional product, of which, since the
manufacturers provided three remaining doses to Liberia,
there is now none. Although this report engenders hope, one
cannot reach a sound conclusion on the basis of two
patients' survival. Moreover, a third patient has now died
despite reportedly having received ZMapp. In addition, the
likelihood that the first two recipients would have died
without therapy may have been significantly less than the
approximately 50% so far noted in the current epidemic.
Surviving beyond the first several days of EV illness may be
predictive of overall survival, as it was in the 1995 Congo
outbreak. Brantly reportedly became ill 9 days before
receiving the product, and Writebol may have been sick at
least as long. Brantly received a transfusion from a
recovered patient, for which there is conflicting evidence
of effectiveness, and high-quality supportive medical care
may well improve survival, an issue that merits further
emphasis. Finally, mortality often decreases over the course
of Ebola outbreaks, perhaps because of enhanced diagnosis
and care. : passive parenteral
immunization with prion protein antibodies in amouse model
induced with scrapie brain homogenate to reduce PrPSc
formationref that is linked to a
second antibody that binds to a particular pathogen. After
administration, the HP antibody rapidly binds the target
pathogens to red blood cells. mAbsovine Fab
(DigiFab®) bind to digoxin and digitoxin greatly enhancing
the renal excretion of these drugs;
FDA-approved in 2001
mAbs prevent crossing the blood-brain
barrier
(BBB) may
help prevent relapse during efforts to abstain from the drug.
They are under development for : passive
transfer of nicotine-specific antibodies (from vaccinated
rabbits) into rats attenuates numerous actions of nicotine:
increases in blood pressure and locomotor activity, the
induction of nicotine dependence, the relief of nicotine
withdrawal by subsequent nicotine and the stimulus
properties that allow rats to discriminate a nicotine from a
saline injectionref;
NIC9D9ref(GNC92H2) :
reduced 'relapse' to drug use in animal model systemsref :
mAbs provide intriguing evidence of sustained protection for
months after single-dose administration mAbs
mAbs :
mAbs :
mAbs : tanezumab / RN624
(formerly known as RI 624; source : Rinat Neuroscience,
now Pfizer) is a recombinant, humanized IgG2 mAb that blocks
the interaction of nerve growth factor with its receptors,
TrkA and p75ref mAbs
mAbs : RN1219
(Rinat Neuroscience,
now Pfizer) is a humanized monoclonal antibody.
Immunotherapy reduces not only extracellular Ab plaques but also intracellular Ab accumulation and most notably leads
to the clearance of early t
pathology. Ab deposits are
cleared first and subsequently reemerge prior to the tau
pathology, indicative of a hierarchical and direct
relationship between Ab and t. The clearance of the t pathology is mediated by the
proteasome and is dependent on the phosphorylation state of
t, as hyperphosphorylated t aggregates are unaffected by the Ab antibody treatment. These findings
indicate that Ab immunization may
be useful for clearing both hallmark lesions of AD, provided
that intervention occurs early in the disease courseref mAbs :
mAbs :
mAbs mAbs :or bone metastases
from breast cancer was well tolerated and reduced bone
resorption for at least 84 days. The decrease in bone
turnover markers was similar in magnitude but more sustained
than with i.v. pamidronateref.
mAbs (-mab)ref
bind to tumor cell surface antigens => killing
by ... by direct signaling via BcR
cross-linking (eg for BCL1 cell lineref)ref1,ref2 (poorly from murine
IgM, IgG2a, and IgG3ref)
: target epitopes proximal to cell membrane are required (murine IgG3
(for NK cells), IgG2a (for monocytes), and IgG2b
: murine IgG1 is generally least efficient in
fixing complement or participating in ADCC). In vivo,
ADCC may be compromised in cancer patients due to a paucity of
appropriate effector cells or to the presence of circulating
immune complexes that occupy or downregulate FcRs. This
mechanism is critical for the antitumor effects of several
therapeutic mAbs, including rituxumab, because mice with
normal Fc receptors exhibit antitumor effects following mAb
treatment, while mice lacking FcgRI
and FcgIII do notref1,
ref2.
Many early studies showed that murine mAbs had limited
potential to elicit a potent antitumor response, because the murine
Fcgregions are less efficient at
recruiting human effector cells than their human
counterparts. This problem has been largely allayed by
the use of chimeric and humanized antibodies. Genetic
engineering techniques have also been used to improve the
immunologic effects of therapeutic mAbs by altering antibody
shape and size, increasing the valency of mAbs, and creating
bifunctional antibodies with 2 antigenic receptors, one to a
tumor antigen and another to an effector cell to increase
efficiency of ADCCref1,ref2.
Stimulation of the immune system may also substantially
improve effector functions in response to mAb therapy. CpG
motifs can act as a potent stimulators of immune responseref1,
ref2,
ref3,
and can synergistically enhance the effect of therapeutic mAbs
by sensitizing malignant cells to ADCCref.
Furthermore, different oligonucleotide sequences were shown to
illicit different immune responses, suggesting that mAbs are
capable of inducing antitumor effects by a variety of
mechanisms.,
inducing active adaptive immunity
coreceptor molecule and joined at its 5' end to a gene
fragment encoding the intracellular moiety of the gc common to the FceRs and FcgRs.
Transfected lymphocytes express the chimeric receptors on
their surface and upon stimulation with hapten immobilized
on plastic they can produce IL-2. Transfectomas activated
in this manner also undergo specific proliferation in the
absence of exogenous IL-2. Moreover, the transduced
lymphocytes could effectively lyse target cells expressing
the TNP hapten on their surfaceref.ref.
treatment, suppressed tumor growth and pre-established
tumor metastases. Synergistic effects of the combination
were observed in several tumor models where the target
tumor was sensitive to DR5-mediated apoptosis. IL-21
promoted tumor-specific CTL activity and enhanced memory
responses to tumor rechallenge. These results indicate
that a rational combination of Ab-based therapy that
causes tumor cell apoptosis and a cytokine that promotes T
cell memory is a useful new strategy for cancer
immunotherapyref
and chronic
lymphocytic
leukemia (CLL)))depletes mature
B cells but does not effectively deplete pre-B or immature
B cells, some B cell subpopulations, antibody-producing
cells, or their malignant counterparts, allowing B-cell
repertoire reconstitution and preventing reduction of
antibody titers. Unfortunately CD20 is expressed only on
macaque cynomolgus monkeys : infusion with doses ranging
from 1.6 mg/kg to 6.4 mg/kg resulted in greater than 98%
depletion of peripheral blood (PB) B cells and 40% to 70%
depletion of lymph node B cells. Recovery of PB B cells
usually started at 2 weeks after treatment and required 60
to greater than 90 days to reach normal levels. As much as
95% depletion of B cells in peripheral lymph nodes and
bone marrow was observed following weekly injections of
16.8 mg/kg antibodyref.
Only later effectiveness of depletion has been proved on
human beings : serum antibody levels were sustained longer
after the fourth infusion than after the first, and were
higher in responders and in patients with lower tumor
burden. The majority of adverse events occurred during the
first infusion and were grade 1 or 2; fever and chills
were the most common events. Only 12% of patients had
grade 3 and 3% grade 4 toxicities. A human antichimeric
antibody (HACA) was detected in only 1/166 patientsref
(++) (response rate 48%)
(+)(+++)
(+++)(+++)
(+++)(++)ref cells, but it
may be present on myeloma precursor cellsrefrefref1,
ref2
(79%) : 17 patients with RA refractory to standard
therapy were treated with rituximab. Patients 1-6
received 4 weekly infusions of rituximab at a dose of
375 mg/m2, and patients 7-17 received 2
infusions of rituximab (1,000 mg), 2 weeks apart.
4-color staining was performed at several time points,
using CD38, IgD, and CD27 in addition to other cell
surface markers. In one patient, the mutational status
of the Ig receptor was examined. The analysis revealed a
distinct pattern of B cell regeneration. The first wave
of repopulating B cells were immature B cells (CD38high,
IgD+,CD10+, CD24high),
the Ig receptors of which were not yet somatically
mutated. In parallel, a recirculation of plasma cells
was observed. Later, the number of naive B cells
increased, and these cells predominated in the
peripheral blood B cell pool. CD27+ memory B
cells showed a slow and delayed repopulation, and the
level of these cells stayed significantly reduced
(<50%) compared with baseline values, for > 2
yearsref.ref1,
ref2
(69%) : a correlation was found between the baseline
frequency and time to repopulation; the fewer B cells
initially, the longer to their return. While the few B
cells remaining after treatment were of memory,
double-negative (IgD-CD27-), and
CD5+ phenotype, the returning B cells were
mainly naive, indicating de novo
production of B cells. Serum levels of IgG and
antibodies against Ro52, Ro60, La44, measles and tetanus
remained unchanged, while decreases in IgM, IgE,
anti-dsDNA and anti-C1q antibodies were observed.
Additionally, a significant increase in activated CD4+
and CD8+ T cells, as well as CD25brightFOXP3+
regulatory T cells was observedref
(66%)
: despite B-cell depletion is effective in all patients,
not all experience a response (involvement of T-cells in
disease or competition of rituximab-CD20 complexes with
autoantibodies-platelet complexes for FcRs, similar to
IVIg ? T cell clones are found in AITP and they change
specificity after rituximab treatment)refref1,
ref2
(1/2)
(1/2)ref1,
ref2,
ref3
(0/1)(1/1)(2/2)
(0/1)ref? and CD59 / MIRL)
via unknown pathwaysref1,
ref2:
fludarabine synergizes with
rituximab by downregulating CD55 membrane
expressionref,
where CDC is easier (lower complement inhibitors
concentrations or structural fragility) : while rituximab,
1F5 and 2H7 induce redistribution, B1 does notref1,
ref2ref1,
ref2
: In mice, B cell depletion is predominantly, if not
exclusively, mediated by monocytes. CD20 mAbs rapidly
deplete circulating and tissue B cells in an antibody
isotype-restricted manner with a hierarchy of antibody
effectiveness: IgG2a/c > IgG1
> IgG2b >> IgG3.
Depending on antibody isotype, mouse B cell depletion
is regulated by FcgRI-, FcgRII-, FcgRIII-,
and FcgRIV-dependent
pathways. The potency of IgG2a/c mAbs for B
cell depletion in vivo results from FcgRIV interactions, with likely
contributions from high-affinity FcgRI.
IgG1 mAbs induce B cell depletion through
preferential, if not exclusive, interactions with
low-affinity FcgRIII, while
IgG2b mAbs interact preferentially with
intermediate-affinity FcgRIV.
By
contrast, inhibitory FcgRIIB-deficiency
significantly increases CD20 mAb-induced B cell
depletion at low mAb doses by enhancing monocyte
function. Thus, isotype-specific mAb interactions with
distinct FcgammaRs contribute significantly to the
effectiveness of CD20 mAbs in vivo. These
results provide a molecular basis for earlier
observations that human FcgRII
and FcgRIII polymorphisms
correlate with the in vivo effectiveness of
CD20 antibody therapyrefref
(through the production of ROS)ref
: cytotoxicity assays showed that untreated patient's
serum with rituximab, but not that of heat-inactivated
patient's serum with rituximab or rituximab alone,
induced potent rituximab-mediated cytotoxicity against
tumor cells in the patient's CSF, suggesting induction
of CDC against PCNSLref through receptor
dimerizationref
and proliferation blockref
is no longer supported. Rituximab
alone is unable to induce a cytotoxic effect on primary
malignant B cells, the addition of a source of
complement being necessary to obtain such an effectref. CDC is more effective than ADCCref.
It has been described that rituximab induces a
caspase-dependent apoptosisref1,
ref2,
ref3.
However, in all these studies in vitro crosslinking
of rituximab with secondary antibodies was performed. The
production of antibodies against rituximab has not been
described in patients treated with rituximab; therefore,
this mechanism is unlikely to occur in vivoref.
Nevertheless, preliminary studies have suggested that
caspases may be activated following in vivo
infusion of rituximabref,
therefore additional mechanisms could be involved in in
vivo caspase activation. Although it has been
described that some anti-CD20 antibodies can promote the
activation of the Src-family kinasesref,
no implication of these kinases has been observed. Late
responses to rituximab treatment could be explained by
antigen cross-presentation (so-called "vaccine effect").
Rituxan has been used to treat >
380,000 patients worldwide. or mantle cell
lymphoma. Management of severe reactions :
the Rituxan infusion should be interrupted for
severe reactions and supportive care measures
instituted as medicaly indicated (e.g. intravenous
fluids, vasopressors, oxygen, bronchodilators,
diphenhydramine, and acetaminophen). In most cases,
the infusion can be resumed at a 50% reduction in
rate (e.g., from 100 mg/hr to 50 mg/hr) when
symptoms have completely resolved. Patients
requiring close monitoring during first and all
subsequent infusions include those with pre-existing
cardiac and pulmonary conditions, those with prior
clinically significant cardiopulmonary adverse
events and those with high numbers of circulating
malignant cells (> 25,000/mm3) with or
without evidence of high tumor burden : rapid reduction in tumor
volume followed by acute renal failure,
hyperkalemia, hypocalcemia, hyperuricemia, or
hyperphosphatasemia, have been reported within 12 to
24 hours after the first Rituxan infusion. Rare
instances of fatal outcome have been reported in the
setting of TLS following treatment with Rituxan. The
risks of TLS appear to be greater in patiets with
high numbers of circulating malignant cells (>
25,000/mm3) or high tumor burden.
Prophylaxis for TLS should be considered for
patients at high risk. Correction of electrolyte
anmormalities, monitoring of renal function and
fluid balance, and administration of supportive
care, including dialysis, should be initiated as
indicated. Following complete resolution of the
complications of TLS, Rituxan has been rolerated
when re-adminstered in conjunction with prophylaxtic
therapy for TLS in a limited number of cases, mucocutaneous reactions,
hypersensitivity reactions, cardiac arrhythmias and
angina, and renal failure. Infusion reactions and
lymphopenia are the most commonly occuring adverse
reactions. Because clinical trials are conducted
under widely varying conditions, adverse reaction
rates observed in the clinical trials of a drug
cannot be directly compared to rates in the clinical
trials of another drug and may not reflect the rates
observed in practice. The adverse rection
information from clinical trials does, however,
provide a basis for identifying the adverse events
that appear to be related to drug use and for
approximating rates. Additional adverse reactions
have been identified during postmarketing use of
Rituxan. Because these reactions are reported
voluntarily from a population of uncertain size, it
is not always possible to reliably estimate their
frequency or establish a causal relationship to
Rituxan exposure. Decisions to include these
reactions in labeling are typically based on one or
more of the following factors: (1) seriousness of
the reaction, (2) frequency of reporting, or (3)
strength of causal connection to Rituxan. Where
specific percentages are noted, these data are based
on 356 patients treated in nonrandomized, single-arm
studies of Rituxan administered as a single agent.
Most patiens received Rituxan 375 mg/m2
weekly for 4 doses. These include 39 patients with
bulky disease (lesions > 10 cm) and 60 patients
who received more than 1 course of Rituxan. 37
patients received 375 mg/m2 for 8 doses
and 25 patients received doses other than 375 mg/m2
for 4 doses and up to 500 mg/m2
single dose in the phase 1 setting.)
(highly expressed on HL and ALCL) : binding of
therapeutic antibodies stimulates a juxtamembrane cleavage
of CD30 leading to a loss of target antigen and an enhanced
release of the soluble ectodomain of CD30 (sCD30). The
extracellular domain of CD30 that is shed from the cells,
can reduce the effects of CD30-targeting agents by
competitive binding. sCD30 binds to CD30L
(CD153)-expressing
non-target cells. Because antibodies bind to sCD30, this
results in unwanted antibody binding to these cells via
sCD30 bridging. Shedding of CD30 can be enhanced by PKC
activation, implicating the disintegrin metalloproteinase
ADAM17 but not free cytoplasmic calcium. However,
antibody-induced CD30 shedding is calcium dependent and PKC
independent. This shedding involved the related
metalloproteinase ADAM10 as shown by the use of the
preferential ADAM10 inhibitor GI254023X and by an
ADAM10-deficient cell line generated from embryonically
lethal ADAM10-/- mouse. In coculture experiments,
the antibody-induced transfer of sCD30 from the human
Hodgkin's lymphoma cell line L540 to the CD30-negative but
CD153-expressing human mast cell line HMC-1 was inhibited by
GI254023X. These findings suggest that selective
metalloproteinase inhibitors blocking antibody-induced
shedding of target antigens could be of therapeutic value to
increase the specificity and reduce side effects of
immunotherapy with monoclonal antibodiesref.
2 epitopes on membrane-associated CD30 are missing on
soluble CD30 probably because of a conformational change
upon shedding. These epitopes are potentially superior
targets for immunotherapy because targeting them should be
free from the competitive effects of soluble CD30. 7
anti-native CD30 mAbs assigned to 8 different topographical
epitopes were studied. Soluble CD30 was prepared from
culture supernatants of L540 cells or Karpas 299 cells. In
an ELISA, the mAbs to 2 epitopes, Ep2 (amino acids 107–153)
and Ep7 (amino acids 282–338), showed < 2% average
cross-reactivity to soluble CD30 compared with a CD30-Fc
fusion protein. In addition, these mAbs bound to CD30 on
cells in the presence of an excess of soluble CD30. These
epitopes (Ep2 and Ep7) are, therefore, more efficiently
presented on cell-associated CD30 than on soluble CD30
(membrane-specific epitopes). Also, soluble CD30 in the sera
of mice bearing L540 tumors did not form immune complexes
with the membrane-specific mAbs analyzed by size-exclusion
chromatography. In contrast, mAbs to the other epitopes
reacted with both soluble CD30 and membrane CD30. These
results suggest that it may be possible to find
membrane-specific epitopes on other immunotherapy target
moleculesref.,
the low affinity inhibitory receptor for IgG on B-cells : a
high-affinity monoclonal antibody (mAb 2B6), from a novel
panel of anti-human CD32B-specific mAbs was chimerized
(ch2B6) and humanized (hu2B6-3.5). Both ch2B6 and hu2B6-3.5
were capable of directing cytotoxicity by peripheral blood
mononuclear cells (PBMC) and monocyte-derived macrophages
against B-lymphoma lines in vitro. In a human B-cell
lymphoma mouse xenograft model, administration of ch2B6 or
hu2B6- 3.5 reduced tumor growth rate and improved tumor-free
survival. Both the in vitro and in vivo activities of 2B6
required an intact Fc, suggesting an FcR-mediated mechanism
of actionref-initiating
cells
(AML-IC) and normal hematopoietic
stem
cells (HSC)
makes identification of an antigen for targeted therapy
extremely difficult. Xenotransplantation assays should be
used wherever possible to test the suitability of candidate
antigens for targeted therapy, including the assessment of
the effect on normal cellsref. : there is now
some clinical experience with anti-CD38 antibody in lymphoma
and myeloma. However, to date, there has been minimal
clinical activity observed. Additional antibodies are
entering clinical trials. A new approach involves the
generation of an anti-CD38 single-chain variable fragment
(scFv) construct that acts as the carrier of a toxin gene
instead of being conjugated directly to the toxin itself. It
is hoped that expression of the toxin by CD38+
plasma cells will promote suicide of the malignant cells
without affecting normal cells or generating an immunologic
response to the toxinref
and advanced, high-risk multiple myeloma, where CD44v6
expression correlates with chromosomal band 13q14 deletions,
a well-known risk factor in MMref1,
ref2),
100% in Waldenstrom's
macroglobulinemia
(WM)ref,
mantle cell
lymphoma
(60%)ref1,
ref2, 5% in multiple myelomaref
(but more represented on clonogenic CD45+38+++
B cells than on the more differentiated CD45-38+++
B-cellsref),
Hodgkin's disease ?), T-cell chronic
lymphocytic leukemia
(100% in T-LGLref,
T-PLL, T-CLL), Langerhans
cell
histiocytosis (LCH)ref,
hypereosinophilic
syndromes
(HES)ref,
enteropathy-associated
T-cell
lymphomaand
aggressive
NK-cell
leukemia
: screening on myeloproliferative disorders ?
cells in vitro, especially in combination with a
cross-linking anti-Fc-antibody, this effect being mediated
by a caspase-dependent pathwayref1,
ref2,
ref3,
ref4.
Treatment with CAMPATH-1H is
associated with a "first dose" reaction, consisting of
fever, rigors, and nausea related to release of TNF-a
and IL-6.
Prolonged depression of normal T- and B-lymphocyte levels
predisposes to opportunistic infection. Infections can be
partly prevented by prophylaxis with acyclovir,
cotrimoxazole,
and itraconazole. ,
because red cells are unaffected by somatic selection;
occurrence is inversely related to the absolute lymphocyte
countref.
mAb : CD70 is a TNF superfamily member whose normal
expression is highly restricted but is aberrantly expressed
in hematologic malignancies including non-Hodgkin's
lymphoma, Hodgkin's
lymphoma
and multiple myeloma. In addition,
solid tumors such as renal
cell
carcinoma,
nasopharyngeal
carcinoma,
thymic carcinoma, medulloblastoma, and glioblastoma
multiforme
express high levels of this antigen. To functionally target
CD70-expressing cancers, a murine anti-CD70 monoclonal
antibody was engineered to contain human IgG1
constant domains. The engineered antibody retained the
binding specificity of the murine parent monoclonal antibody
and was shown to induce Fc-mediated effector functions
including antibody-dependent cellular cytotoxicity,
complement dependent cytotoxicity and antibody-dependent
cellular phagocytosis in vitro. Further, administration of
this antibody significantly prolonged survival of SCID mice
bearing CD70+ disseminated human NHL xenografts. Survival of
these mice was dependent upon the activity of resident
effector cells including neutrophils, macrophages and NK
cells. These data suggest that an anti-CD70 antibody, when
engineered to contain human IgG1 constant
domains, possesses effector cell-mediated antitumor activity
and has potential utility for anticancer therapyref.
cells, 2 cell types with a high level of CD99 expression.
Engagement of the molecule induces a rapid death signal that
appears to be related to the level of expression of this
antigen. The rapid apoptosis induced by agonistic anti-CD99
mAbs is of clinical interest in ES, a tumour for which no
new drugs have been described as clearly effective in the
last 10 years. An anti-CD99 mAb can be used to advantage in
association with doxorubicin. Striking
effectiveness was observed against local tumours and
metastases. No remarkably toxic effects of anti-CD99 mAb
were found in bone marrow against blood precursors. These
results provide the necessary rationale and support for a
novel modality of therapeutic intervention, which may have
application in the care of patients with ESref :
mAbs : mAbs :
expression of CCR4 by tumor cells is associated with skin
involvement, but CCR4 also has an important role in normal
and tumor immunity. In a subset of patients with CCR4+
T-cell leukemia/lymphoma, the tumor cells themselves
function as regulatory T (Treg) cells,
contributing to tumor survival in the face of host antitumor
immune responses. In other types of cancers, the chemokines
TARC/CCL17 and MDC/CCL22, specific ligands for CCR4 that are
produced by tumor cells and the tumor microenvironment,
attract CCR4+ Treg cells to the tumor,
where they create a favorable environment for tumor escape
from host immune responses. Expressed on 23.8% of Hodgkin's lymphomaref
,
vasopressin,
and histamine
(little effect),
resulted in an increased perfusion of chemotherapeutic
agents into tumors that was associated with improvements
in overall tumor response ratesref1,
ref2,
ref3,
ref4,
ref5., physalaemin, histamine,
bradykinin, and LT-B4ref.
Furthermore, targeted delivery of TNF-a conjugated to the
CNGRC peptide of aminopeptidase N (CD13), which targets
tumor neovasculature, resulted in an 8- to 10-fold
potentiation in the activity of doxyrubicin in mouse lymphoma
and melanoma models without a significant increase in overall
toxicityref1,
ref2.
In addition to the benign and malignant prostatic epithelium,
PSMA is also expressed on the neovasculature associated with a
variety of solid tumors, but not in the normal endotheliumref1,ref2.
By conjugating vasoactive agents to mAbs
raised
against PSMA, a localized increase in vascular
permeability could be achieved exclusively at sites proximal
to tumors. Using such a treatment before the administration of
therapeutic agents, including antibodies, would substantially
enhance the dose received by the tumor. In addition to
enhancing vascular permeability, additional approaches have
attempted to exploit intracellular transport pathways as an
alternative means by which to bypass the endothelial barrier.
McIntosh et al.ref
have used mAbs against antigens found within caveolae of the
lung microvasculature to achieve selective and efficient drug
delivery to underlying lung tissue.
agentsref,
in which antibodies are labelled with molecules that
only become active on illumination mAb : HGS-TR2J
(source : Human Genome
Sciences) is in phase I clinical trial. Like anti-Fas
mAbs, some mAbs against TRAIL receptors have tumoricidal
activity too. A novel mouse anti-human DR5 monoclonal
antibody, AD5-10, induces apoptosis of various tumor
cell lines in the absence of second cross-linking in
vitro and showed strong tumoricidal activity in
vivo. AD5-10 does not compete with TRAIL for binding
to DR5 and synergizes with TRAIL to induce apoptosis of
tumor cells. AD5-10 induces both caspase-dependent and
caspase-independent cell death in Jurkat cells, while TRAIL
induces only caspase-dependent cell death. DR5 can mediate
caspase-independent cell death and DR5 can mediate distinct
cell signals when interacting with different extracellular
proteins. Studies on AD5-10 help us to understand more on
the functions of DR5 and may provide new ideas for cancer
immunotherapyref.
(in pancreatic
adenocarcinomas, NSCLC,
colorectal
carcinomas, and head and
neck cancers), but not of normal tissuescoreceptor (overexpressed in 25-30 % of
metastatic breast
carcinomas, but also in ovarian
carcinomas,NSCLCref1,
ref2,
ref3, stomach and androgen-dependent prostate
adenocarcinoma)
(1-4%) because it acts as a modifier of
anthracycline-induced cardiotoxicityref1,
ref2,ref3.
Left ventricular function should be evaluated in all
patients prior to and during treatment with Herceptin.
Discontinuation of Herceptin treatment should be
strongly considered in patients who develop a clinically
significant decrease in left ventricular function. The
incidence and severity of cardiac dysfunction was
particularly high in patients who received Herceptin in
combination with anthracyclines and cyclophosphamide; it appears to utilize Fc
receptors and angiogenesisref
activation contributes to
tumor inhibition by trastuzumab (increased PTEN membrane
localization and phosphatase activity by reducing PTEN
tyrosine phosphorylation via Src inhibition), and loss of
PTEN predicts trastuzumab resistance in patientsref.
HER2 exon 20 mutation (G776L) conferring resisance to
trastuzumab has been reported in a NSCLC patientref
with ERBB3 / HER3,
so effective also against androgen-independent prostate
adenocarcinoma; distribution phase of <1 day,
terminal half-life of approximately 10 days, and volume
of distribution of approximately 40 mL/kg that
approximates serum volume. With the exception of
diarrhea, pertuzumab was generally well tolerated in
cynomolgus monkeys.,
melanomas
and lymphoma : IgG2a and IgM anti-GD2 therapy
protected mice challenged with the EL4 syngeneic mouse model
of metastatic lymphoma from metastases and prolonged
survival. Expression of CD59, an inhibitor of CDC,
effectively protected EL4 cells from CMC in vitro but
did not affect the outcome of mAb therapy. Protection by IgG
therapy was also unaffected in mice deficient in C3 or CR3
but was almost completely abrogated in FcgRI/III-deficient
mice. These data indicate a crucial role for ADCC. However,
at lower doses of IgG, therapeutic effect was partially
abrogated in C3-deficient mice, indicating
complement-mediated enhancement of ADCC at limiting IgG
concentration. In contrast to IgG, the therapeutic effect of
IgM was completely abrogated in C3-deficient mice. High
level expression of CD59 on EL4 did not influence IgM
therapy, suggesting IgM functions by complement-dependent
cell-mediated cytotoxicity (CDCC), a mechanism thought to be
inactive against tumor cells. Thus, IgG and IgM can operate
via different primary mechanisms of action, and CDCC and
complement-dependent enhancement of ADCC mechanisms are
operative in vivo. The effects of complement can be
supplemental to other antibody-mediated mechanisms and
likely have increased significance at limiting antibody
concentration or low antigen densityref))
=> stimulation of IL-12 secretion by macrophages. They
can also bind to GD3 on the surface of T cells,
enhancing their cytotoxic and proliferative responses.
and NSCLC) and colorectal
carcinomas))
:antigen,
8/16 (50%) esophageal carcinomas, and 4/17 (23%) ovarian
carcinomas,
but not in lung, breast or colon
carcinomas.
A murine monoclonal antibody (mAb A34) was generated to
the extracellular domain of the A34 protein and used to
biochemically and immunohistochemically characterize the
A34 antigenic system. The mAb A34 specifically recognized
glycoproteins ranging in apparent size from 55-70 kDa,
present in normal gastric mucosa and in COS-7 cells
transfected with A34 cDNA. Of 31 different normal tissues
examined by immunohistochemistry, GPA34 protein expression
was detected primarily in normal stomach mucosa and
testicular germ cells, and in the tumor cells of 5/17
(29%) gastric cancers, 7/11 (63%) esophageal cancers, and
2/21 (9%) ovarian cancers, in agreement with gene
expression results. The A34 antigen and monoclonal
antibody may be of considerable value for immunotherapy of
different types of cancerref
(not effective against ovarian
carcinomas),
gastric
carcinomas,
pancreatic
adenocarcinomas,
NSCLC) :
mAb :
tumor establishment in nude mice. It also delayed and
inhibited tumor progression of established tumors,
prolonging mice survival. In vitro, A24 induced up
to 85% reduction of MCL cell proliferation (IC50
= 3.75 nmol/L) independently of antibody aggregation, CDC
or ADCC. A24 induced MCL cell apoptosis through caspase-3
and caspase-9 activation, either alone or synergistically
with chemotherapeutic agents. A24 induced TfR endocytosis
via the clathrin adaptor protein-2 complex pathway
followed by transport to lysosomal compartmentsref
(ovarian
carcinomas)ref
:,
colorectal
carcinomas,
ovarian
carcinomas
and lung
carcinomas)
:,
breast
carcinomas,
lung
carcinomas,pancreatic
carcinomas, and gastric
carcinomas) : (TAA in melanoma))
: Melimmune© is a dual preparation of 2
murine anti-idiotypic antibodies (anti-Ids) which mimic
separate epitopes)
:
and
95% of ovarian
carcinomas, osteosarcoma)
:
:)
:)
:(breast
carcinomas,
lung
carcinomas
and prostate
adenocarcinoma) mAbs :)
:,
overexpressed by colorectl cancers, protecting tumour
cells from attack by complementref;
it stimulates strong in vitro T-cell
proliferation, IFN-g
secretion and redirected cytotoxicity in unprimed T
cells from healthy donors. However, removal of the Fc
region of the anti-idiotype reduced the sensitivity of
the assay 1,000-fold, as did inhibiting Fc uptake of the
anti-idiotype by an excess of human IgGref;
it initiated phase II clinical trials in May 2000ref:, colorectal
adenocarcinomas (lengthens the average survival times for
patients with metastatic CRC by almost 5 months (20.3
months vs. 10.6 months)) and breast
carcinomas, and in phase II trials for various other
solid tumor types (including renal
cell
carcinoma (RCC)).Side effects :
easily manageable systemic
arterial
hypertension (11%); 2-fold risk (5% vs.2.5% in controls)
of blood clots, which can lead to stroke, heart attack
and chest pain, reversible
posterior
leukoencephalopathy syndrome (RPLS)ref.
Ranibizumab has been genetically engineered from
bevacizumab through a process of selective mutation to
increase its affinity for binding and inhibiting the
growth factor. The Fab domain of ranibizumab differs from
the Fab domain of bevacizumab by 6 amino acids, 5 on the
heavy chain (4 of which are in the binding site) and 1 on
the light chain
but not to VEGR1
/ Flt-3 to produce a new chemotactic
BsAb, named 18TBHL. It was expressed in soluble form in
cytoplasm of E.coli and purified with DEAE anion
chromatography and IMAC (Immobilized Metal Affinity
Chromatography). The chemotactic ability of 18TBHL to PBLs
(peripheral blood lymphocytes) was detected by Boyden chamber
chemotaxis assay. The specific ability of its binding to
ovarian carcinoma cells, SKOV3, and PBLs was tested by ELISA
and flow cytometry. Of the most importance, the enhanced tumor
specific cytolysis of 18TBHL was validated by MTT method and
flow cytometry just as expected. The results indicated that
fusion of chemotactic peptide to BsAb potentiated its
cytotoxicity to tumor cells in vitro. It suggested that
18TBHL would be a promising candidate agent in cancer
immunotherapyref.
mAbs
mAb for the treatment of allergic asthma mAb for the treatment of
allergic asthmaref,
atopic
dermatitisref1,
ref2,
angiolymphoid
hyperplasia
with eosinophiliaref,
hypereosinophilic
syndromesref1,
ref2,
ref3,
ref4,
mepolizumab had few side effects and lowered blood
eosinophil levelsref1,
ref2,
ref3,
ref4,
ref5.
Subsequent studies suggested that mepolizumab may have
clinical value in patients with the hypereosinophilic
syndromeref1,
ref2,
ref3,
ref4
mAb,
is in early-stage clinical trials. mAbsmAb
(reduce the use of systemic steroid bursts in patients with
moderate to severe asthma who are using other controller
medications) 
mAb : AMG
157 is a human anti-TSLP monoclonal IgG2 λref
mAbs
: CD25 is expressed by activated but not resting, T
lymphocytes. However, it might not readily discriminate
between IL-2R+ activated pathogenic T cells and
regulatory T cells. Used for acute graft versus
host disease (aGvHD)ref.
CD25 shedding impairs responsesref mAb : BG9588
for the treatment of SLEref:
the study was terminated prematurely because of thromboembolic
events (CD40L is expressed also on platelets) occurring in
patients in this and other BG9588 protocols (2 myocardial
infarctions in this study). mAb : for multiple
myeloma
and the
lymphoproliferative disorder giant
lymph
node hyperplasia (Castleman's disease)ref mAb,
Crohn's
disease,
multiple myeloma and the
lymphoproliferative disorder giant
lymph
node hyperplasia (Castleman's disease)refref1,
ref2 and rheumatoid
arthritis
(RA)ref1,
ref2
mAb : B-N10 murine mAb for the treatment of SLEref/IL-23
mAb : monoclonal antibody which binds the shared p40
protein subunit of human interleukins 12 and 23 with high
affinity and specificity and thus preventing interaction with
their cell-surface IL-12 Ra
receptor
and inflammatory disease; no development reported for
autoimmune disease or rheumatoid arthritis
mAb : for rheumatoid
arthritis
(RA)ref
mAb : mAbs
: mAbsref1,
ref2,
ref3.. Do not associate with IL-1ra 7.5 mg/wk to prevent human
anti-chimeric antibodies (HACA)ref
and Crohn
diseaserefref
and histoplasmosisref,
and cerebral toxoplasmosisref.
Granulomatous infections were reported at rates of
approximately 239 per 100,000 patients who received
infliximab and approximately 74 per 100,000 patients who
received etanercept (P<.001). Tuberculosis
was the most frequently reported disease, occurring in
approximately 144 and approximately 35 per 100,000
infliximab-treated and etanercept-treated patients,
respectively (P<.001). Candidiasis, coccidioidomycosis,
histoplasmosis,
listeriosis, nocardiosis, and infections
due to nontuberculous
mycobacteriawere
reported
with significantly greater frequency among
infliximab-treated patients. 72% of these infection occurred
< or =90 days after starting infliximab treatment, and
28% occurred after starting etanercept treatment (P<.001).
These data indicate a risk of granulomatous infection that
was 3.25-fold greater among patients who received infliximab
than among those who received etanercept. The clustering of
reports shortly after initiation of treatment with
infliximab is consistent with reactivation of latent
infectionref.
Tuberculosis should be assessed prior to therapy with
Mantoux intradermoreaction and chest X-rays and eventually
treated.-Ig fusion protein
reduces airway occlusion and immunopathology by bystander
activated T cells without preventing virus clearance from
naive antigen-specific T-cells in a murine mouse influenza
modelref mAbs :, BCMA, and BAFF-R / BR3. LymphoStat-B
potently inhibited BLyS-induced proliferation of B cells in
vitro, and administration of LymphoStat-B to mice prevented
human BLyS-induced increases in splenic B cell numbers and
IgA titers. In cynomolgus monkeys, administration of
LymphoStat-B resulted in decreased B cell representation in
both spleen and mesenteric lymph nodesref.
It has been approved for therapy of rheumatoid
arthritis
(RA)ref
mAb :
and nephritisref,
and for reducing intravascular hemolysis, hemoglobinuria,
and the need for transfusion, with an associated improvement
in the quality of life in patients with paroxysmal
nocturnal
hemoglobinuria (PNH)refref1,
ref2,
ref3,
ref4,
ref5,
ref6,
ref6
and coronary
artery
bypass graft surgeryref
and investigated for Crohn's
disease
and rheumatoid
arthritis
(RA).
Side effects : JC virus–induced PMLref1, ref2
in 1 in 1000 patients with Crohn's
diseaseref1,
ref2,
in 11 cases to date of about 50,000 persons under
treatment for relapsing–remitting multiple
sclerosis
(MS)ref1,
ref2
treated for a mean of 17.9 monthsref;
uncommon CNS or peripheral infections, such as ocular
toxoplasmosisref.
Neoplastic complications of natalizumab have been limited
so far to 2 cases of melanomaref
and 2 cases of PCNSLref
exposure, which is consistent with the expected background
rate in the general US population. The biologic effects of
natalizumab remain for approximately 3 months after the
end of dosing, and changes in the cytology of
cerebrospinal fluid remain for up to 6 months after
withdrawal. The absolute number of cerebral perivascular
spaces (CPVS) in the patient with MS treated with
natalizumab was significantly lower than in the control
groups owing to extensive destruction of the tissue
architecture. The expression of MHC class II molecules and
the number of CD209+ dendritic cells were
significantly decreased in the CPVS of the patient with MS
treated with natalizumab. No CD4+ T cells were
detectableref.
Albeit Foxp3+ Tregs had lower levels
of CD49d and bind less natalizumab, natalizumab blocked
the transmigration of Foxp3+ Tregs
similar to non-regulatory T cells. The frequency of
peripheral blood Tregs was unaffected by
Natalizumab treatment. Natalizumab does not alter the
suppressive capacity of CD4+CD25highCD127lowFoxp3+
Tregs under in vitro conditions.
Furthermore, the impaired function of Tregs in MS patients
is not restored by natalizumab treatmentrefref mAbs has been
documented, and its activation alone results in the
secretion of IFN-gref.
A subgroup of human neutrophils expresses the TcR.
Costimulation of the TcR and CD28 triggers the release of
IL-8 in these cellsref.
Therefore, neutrophils may represent a quantitatively
important target of TGN1412 and may link the pathogenesis
of the cytokine-release syndrome to the innate immune
system. Expression of the T-cell receptors and
(indicated in red) is shown in a normal human
peripheral-blood neutrophil (Panel A) and in a lymphocyte
from the same person, used as a control cell (Panel B).
Cells were double-stained with a monoclonal antibody
against the T-cell receptors and (red) and the
neutrophil marker CD15 (green); the nuclei were
counterstained with 4',6-diamidine-2-phenylidole
dihydrochloride (blue). Panel C shows the presence of the
T-cell receptors and on the surface of a
peripheral-blood neutrophil on immunogold electron
microscopy. Arrows indicate T-cell receptor heterodimers
(pairs).
monoclonal
antibody. Previous studies in nonhuman primates have
demonstrated that this isotype is ineffective in engaging
FcRsref.
Although TGN1412 was used at 1/500 of the dose that was
used safely in nonhuman primates, increased avidity of the
IgG4 Fc region for human FcRs may have caused
sufficient cross-linking of CD28 to elicit a cytokine
storm. Such a hypothesis could be tested experimentally.
Species differ greatly with respect to the number of FcR
genes and specific cell-type expression patternsref
There is a need for better models to address these issues.
It should be noted that differences in both the antibody
subclass and the glycosylation status of the antibody
influence the profile of FcR binding. In addition, human
IgG4 has a low level of binding to FcRs but is
not a nonbinder. Clearly, it is important to consider the
nature of the Fc portion of antibodies as they are
developed for clinical use as therapeutic agentsref.,
which is induced by a staphylococcal superantigen that
activates T cells, through their antigen receptor, to
induce massive cytokine releaseref.
However, mice with deletion of the CD28 gene are protected
from superantigen-induced, toxic shock syndrome–like
diseaseref.
In addition, peptides that block binding to CD28 also
block superantigen-dependent cytokine secretion by T cellsref.
Together, these observations indicate that T cells
activated by superantigen binding to both CD28 and the
antigen receptor mediate the toxic shock syndrome.
CD28-deficient mice are protected from lethal superantigen
challenge through the selective abrogation of the release
of TNFref.
Because TNF is also strongly induced by anti-CD28
antibodies and TNF infusion can induce toxic shock
syndrome–like diseaseref,
short-term use of anti-TNF agents may benefit patients
with the toxic shock syndrome and related syndromes
involving "cytokine storm." When transfer to hospital care
took place 12 to 16 hours after TGN1412 infusion, the TNF
peak was likely to have passed and that downstream events
had already been triggered. As cytokine data became
available, showing persistently high TNF levels, this
decision was reviewed. In the setting of severe
lymphopenia and monocytopenia of unknown duration,
treatment with high-dose corticosteroids and IL-2R
antagonist antibody, and the increased risk of secondary
infections in the patients, the risk of anti-TNF therapy
still outweighed its potential benefit. They would be yet
more cautious with a cause associated with infection, such
as the toxic shock syndrome.,
a severe and sometimes fatal conditionref1,
ref2.
Unfortunately, important diagnostic criteria for the
macrophage activation syndromeref
(Henter JI, Horne A, Arico M, et al. HLH-2004: diagnostic
and therapeutic guidelines for hemophagocytic
lymphohistiocytosis. Pediatr Blood Cancer (in press)) such
as increased ferritin and soluble CD25 levels, enhanced
activity of NK cells, and specific histologic
characteristics of bone marrow, were not reported in these
patients. Also, hepatosplenomegaly and lymphadenopathy,
which are classic features of the MAS, were absent in the
volunteers exposed to TGN1412, perhaps because of the very
acute nature of the reaction. If characterized further,
TGN1412 may provide insight into the mechanisms that lead
to the MAS in other clinical settings, such as juvenile
idiopathic arthritis or sepsis. Although T cells were the
intended targets of TGN1412, it is still unclear whether
T-cell activation induced the cytokine storm. The trigger
may have been on another type of cell expressing CD28 or
may have involved ligation of FcRs. Hence the macrophage
activation syndrome was also considered. Since the
neutrophil count was preserved and there was neither
hepatosplenomegaly nor lymphadenopathy, the macrophage
activation syndrome as a separate entity should be ruled
out,
which selectively removes high-molecular-weight
substances, including immunoglobulins and immune
complexes, has been proven to be effective for the
clearance of circulating monoclonal antibodiesref,
and some studies have shown the usefulness of continuous
hemofiltration or continuous hemodiafiltration for
ameliorating cytokine stormref1,
ref2.
Plasmapheresis was considered, but the known
pharmacokinetics of a low IgG4 antibody level
suggested that the drug would be tissue-bound. There may
be a role for continuous venovenous hemodiafiltration in
blunting the cytokine storm associated with a severe
systemic inflammatory response. Furthermore, high
dialysate ratesref
and high-cutoff filtersref
have previously been suggested for use in treating sepsis.
The patients received continuous venovenous
hemodiafiltration at an initial dialysate rate of 1 liter
per hour, which was subsequently raised to 4 l/hr, using
standard membranes. Aggressive multiorgan support and
early institution of high-volume diafiltration and
treatment with high-dose corticosteroids, combined with
the physiological resilience of these 6 young, healthy
research volunteers, were key to their survivalref1,
ref2
and moderate to severe chronic
plaque
psoriasisref1,
ref2.
Primatized antibodies, genetically engineered from cynomolgus
macaque monkey and human components, are structurally
indistinguishable from human antibodies. They may, therefore,
be less likely to cause adverse reactions in humans, making
them potentially suited for long-term, chronic treatment. IDEC
has signed an antibody humanization patent licensing agreement
with Protein Design Labs. IDEC is also collaborating with
Mitsubishi-Tokyo (formerly Mitsubishi Kasei) on the
development of this antibodyrefmAbs.
Anti-CD154 treatment at the time of transplantation allows the
development of CD4+CD25+
TReg cells,
which, if present in sufficient numbers, can supress rejection
mediated not only by CD4+ T cells, but also by CD8+
T cells (linked unresponsiveness). TReg
cells in this system become primed by and are specific for
donor antigens that have been processed and presented on host
antigen-presenting cells : animals rendered tolerant to a
type-A graft reject third-party (type-B) grafts, but accept
grafts from (A x B) F1 donors. In time, hosts that
have accepted (A x B) F1 grafts become tolerant of
type-B grafts. Naive T cells introduced into the
tolerant environment become tolerant of graft B and can
develop the ability to regulate in their own right (infectious
tolerance)., HHV-5
/ CMV, polyomavirus, HCV).
mAbsref.
Skin rashes and gut reactions have been seen during tests, as
for the anti-CD28 mAb. This
also boosts the effects of the CD28 receptor, but to a lesser
degree than TGN1412. Scientists have continued to work with
anti-CTLA4 antibody and it is now in large-scale clinical
trials to treat cancer
is commenced, immune activation is reduced, but CTLA-4
levels do not decline. In individuals with undetectable
viral load, CD4 cell increases are directly correlated with
CTLA-4 levels: those with the highest CTLA-4 levels enjoy
the smallest CD4 cell increases. CTLA-4+ cells
express a higher level of the HIV-1coreceptor
CCR5,
so downregulating CTLA-4 expression might also be expected
to downregulate CCR5 expression and thus reduce
opportunities for HIV infection.
to act against HIV’s gp41 region as it prepares for
fusion. When CCR5 expression on the cell surface is low,
HIV’s gp41 region remains exposed for much longer whilst
the virus seeks to engage with multiple receptors on the
surface of the cell.Side effects : hypophysitis
-Ig fusion protein
and OX40-specific antibody
augments CD4 T-cell response in sarcomaand melanomaref,
CD4 and CD8 T-cell response in sarcoma and gliomaref,
and CD4 T-cell response in colon
carcinomarefref-specific
antibody augments CD8 and CD4 T-cell response in
sarcoma, mastocytomaref,
and melanomarefand
CD8
T-cell response in melanomaref-depleting
mAbs
mAbs : known to elicit CTL responses against murine
lymphoma models. In the syngeneic tumor model BCL1,
timing of cyclophosphamide relative to mAb is
critical to therapeutic outcome. Pretreatment with
cyclophosphamide 7 to 10 days prior to mAb results in markedly
reduced survival levels, similar to that achieved with
cyclophosphamide alone. Conversely, when anti-CD40 is given
before cyclophosphamide, the level of tumor protection was
moderately increased. In vivo tracking experiments
reveal that pretreatment with cyclophosphamide leads to
diminished CTL expansion, as well as an increased number of
CD11b+ cells that display an activated phenotype.
These latter cells are able to inhibit T-cell proliferation,
at least in part via production of NO, but do not induce
T-cell apoptosis. Furthermore, adoptive transfer of the
induced CD11b+ cells is sufficient to inhibit
anti-CD40 therapy in tumor-bearing recipientsref.
Usedmainly for multiple
myeloma.
ligands/p75
linked
to Fcg1, which doesn't
fix complement, Ka = 1010,
half-life = 4-5 days, s.c. (25 mg/biweekly)
by selectively inducing apoptosis of CD2+ (both
CD4+ and CD8+) human memory-effector T
cells through binding to CD2and CD16a / FcgRIIIAand
inducing
signaling via CD16 / FcgRIII.ref1,
ref2,
ref3
and chronic
plaque
psoriasisref,
it is an ineffective means of prophylaxis against
rejection in nonhuman primate models of transplantationref.–immunoglobulin
fusion proteins block the interaction of OX40 with its
ligand on APCs and eliminate weight loss and cachexia
without preventing virus clearance–immunoglobulin
fusion
protein–immunoglobulin
fusion
protein
-FcIgG1fusion
protein ligands encoded by DNA-based
vaccines together with tumor antigens such as survivin or
CEA, markedly activate both innate and adaptive antitumor
immunity. Such vaccines result in highly effective, NK- and
CD8+ T cell-mediated protection against either
breast or colon carcinoma cells in prophylactic and
therapeutic settings. Notably, this protection was
irrespective of the NKG2D ligand expression level of the tumor
cells. Hence, this strategy has the potential to lead to
widely applicable and possibly clinically useful DNA-based
cancer vaccinesref.
ligands (see also xenobiotic TLR
ligands used as immunostimulants) :
- also experienced tumour regression by inducing hemorrhagic
necrosis in 50% of cases. To explore whether this
association was causal, Coley deliberated injected live S.pyogenes
into the skin of patients with advanced cancer. A severe
local infection with high fevers ensued, but after the
illness subsided, the patient experienced marked tumour
regression and prolonged survival. As this pproach was
associted with significan toxicity, Coley later explored the
use of bacterial extracts that were derived from a mixture
of culture supernatants from inactivated S.pyogenes
(Serratia marcescens) and Serratia marcescens. The
detailed clinical reports of many patients who experienced
durable tumour regressions in response to the bacterial
extracts were collated and reported after Coley's death by
his daughter, Helen Coley Nauts [Nauts, H., Fowwler, G &
Bogatko, F. A review of the influence of bacterial infection
and of bacterial producs (Coley's toxins) on malignant
tumours in man ; a critical analysis of 30 inoperable cases
treated by Coley's mixed toxins, in which diagnosis was
confirmed by microscopic examination selected for special
study. Acta Med. Scand. 144, S1-S103 (1953)]. Her
careful scholarship led to a broader appreaciation of the
connections between the immune system and cancer. Although
Coley's toxin did not become a standard cancer therapy, the
instillation of Mycobacterium bovis Bacillus
Calmette-Guérin
(BCG)
into the bladder of patients with early-stage bladder
carcinoma
was shown to be highly efficacious and is still used as a
treatment for bladder cancerref.
The intravesical administration of BCG vaccine for
high-risk, non–muscle-invasive bladder cancer and carcinoma
in situ is one of the most successful immunotherapies to
dateref.
Studies in animals have shown that the generation of
effector cells, called BCG-activated killer cells, depends
on Th1–type cytokinesref.
In humans, a high Th1-biased urinary cytokine
profile after several weekly intravesical instillations of
BCG correlates with a better clinical response : urinary
IL-2 at the 8th instillation of BCG may serve as a valuable
prognostic factor of treatment efficacy as well as tumor
recurrence after treatmentref.
The discontinuation of statin therapy, which promotes the
development of Th2 cellsref1,
ref2.
during BCG immunotherapy might improve the clinical outcome,
since the use of statins was significantly associated with
an increased risk of tumor progression and a subsequent need
for radical cystectomyref
ligands : MALP-2, tested in pancreatic
adenocarcinoma and CDC
=> cell lysis, are routinely
introduced by direct halogenation of tyrosine residues of
the protein or 90Y,
require chelation of the metal through a suitable ligand.
This chelating agent is routinely introduced through a
reactive functional group that targets N-terminal
and e-amines of lysine
residues. Such linking moieties include
or 90Yfor therapy
: various radioprotectants (eg vitamin C) can successfully
prevent the loss of immunoreactivity or breakdown as a result
of radiolysis during the labeling process. Radiolabeled
antibodies are important clinical reagents for both tumor
imaging and therapy and also provide an effective evaluation
of pharmacokineticsref.
The choice of radionuclide dictates the application.
Therapeutic administration requires a balance between long
dissociation rates at the target site and slow blood
clearance, which can lead to accumulation in the liver and
high radiation exposure of other tissues. Renal and hepatic
localization of intact radiolabeled antibody fragments
constitutes a major problem. An important study demonstrated
that a previously undescribed radio-iodination reagent could
liberate radionuclide from the antibody fragment before
incorporation into renal cells. The radionuclide is excreted
rapidly, thus decreasing the total renal radiation doseref.
The development of new metabolizable chelates will further
improve the pharmacokinetics of recombinant antibodies for
cancer targeting. Modifications to surface charge designed to
alter the isoelectric point (pI), such as glycolation (e.g. PEGylation),
can also reduce the tissue (kidney) uptakeref.
Longer half life permits imaging and decay at later times
after injection, when contrast between tumor and surrounding
normal tissues is generally more favorable.
=>
a-particles have a
range in tissue of only 55-80 mm
and are radiation of high linear energy transfer (LET)
=> effectiveness does not require the presence of
oxygen nor is it dependent on dose rate. As a result,
their relative biologic effectiveness (RBE) is much higher
than that of b-emitters.
However, there are several obstacles to the use of a-particle
immunotherapy, including problems with chelation chemistry
and nontarget tissue toxicity.
=> b-particles (when
localized in the cell nucleus, also b-emitters
can act as high-LET radiation and they have been
explored for use in tandem with antibodies that become
internalized into the tumor cell after binding) and anaplastic
astrocytoma has advantages over other
available b-particle
emitters as a therapeutic agent, but its efficacy in
the treatment of micrometastases seems to be less than
that of low-energy electrons (LEE) emitters 111In
and 67Ga, due to greater nonspecific
toxicity. This conclusion, however, may not apply to
therapy of macroscopic tumorsref 40 to 60
mg/kg (day -4) and cyclophosphamide 100 mg/kg (day -2) followed by autologous
stem
cell transplantation (ASCT) in 31 patients with CD20+
NHL without an increase in transplant-related
toxicity or delayed engraftmentref.
In a randomized clinical trial comparing Zevalin
with rituximab, the overall response rate was 80%
and 56%, respectivelyref.
Response was determined by assessing the size of
lymph nodes on CTscans. FDG
PET has been well accepted as an
accurate imaging study for staging non-Hodgkin
lymphoma and evaluating response to treatment.
Simultaneous FDG PET and CT imaging (PET CT)
provides coregistered functional PET images with
anatomic CT imagesref.
Cerebrospinal fluid diffusion of Zevalin after
high-activity treatment and stem cell support in a
patient affected by DLBCL with CNS involvementref.
90Y ibritumomab tiuxetan treatment is
safe for use in patients with prior myeloablative
therapy when the general inclusion criteria are
fulfilled. In this small series, the response rates,
however, are limited. Nevertheless, 90Y
ibritumomab tiuxetan treatment may provide clinical
benefit in carefully selected extensively pretreated
patientsref. conditioning : encouraging
results with 2 relapsed lymphoma patients (1
transformed MZL and 1 MCL) who underwent
allogeneic HSCT from HLA-matched donors. The
conditioning regimen consisted of rituximab 250
mg/m2 on days -21 and -14, 0.4 mCi/kg
body weight 90Y-ibritumomab tiuxetan
on day -14 and fludarabine (30 mg/m2))
plus cyclophosphamide (500 mg/m2) on
days -7 to -3. The data demonstrate that
engraftment is fast and reliable with leukocytes
>1 x 109/L on day 12 and platelets
>50 x 109/L on day 10. Thus, the
incorporation of radioimmunotherapy into
allogeneic HSCT protocols combines established
modalities with proven anti-lymphoma activity and,
hence, offers an attractive new therapeutic option
for relapsed lymphoma patientsref
and radioimmunoguided surgery (RIGS)
for AML,
but toxicity remains high, and cure rates are only 25% to
30% for relapsed AML. Pretargeted radioimmunotherapy
(PRIT) is superior to conventional radioimmunotherapy
using a recombinant tetravalent single-chain
Ab-streptavidin (SA) fusion protein (scFv4SA)
directed against human CD45, administered sequentially
with a dendrimeric N-acetylgalactosamine-containing
clearing
agent and radiolabeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic
(DOTA)-biotin.
The scFv4SA construct was genetically
engineered by fusing Fv fragments of the human
CD45-specific BC8 Ab to a full-length genomic SA gene and
was expressed as a soluble tetramer in the periplasmic
space of Escherichia coli. The fusion protein was
purified to >95% homogeneity at an overall yield of
approximately 50% using iminobiotin affinity
chromatography. The immunoreactivity and avidity of the
fusion protein were comparable with those of the intact
BC8 Ab, and the scFv4SA construct bound an
average of 3.9 biotin molecules out of 4 theoretically
possible. Mouse lymphoma xenograft experiments showed
minimal toxicity, excellent tumor-specific targeting of
the fusion protein and radiolabeled DOTA-biotin in vivo,
marked inhibition of tumor growth, and cured 100% of mice
bearing CD45-expressing tumors. These promising results
have prompted large-scale cGMP production of the BC8
fusion protein for clinical trials to be conducted in
patients with hematologic malignanciesref=>
immuno-PET
can be divided into melanin binders, melanin
precursors and binders to melanogenesis-related
proteins. Therapeutic agents targeting melanin to deliver
radionuclide therapy could appear in the clinic within a decaderef.
can produce tumor
responses at sites of NHL that relapse after RIT with acceptable
toxicityref
Stx binds to the cell surface glycolipid CD77 /
Gb3, which is expressed by B cells in certain
B-cell lymphomas. This finding led to studies that
showed that Stx can purge bone marrow of malignant CD77+
B cells before an autologous bone marrow transplant. toxin A chain; despite routine
diphtheria toxoid vaccinations, most children and
adolescents with AML have anti-DT388-GM-CSF
IgG concentrations (< 2 mg/mL)
that
do not preclude in vivo activity of DT388-GM-CSF exotoxin A truncated form
(PE38)
patients who were resistant to cladribine, 11 had a
complete remission and 2 had a partial remission with
BL22. The three patients who did not have a response
received low doses of BL22 or had preexisting
toxin-neutralizing antibodies. Of the 11 patients in
complete remission, 2 had minimal residual disease in
the bone marrow or blood. During a median follow-up of
16 months (range, 10 to 23), 3 of the 11 patients who
had a complete response relapsed and were retreated; all
of these patients had a second complete remission. In 2
of the 16 patients, a serious but completely reversible
hemolytic-uremic
syndrome
developed during the second cycle of treatment with
BL22. Common toxic effects included transient
hypoalbuminemia and elevated aminotransferase levels.
BL22 can induce complete remissions in patients with
hairy-cell leukemia that is resistant to treatment with
purine analoguesref
and also has activity in other hemological malignancies;
however, a neutralizing Ab response to PE38 in patients with
solid tumors prevents repeated treatments to maximize the
benefit. The murine Ab response was analyzed as a model to
study the B cell epitopes associated with PE38. Sixty
distinct mAbs to PE38 were characterized. Mutual competitive
binding of the mAbs indicated the presence of 7 major
epitope groups and 13 subgroups. The competition pattern
indicated that the epitopes are discrete and could not be
reproduced using a computer simulation program that created
epitopes out of random surface residues on PE38. Using sera
from immunotoxin-treated patients, the formation of human
Abs to each of the topographical epitopes was demonstrated.
One epitope subgroup, E1a, was identified as the principal
neutralizing epitope. The location of each epitope on PE38
was determined by preparing 41 mutants of PE38 in which
bulky surface residues were mutated to either alanine or
glycine. All 7 major epitope groups and 9 of 13 epitope
subgroups were identified by 14 different mutants and these
retained high cytotoxic activity. A relatively small number
of discrete immunogenic sites are associated with PE38, most
of which can be eliminated by point mutationsref.
is a cytotoxic
ribonuclease (RNase) isolated from frog oocytes.)
:) can
eventually induce human anti-dgA antibodies (HARA). =alliinase-conjugated
anti-TAA-mAb=>
allicin :)
conjugated to ...
: immunoconjugates targeted against TAAs exhibit
tumour-specific cytotoxic effects and cause regression of
established human tumour xenografts in nude mice)-peptide
complexes in
tumors which have down-regulated its expression. derivative after
pretargeting of a streptavidin-antibody conjugate
: combinations of clinical-grade, isolated human gdT cells with the humanized anti-GD2 antibody
hu14.18 and a novel fusion cytokine, Fc-IL7. gdT cells were large-scale enriched from
leukapheresis product obtained from G-CSF–mobilized
donors. T cell cytotoxicity was tested in a europium-TDA
release assay. The effect of Fc-IL7 on gd
T-cell survival in vitro was assessed by flow cytometry.
NOD.CB17-Prkdcscid/J mice received 1 x 106 NB-1691
neuroblastoma cells via the tail vein 5 to 6 days before therapy
began. Treatment, for 5 consecutive weeks, consisted of
injections of 1 x 106 gd
T cells weekly, 1 x 106 gd
T cells weekly, and 20 µg hu14.18 antibody 4 times per week, or
1 x 106gd T cells weekly
with 20 µg hu14.18 antibody 4 times per week, and 20 µg Fc-IL7
once weekly. The natural cytotoxicity of gdT
cells to NB-1691 cells in vitro was dramatically
enhanced by hu14.18 antibody. Fc-IL7 effectively kept cultured gd T cells viable. Combination therapy
with gd T cells and hu14.18 antibody
significantly enhanced survival (P = 0.001), as did treatment
with gd T cells, hu14.18 antibody,
and Fc-IL7 (P = 0.005). Inclusion of Fc-IL7 offered an
additional survival benefit (P = 0.04)ref
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