ToC | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9
Immunization of HLA-A2+ Melanoma Patients in the Adjuvant Setting with a Mutated gp100 Peptide (209-2M) Induces Specific T Cell Immune ResponsesVaccination of metastatic melanoma patients
with gp100 (209-2M) peptide produced immune responses in nearly
all patients but no clinical responses unless the vaccine
was followed by high-dose IL-2. We reasoned that melanoma
patients with a low tumor burden might have a greater immune
response because of their good performance status and their
ability to receive six months of vaccinations. Thirty patients
with cutaneous melanomas at least 1 mm thick or metastatic
to regional lymph nodes, were randomized to six months of
vaccinations on an every two week (13 vaccines) or every three
week (9 vaccines) schedule. Montanide ISA 51 was mixed with
the gp 100 peptide. Peripheral blood mononuclear cells (PBMCs)
were obtained pre and post study by leukapheresis and monthly
by peripheral blood draws. PBMCs were frozen and then analyzed
freshly thawed or after in vitro culture using tetramers,
intracellular cytokine staining and ELISPOT assays.
Patient characteristics: median age, 51 years, range, 23-85
years, male/female 17/13. Seventeen patients (57%) had positive
lymph nodes, 14 patients (47%) received interferon alfa (IFN)
during their vaccines. Patient characteristics were well balanced
in both arms. Of the 25 patients analyzed to date, 22 had
significant increases in the number of gp100 tetramer positive
CD8 T cells. Overall, the median baseline percent tetramer
gp100 positive CD8 T cells was 0.08% and increased four fold
to 0.32% after vaccination, the average increased 19 fold
from 0.06 to 1.12%. Three patients had a striking response
to vaccination with 3%, 5%, and 11% respectively, tetramer
positive CD8 T cells after vaccination. The group of patients
that received vaccinations every two weeks had a higher median
of gp 100 tetramer positive CD8 T cells, 0.51% vs 0.26%. Patients
who received IFN during vaccination had no impairment of the
ability to generate a specific T cell response.
BACKGROUND: Single epitope immunistaion
is limited in efficacy due to escape by mutation and modulation.
Furthermore, many tumour antigens have unknown or transforming
activities. Nucleic acid vaccines overcome some of the difficulties
with peptide vaccines such as peptide-clearance and conformational
differences between exogenous peptide and endogenously processed
antigens. The polyepitope approach to immunisation represents
a means to overcome these problems yet elicit cytotoxic T-cell
(CTL) responses against a wide range of target antigens and
epitopes.
OBJECTIVE: To examine the CTL responses to a melanoma
polyepitope using human T-cells and dendritic cells (DC) from
naïve donors.
METHODS: A melanoma polyepitope was generated which
encoded 6 HLA-A2-restricted epitopes from human tumour antigens.
Human dendritic cells were transduced in vitro with melanoma
polyepitope cDNA and autologous T-cells from naïve donors
were stimulated. CTL were restimulated either with polyepitope
or with multiple peptides before assay for specific activity.
For comparison A2Kb transgenic mice were immunised with melanoma
polyepitope cDNA and CTL responses determined.
RESULTS: Polyepitope transduced human DC simultaneously
primed up to 6 HLA-A2 CTL responses. However, repeated exposure
of DC-primed CTL to polyepitope resulted in immunodominance
to a sole epitope (MART127-35). This did not correlate with
precursor frequency. Furthermore, DC-polyepitope only restimulated
MART-reactive CTL lines but not CTL lines to gp100, tyrosinase
or MAGE3. In contrast to the human system, repeated immunisation
of A2Kb mice with polyepitope DNA resulted in multiple CTL
responses.
DISCUSSION: Polyepitopes are a promising approach to
cancer vaccines. However, repeated stimulation of human T-cells
with a melanoma polyepitope results in immunodominance. A
number of possible explanations exist however, our data demonstrates
that this immunodominance may relate to preferential release
of MART1 epitope by the antigen processing machinery. Studies
of this kind using human T-cells may offer more informative
pre-clinical information than some animal models. This may
reflect differences in T-cell repertoire and in the antigen-processing
machinery.
Introduction: There is considerable controversy
as to whether necrotic (lysate) or apoptotic tumor cells (Apo)
serve as the superior source of multiple tumor-associated
antigens (TAAs) to pulse dendritic cells (DCs) for immunotherapeutic
applications. Here, we used highly enriched apoptotic cells
vs. lysate of B16 melanoma to examine whether important distinctions
exist between these two forms of TAAs.
Methods: Apo melanoma cells generated by UVB light were highly
enriched by FACS sorting. Lysate was generated by four cycles
of rapid freeze/thaw. Levels of apoptotic and necrotic cells
were determined by the Annexin V-PI FACS assay and their content
of heat shock proteins (HSPs) was examined by immunoblotting.
DCs and killed tumor cells were co-cultured and analyzed for
uptake by two-color FACS analysis, electron microscopy, and
confocal microscopy. Also, IL-12 production by and changes
in phenotypic markers as a measure of maturation of the tumor-loaded
DCs was determined by ELISA and FACS, respectively. Apo- and
lysate-pulsed DCs were compared for immune priming and antitumor
therapeutic efficacy in vivo. For immune priming, naive B6
mice were immunized s.c. twice at 7 day intervals and were
then challenged with a lethal dose of viable B16 melanoma
cells. For therapy, B6 mice received a lethal dose of viable
melanoma cells 7 days before immunization. These mice were
then immunized s.c. with either Apo- or lysate-pulsed DCs
twice, 7 days apart. Tumor growth was measured.
Results: After FACS sorting, UVB-irradiated B16 melanoma cells
were about 90% apoptotic. The lysate contained essentially
100% necrotic cells. DCs could efficiently engulf both forms
of killed tumor cells within 18 hr. For IL-12 production,
DCs that had engulfed sorted Apo produced 528+/- 20 pg/ml
vs. 255 +/- 65 pg/ml when exposed to lysate. DCs pulsed with
either Apo or lysate could equally promote immune priming
and inhibit tumor growth in vivo.
Conclusions: Although some differences existed in HSP content
and IL-12 production with the two forms of TAAs, their respective
capacities to mature DCs phenotypically and to elicit both
effective immune priming and antitumor therapy in vivo when
presented by DCs were equivalent.
The industrialization of tools for rapid DNA sequencing and global gene expression analysis has been a major technological scientific advancement that is being applied from target discovery to surveying of clinical populations (Pharmacogenomics). The ultimate goal is to improve the efficiency of drug development by prioritization of lead compounds to the clinic with a decrease in attrition due to lack of efficacy and unforeseen toxicity. This presentation will focus on specific case-study examples providing experimental support to the utility of genomics to from target discovery, lead prioritization and association of genetic polymorphisms with disease.
Directed molecular evolution by DNA shuffling
followed by screening is a technology that allows for rapid
evolution of genes and genomes in vitro. DNA shuffling of
natural diversity is used to create large libraries of chimeric
sequences of genes of interest. The libraries are subjected
to a multi-tiered high throughput screening process using
cell-based robotic assays, FACS selection and/or in vivo assays.
If desired, a pool of the best sequences from one round of
screening is shuffled and rescreened to obtain further optimization
and the best hits are subjected to detailed biological characterization.
We have used DNA shuffling and screening to generate libraries
of vaccine antigens, cytokines and viruses. We have generated
libraries of chimeric antigens that provide crossreactive
immune responses in contrast to the parental antigens. In
addition, we have used mammalian cDNAs encoding cytokine genes
as the starting sequences to generate cytokine libraries and
several chimeras with improved expression and specific activity
have been identified. We have also evolved immunomodulatory
molecules with altered ligand binding specificities, and hence,
with altered biological properties. Moreover, DNA shuffling
and screening of MLV viruses resulted in variants with improved
resistance to ultracentrifugation. Directed molecular evolution
by DNA shuffling and screening is a powerful platform technology
to generate novel and improved vaccines and immunotherapeutics.
The LifeSeq® Gold database is the best available resource for the preliminary identification of genes associated with disease processes. The selection of potential antibody targets, drug targets, diagnostics, and therapeutic proteins for further investigation can be facilitated by additional complementary knowledge such as protein structure analysis and gene expression network information obtained by microarray analysis. We will describe the process by which all the potential targets can be rapidly characterized at multiple levels, stratified, and functionally assessed, by incorporating specialized and integrated technological and analysis capabilities.
