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• Cancer Immunotherapy A potentially specific
  therapeutic approach
• Background of the immune system evolved to
  distinguish foreign pathogens from self
• Antibody and cellular receptors and what they
  recognizeinclude NK cells
• Class I and II peptide complexes are what the
  immune system sees as self and foreign
• Preventing tumors by immunizing against tumor
  viruses
• Immune effector mechanism that can kill tumors
• Experiments with mouse tumors showing tumor
  immunity can be specific and effective
• Immunosurveillance
• What is foreign in tumors that could lead to
  recognition by T cells or NK cells?
• Mutant proteins from DNA changes
• Oncogene products Ras, BCR/Abl
• Tumor Suppressors p53
• Aberrently expressed proteins
• Embryonic genes not normally expressed
• Intron-encoded antigens
• Abnormal reading frame transcripts
• Abnormal glycosylation
• Lack of class I MHC molecules (leading to NK cell
  recognition)

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Mice can be immunized against tumors
MCA

Tumor cells
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Tumor immunity in mice is mediated by T
lymphocytes
MCA
Surgicallycured mouse
control mouse
T cells
T cells
Tumor cells
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Cytotoxic T cells rapidly induce apoptotic death
in target cells in vitro
K. Hahn et al Virology 201330 (1994)
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Cytotoxic lymphocytes kill target cells in vitro
by two death pathways
Cytotoxic T Lymphocyte (CTL)
FasL mRNA
Transcription
Granule Exocytosis
FasL
Translation
TcR
FasL
Fas
Perforin Pore
Granzymes
Target Cell
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CTL recognizing a melanocyte antigen peptide can
cure melanoma in vivo
2x105 B16 cells iv
CTL iv
Kill, count pulmonary tumors
IL-2, 2x daily
Day 0 3 8
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Anti-gp100
CTL, cloned in vitro
Overwijk et al 188277 (1998)
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NK cells are a second type of cytotoxic
lymphocyte recognizing cells lacking Class I MHC
antigen
Activating Ligand
Activating Ligand
Class I MHC
Activating Receptor
Perforin, granzymes
Activating Receptor
Inhibitory Receptor
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Does the immune system provide a surveillance
system which eliminates newly-arising tumors in
normal people?
After it became clear that T cell immunity
can prevent cancer, it was proposed that
immunosurveillance was routinely preventing many
nascent tumors in normal people. However, in the
1970s immunosuppressed people and T cell
deficient nude mice were found to have no
increased tumor incidence, casting doubt on this
idea. By the 1990s it was recognized that
nude mice have potent NK cells that might protect
them against nascent tumors. Knockout mice
specifically lacking important immunological
molecules were developed and showed increased
susceptibility to develop tumors. Knockout
Defect
Increased tumor susceptibility RAG
TcR and Ig gene rearrangement
MCA-induced sarcomas
Spontaneous intestinal tumors Perforin
CTL NK cytotoxicity
MCA-induced sarcomas
Spontaneous lymphomas IFN-g NK/T
cell secretion of IFN-g MCA-induced
sarcomas Spontaneous lymphomas STAT1
Response to IFNs MCA-induced
sarcomas Misc spontaneous tumors
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Perforin contributes to anti-tumor surveillance
in normal and p53 deficient mice
Smyth, et al J.Exp.Med. 192 755 (2000)
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Because tumor immunity in mice can be transferred
with T lymphocytes, and immune defects make mice
more susceptible to tumor development, the immune
system must recognize tumors as foreign. Why not
cure cancer by exploiting the immune system?
Since the immune system classically
distinguishes foreign from self, immunotherapy
should have fewer toxic side effects than other
forms of cancer therapy.
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Strategies in cancer immunotherapy
Adoptive (passive) immunotherapy Transfer of
effector cells Sensitized ex vivo pulsed
antigen-presenting cells transduced
effectors cellular ag or restricted
epitopes Expanded ex vivo bulk
populations cloned populations Transfer of
anti-tumor mAbs to induce ADCC coupled to
toxin or isotope
Active immunotherapy (vaccines) Immunize in vivo
with Purified antigen immunodominant
peptide DNA encoding tumor Ag recombinant virus
encoding tumor Ag pulsed antigen-presenting
cells intact tumor cells or extract Adjuvant
treatments in vivo IL-2, other cytokine
proteins APC/tumor cell cytokine
pretreatment Expression of costimulators/cytokine
s
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The good news Vaccines against tumor viruses
can prevent cancer by raising anti-virus
antibodies
Feline Leukemia Virus Pet kittens are now
routinely vaccinated with heat-killed FLV or a
recombinant subunit vaccine, drastically reducing
infections by feline leukemia virus, which was
previously caused the leading fatal disease of
cats. Hepatitis B A recombinant hepatitis B
subunit vaccine is now routinely given to
children in many countries, preventing the
chronic hepatitis caused by viral infection. The
500,000-1,000,000 cases/year of hepatocellular
carcinoma arising from chronic HBV infection in
China will likely be prevented. Human Papilloma
Virus HPV-16 and HPV-18 infections are
implicated as the cause of most cervical cancers.
Vaccines made from recombinant capsid proteins
that form virus-like-particles (including at NCI
by Doug Lowys group) are currently in Phase III
clinical trials to prevent cervical cancer.
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The bad news Most human cancer is not caused
by viruses. In mouse model systems, various
tumor vaccine treatments can prevent tumors from
developing after subsequent injection of tumor
cells (prophylaxis). In most of these cases,
these treatments do not cause regression of
established tumors. Recently examples of
therapeutic vaccines for mouse tumors have begun
to appear. After gt30 year of intense efforts,
there is no approved vaccine for treating any
type of human cancer, and only a handful of
vaccine-induced tumor regressions have been
reported. Basically, vaccine-based approaches to
human cancer are still a hope.
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New approaches to cancer vaccine development
offer real hope

• Conceptual Advances
• Identification of tumor Ags and use of
  recombinant/peptide Ag in vaccines
• Recognition that tumor immunity can be a limited
  form of autoimmunity
• Understanding the function of antigen presenting
  cells
• New approaches to vaccine design
• Use of peptides optimized for binding to both MHC
  and TcR
• Multi-epitope vaccines containing both helper and
  CTL epitopes
• Co-treatment with protein cytokines
• Injection of vectors to express Ag, cytokine, and
  costimulatory proteins in vivo
• Blocking negative modulators aCTLA-4 abs and
  depleting Tregs
• Injection of in vitro Ag-pulsed and activated
  autologous dendritic cells
• Monitoring patient cellular responses to optimize
  responses

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Tumor-infiltrating lymphocytes can be isolated
from surgically resected tumors, cultured, and
their antigens identified
Mutant oncoproteins/tumor suppressors
b-catenin (melanoma) cyclin-dependent
kinase 4 (melanoma) caspase 8 (squamous
cell carcinoma) Intron-encoded proteins
gp100-in4 (melanoma) Abnormally glycosylated
MUC-1 (breast, pancreas) Embryonic proteins
MAGE, BAGE, GAGE (melanoma, breast,
glioma) Abnormally expressed HER-2/neu
kinase (breast, ovary) Normal tissue specific
differentiation antigens tyrosinase
(melanoma) gp100 (melanoma) Mart-1
(melanoma)
Foreign proteins
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(No Transcript)
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Receptors on T lymphocytes that bind foreign
peptides complexed by ubiquitously expressed
self MHC molecules when microorganisms are
inside cells
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Peptide antigens can be modified to maximize MHC
and TcR binding
Berzofsky, et al, Nature Reviews Immunology 1209
(2001)
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Published studies of HLA Class I tumor antigen
peptides used in cancer clinical trials
Cancer/testis antigens
Melanocyte differentiation antigens
Other Shared antigens
I.D. Davis et al, J. Leukocyte Biol 733 (2003)
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Peptide-expressing vaccine stimulating CTL can
lead to regression of established subcutaneous
melanoma
Vacccine recombinant Fowl Pox expressing gp100
peptide with enhanced MHC binding
Treatment Day 0 Day 0-5 None
None Vac IL-2 CTL
None CTL IL-2 CTLVac None
CTLVac IL-2
B16 melanoma cells injected subcutaneously at Day
-13
Overwijk et al J. Exp. Med. 198569 (2003)
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Autoimmunity against some tissue specific
antigens may be an acceptable approach to cancer
therapy if the tissue is not critical to life
Overwijk et al, Proc Natl Acad Sci U S A. 962982
(1999)
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Mature lymphocytes clonally express rearranged
receptors on their surface,but most
self-reactive lymphocytes die during development
Negative
High affinity receptor for self protein expressed
in thymus
Selection
Self-Ag
Development in thymus
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Vaccines can drive clonal expansion of
self-reactive T cells that escape negative
selection
Tumor cells bearing self-Ag
Vaccine-
Negative
Driven
Selection
Clonal
Expansion
Self-Ag
Development in thymus
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Immature dendritic cells capture antigen but need
to mature to become efficient antigen presenting
cells
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Making dendritic cell vaccines
IL-4 GM-CSF
Peptide
LPS Poly IC CpG oligo TNFa CD40L
TLR ligands
Mature Dendritic cells
Blood Monocytes
Immature Dendritic cells
Inject i.v.
Freeze for boosts
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Responses to peptide-pulsed dendritic cell
vaccine in melanoma patients Enhanced T cell
reactivity to melanoma antigens correlates with
clinical response
Overnight culture culture with peptide
Banchereau et al, Cancer Res 616451 (2001)
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CTLA-4 blockade powerfully enhances
vaccine-mediated tumor immunity
Treatment Day_______________

Days after SQ B16 tumor injection
B16/GMCSF Hamser IgG
Hamster IgG
B16/GMCSF aCTLA-4
aCTLA-4
Therapy 1 day after iv injection of metatastic B16
Van Elsas et al, J.Exp.Med.190355 (1999)
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Melanoma patient responses to gp100 peptide
vaccine anti-CTLA-4
Phan, et al Proc Natl Acad Sci U S A. 1008372
(2003) (NCI Surgery Branch)
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Approaches to evaluate patient responses to tumor
vaccines
Traditional in vitro functional assays
Cytotoxicity to detect CTL Ag-induced
proliferation TcR frequency in blood
MHC-peptide tetramers (flow cytometry)
PCR Ag-induced cytokine secretion assays
Bulk culture Elispot Intracellular
cytokine in permeabilized cells
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Soluble MHC-peptide tetrameric complexes can be
used to detect specific T cells
Peptide
Natural Class I MHC
Soluble Class I MHC
a1
a2
a1
a2
a3
b2M
a3
b2M
Membrane Domain
Biotin
Fluorescent MHC-peptide Tetrameric complex
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MHC-peptide tetramers can be used to follow
patient vaccine responses
Vaccine MART-1 peptide pulsed dendritic cells
Butterfield et al, Clin. Cancer Res. 9998 (2003)