Patrick Hwu, M.D.

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Introduction to Cancer Vaccines and Cancer
Immunotherapy
Patrick Hwu, M.D.
Professor Chairman, Melanoma Medical
OncologyAssociate Director, Center for Cancer
Immunology Research (CCIR)
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Antibody-based therapies
Antigen
Tumor cell
Antibody
Toxin Radioisotope Immune cells
3
The cellular immunity team
CTL
Tumor Cell
CytotoxicT Lymphocyte
T Helper
T helper
T Helper
Antigen
IL-2
DendriticCell
Dendritic Cell
4
Cytotoxic T-lymphocytes can recognize and kill
tumor cells
(From UVA)
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Cellular immunity

• Viral infections
• Cancer
• Organ rejection

6
Virus activates innate immunity
T-cell Activation
Virus
and Proliferation
Activation of Innate Immunity
Inflammation at Site
mDC
of Viral Infection
Migration, Effector Function, andViral
Elimination
pDC
NK Cell
B Cell
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Tumor cells fail to activate innate immunity
Suboptimal T-cell Activation
Tumor
and Proliferation
No Activation of Innate Immunity
No Inflammationat Tumor Site
mDC
Limited Migration and Effector Function
pDC
NK Cell
B Cell
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Plasmacytoid dendritic cells play a key role in
both innate and adaptive immunity
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Adjuvant therapy with high-dose IFN-?2b
Relapse-Free Survival
Overall Survival
100
100
80
80
60
60
IFN-?2b
IFN-?2b
Probability
Probability
40
40
Observation
Observation
20
20
0
0
0
2
4
6
8
10
0
2
4
6
8
10
Years
Years
P1 0.002
P1 0.02
ECOG 1684, Kirkwood et al. J Clin Oncol. 1996
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How does Interferon work?
Immune Cell Activation
IFN
mDC
T-cells
Antivascular Effect
Tumor Inhibition
NK
Tumor Cells
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Development of autoimmunity correlates with
survival in melanoma patients treated with
interferon alpha
Source Gogas et al N Engl Med, February 2006
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How is IFN working to enhance immunity?
DC Antigen Presentation and Activation
IFN
Upregulation ofClass I on Tumor Cells
mDC
T-Cell Costimulation
Tumor Cells
CD8 T-cell
CD4 T-cell
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Virus activates innate immunity
T-cell Activation
Virus
and Proliferation
Activation of Innate Immunity
Inflammation at Site
mDC
of Viral Infection
Migration, Effector Function, andViral
Elimination
pDC
NK Cell
B Cell
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The cellular immunity team
CTL
Tumor Cell
CytotoxicT Lymphocyte
T Helper
T helper
T Helper
Antigen
IL-2
DendriticCell
Dendritic Cell
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Metastatic melanoma treated with IL-2
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Metastatic melanoma treated with IL-2
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IL-2 therapy is effective in some patients with
metastatic melanoma
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Responses are Durable Following Treatment with
High Dose IL-2
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Cytokine therapy

• The IL-2 experience demonstrates that it is
  possible to obtain dramatic clinical results in
  some patients by activating the immune system.
• Challenge Response rates are low and toxicity is
  high.
• Plan
• Understand the molecular nature of responders vs.
  non-responders.
• Develop immune therapies with improved
  effectiveness and less toxicity.

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The cellular immunity team
CTL
Tumor Cell
CytotoxicT Lymphocyte
T Helper
T helper
T Helper
Antigen
IL-2
DendriticCell
Dendritic Cell
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Vaccine therapy

• Whole Tumor Cell Vaccines
• Antigen-Specific Vaccines
• Peptide/protein adjuvant
• Recombinant Viral Vaccines
• DNA vaccines

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T-cells can recognize intracellular peptides
presented by MHC molecules
EndoplasmicReticulum
Peptide
MHC-I
TCR
MHC-I
CD8 T Cell
CD8
EndogenousAntigen
Nucleus
Cancer J Sci Am, 1995
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T-cells can recognize intracellular proteins
presented on the cell surface in the cleft of
HLA molecules

• Differentiation antigens
• Mutated proteins
• Oncogene products
• Abnormal signaling proteins

The immune system can take advantage of the
large amounts of molecular information we have
in the post-genomic era.
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Tools for the development of immune-based
therapies Melanoma tumor antigens recognized by
T-cells

• Melanocyte lineage proteins
• gp100, MART-1/MelanA, TRP-1 (gp75), TRP-2,
  Tyrosinase
• Tumor-specific, widely shared
• MAGE-13, BAGE, GAGE-12, p15
• Tumor-specific mutated antigens
• ?-catenin, MUM-1, CDK4

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• Candidate tumor antigens for common cancers
• Antigen over expressed in cancers of the
• Her-2/neu breast, ovary, bladder, colon,
  lung-NSC
• SSX-2 melanoma, prostate, headneck, lymphoma,
  bladder
• PSA prostate
• PSCA prostate ?metastases
• NY-ESO-1 melanoma, prostate, breast, bladder,
  ovary
• PTH-rP breast, lung-spindle cell, prostate
  ?metastases
• CEA colon/rectum, stomach, pancreas, lung-NSC,
  breast
• adipophilin melanoma, kidney
• SART-3 breast, colon/rectum, pancreas, many
  others
• EphA2 melanoma, breast, prostate, colon/rectum,
  esophageal, lung
• Flu-M1 influenza, positive control
• MART-1 melanoma, positive control
• proteinase-3 CML/AML, positive control

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Vaccine trials

• Adenoviral vaccines (MART, GP100)
• Pox virus (MART, GP100, Tyrosinase)
• DNA (GP100)
• Peptide vaccines (MART, GP100, Tyr, ESO, TRP-2)
• Peptide anti-CTLA-4
• Dendritic cells (pulsed with MART, GP100 peptides)

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Peptide immunizations for melanoma patients in
adjuvant setting

• Eligibility
• Primary gt1.5 mm
• Positive lymph nodes
• Resected metastases
• Treatment I mg peptide in IFA s.c.
• gp100209-217(210M)
• tyrosinase 368-376
  (370D)
• Schedule Randomized to one of three
  arms
• weekly (x10) followed by three week break (40
  cycles)
• every three weeks (x4)
  (16 cycles)
• daily (x4) every three weeks (x4)
  (64 cycles)
• Pheresis at 12 weeks (after each course)
• Plan total of four courses
  (48 weeks)

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Immunologic response ELISPOT Assay
To evaluate the frequency of T cells reactive
against gp100209-217
3
2nd anti-IFN-?-Ab
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Immunologic responseTetramer assay
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Active immunization of patients with metastatic
melanoma
Objective Vaccine Total Response
Recombinant viruses (number of
patients) Adenovirus (MART-1 or
gp100) 24 1 Vaccinia (MART-1 or
gp100) 21 0 Fowlpox (MART-1 or
gp100) 48 1 Fowlpox
(ESgp100209-2M) 36 0 Vaccinia
Fowlpox (tyrosinase) 13 0 Naked
DNA 23 1 Dendritic cells (IV peptide
pulsed) 10 1 Peptides MART-1 23
1 gp100 (154, 209, 280) 28
1 gp100209-2M 79 0 Her-2/neu 7
0 gp100ES-209-2M 9 0 Non A2
peptides (A1, A3, A24, A31, Cw7) 65
2 NY-ESO-1 33 0 Class I II
gp100 27 2 Telomerase 14
0 TRP-2 21 0 MART-1 gp100
(multiple) 58 2 gp100 MART
Flt3L 31 0 alone or with GMCSF or
IL-12 Total 570
12 (2.1)
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Afferent phase of immune response
T-Cell Activation and Proliferation
Dendritic Cell
T Cell
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Efferent phase of immune response
Leukocyte
Rolling
Capture
SelectinLigand
Chemokine Receptor
Inflammation
Endothelial Activation
Chemokine Synthesis
Endothelial Cell
Extravasation
P-selectin
?L ?2 Integrin
ICAM-1
Leukocyte Adhesion
(integrin bindsto ICAM-1)
Adhesion
Chemokines
Effector Function
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Strategies to improve anti-tumor immune responses

• Afferent phase
• Enhance thymic activity
• Stimulate CD4 T-cells
• Efferent phase
• Improve migration of T-cells to tumor and
  function of T-cells at the tumor site.
• Decrease regulatory elements
• Adoptive cell transfer (ACT) with activated
  T-cells

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The cellular immunity team
CTL
Tumor Cell
CytotoxicT Lymphocyte
T Helper
T helper
T Helper
Antigen
IL-2
DendriticCell
Dendritic Cell
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Mediators of immune suppression

• Regulatory Immune Cells
• Soluble Factors
• Inhibitory Receptors on activated NK and T-cells

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Immune cells implicated in suppression of
antitumor immunity
Cell Type
Effector Functions
Reference
Sakaguchi,


Inhibition of CD4
and CD8
T-cell proliferation via direct
T-reg (CD4CD25)
Nature Immunology
cell-to-cell interactions (involving CTLA-4,
GITR?)
2005
Levings et al.,
Suppression of naïve and memory T-cell responses

Tr1 (CD4CD25-)
J Experimental Medicine

?
through production of high levels of IL-10 and
TGF-
2002
Grabilovich,

Inhibition of IFN-?
production by CD8
T cells
Immature myeloid
Nature Rev Immunology
mediated by reactive oxygen species (eg H2O2)
2004
Wilson and Delovitch,
Cytokine release (diverse Th1 and Th2)

Invariant NKT
Nature Rev Immunology
May prevent
or
activate antitumor immunity
2003
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Inhibition of CD8 response after addition of
class II restricted peptide
Source Phan GQ et al. Journal of
Immunotherapy, July/August 2003, 26(4)349-356
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Mediators of immune suppression

• Regulatory Immune Cells
• Soluble Factors
• Inhibitory Receptors on activated NK and T-cells

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Potentially targetable immunoregulatory molecules
Molecule
Cellular expression
Mechanism of action
Provides co-inhibitory signaling
Helper T, Cytoxic T
during naïve T-cell priming
CTLA-4
Membrane bound
Induces local tryptophan metabolism
T-reg
by DCs, inhibiting T-cell proliferation
Inhibits T-cell proliferation, cytokine
Helper T, Cytoxic T
PD-1
production and cytotoxicity
Regulates growth and differentiation of
Tumor, TR1
IL-10
a wide variety of immune cells
Induces immature myeloid cells
iNKT
IL-13
to produce TGF-b
Tumor, TR1,
Directly suppresses proliferation
TGF-b
Treg, Immature myeloid
of antigen-activated T cells
Soluble
Blocks DC differentiation and maturation,

Tumor
VEGF
leading to accumulation of iDC and iMC

Depletes local tryptophan,
Tumor, Dendritic
IDO
inhibiting T-cell proliferation
Depletes local arginine, inhibiting

Tumor, Immature myeloid
ARG1
CD3z expression and T-cell activation
Generates nitric oxide, inhibiting T-cell
Tumor, Immature myeloid
priming, proliferation, and cytotoxicity
iNOS
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Source Hwu P et al. The Journal of Immunology
1643596-3599, 2000
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Inhibition of IDO by 1-MT results in enhanced
T-cell proliferation
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IDO inhibition can enhance antitumor immune
response
Uyttenhove et alNature Medicine  9, 1269 1274,
2003
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Potentially targetable immunoregulatory molecules
Molecule
Cellular expression
Mechanism of action
Provides co-inhibitory signaling
Helper T, Cytoxic T
during naïve T-cell priming
CTLA-4
Membrane bound
Induces local tryptophan metabolism
T-reg
by DCs, inhibiting T-cell proliferation
Inhibits T-cell proliferation, cytokine
Helper T, Cytoxic T
PD-1
production and cytotoxicity
Regulates growth and differentiation of
Tumor, TR1
IL-10
a wide variety of immune cells
Induces immature myeloid cells
iNKT
IL-13
to produce TGF-b
Tumor, TR1,
Directly suppresses proliferation
TGF-b
Treg, Immature myeloid
of antigen-activated T cells
Soluble
Blocks DC differentiation and maturation,

Tumor
VEGF
leading to accumulation of iDC and iMC

Depletes local tryptophan,
Tumor, Dendritic
IDO
inhibiting T-cell proliferation
Depletes local arginine, inhibiting

Tumor, Immature myeloid
ARG1
CD3z expression and T-cell activation
Generates nitric oxide, inhibiting T-cell
Tumor, Immature myeloid
priming, proliferation, and cytotoxicity
iNOS
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Antibodyrecognition
MHC class Irecognition
B7 family memberrecognition
ClassicalMHC class I
HLA-EQa-1
IgGFc
B7-1B7-2
B7-H1B7-DC
B7-H4
KIR
CD94-NKG2A/B
FC?RIIB
Ly49
CTLA-4
PD-1
BTLA
ITIM
SHP-2
SHP1/2
SHP-1
SHP-1/2
SHIP
? SHP-2
SHP-1/2
NK cells,some T cells(human)
NK cells,some T cells(murine)
NK cells,some T cells(human and murine)
B cells,myeloid cells,mast cells
ActivatedT cells
ActivatedT cells, B cells, myeloid
ActivatedT cells, B cells
Source Leibson PJ. Current Opinion in
Immunology 16328-336, 2004
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Question

• If CTLA-4 engagement by B7 induces T-cell
  suppression and tolerance and prevents
  CD28-mediated T-cell activation
• Would administration of anti-CTLA-4 antibodies
  (Ab) to patients simultaneously receiving
  anti-tumor vaccination

• increase tumor immunization
• induce tumor regression

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Response to anti-CTLA4
Attia et al, JCO 23, 2005Phan et al, PNAS 100,
2003Ribas et al, JCO 23, 2005
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Anti-CTLA-4 vaccine can induce tumor regression
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Autoimmune toxicities of anti-CTLA4Grade II/IV
toxicities
Colitis8 (14)
Dermatitis4 (7)
Hepatitis1 (2)
Uveitis1 (2)
Hypophysitis1 (2)
Attia et al, JCO 23, 2005Phan et al, PNAS 100,
2003
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Correlation of autoimmunity with response to
anti-CTLA-4
P0.008
Source Attia et al JCO 23, 2005
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Immune regulation

• The immune system is highly regulated with
  multiple redundant systems, which may inhibit the
  generation of a successful antitumor immune
  response.

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Overcoming immune regulation Current challenges

• Improved methods are needed to measure and
  quantify regulatory immune cells.
• Methods need to be developed to determine the
  rate-limiting inhibitory pathways for particular
  tumors.
• Clinical-grade reagents are needed to block
  inhibitory pathways.

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Strategies to improve anti-tumor immune responses

• Afferent phase
• Enhance thymic activity
• Stimulate CD4 T-cells
• Efferent phase
• Improve migration of T-cells to tumor and
  function of T-cells at the tumor site
• Decrease regulatory elements
• Adoptive cell transfer (ACT) with activated
  T-cells

58
Adoptive cell therapy (ACT) with antigen
specific T-cells
Surgical Removal of Cancer Nodule
Single Cell Suspension Incubated with IL-2
T CellsProliferate
IL-2
T Cells
Cancer CellsDie
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Making space and eliminating regulatory cells
with chemotherapy
Normal Lymphocytes
Tumor Reactive Cytotoxic T-Lymphocytes
Chemotherapy
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Infused T-lymphocytes persist when administered
following lymphodepletion with chemotherapy
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Clinical response following lymphodepletion
T-lymphocyte infusion
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Clinical response following lymphodepletion
T-lymphocyte infusion
Day -108
Day -45
Day -25
Day 34
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Clinical response following lymphodepletion
T-lymphocyte infusion
Science. 298(5594), Oct 25, 2002Dudley et al, J
Clin Oncol 23(10)2346-57, 2005
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The cellular immunity team
CTL
Tumor Cell
CytotoxicT Lymphocyte
T Helper
T helper
T Helper
Antigen
IL-2
DendriticCell
Dendritic Cell
65
Summary
The cellular immune system can be activated to
induce significant tumor regression in some
patients. GoalsIncrease response rates in
melanoma. - Improve afferent immune response -
Optimize lymphocyte migration and function at
the tumor site Apply principles learned from
melanoma to other cancers.