Old problems, new directions Eli Gilboa

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Old problems, new directionsEli Gilboa

• ESMO International Symposium on Immunology
• Nov 15-17, 2007
• Athens, Greece

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Cancer immunotherapy with mRNA transfected
dendritic cellsA personalized form of cell
therapy
Antigen Loading (mRNA transfection)
GM-CSF, IL-4
Immature DC
Monocytes
DC maturation
IL-1b, IL-6, TNF-a, PGE2
Tumor antigens (mRNA)
Antigen-Loaded Mature DC
Cryopreserved DC Vaccine
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The underlying premise of developing
patient-specific vaccination protocols
Added complexity and cost associated with such
interventions will be offset by a substantial
added benefit to the patient.
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Promising vaccination strategies

• DNA vaccines
• Poxvirus vector-based vaccines
• GM-CSF transduced tumor vaccines
• Idiotype GM-CSF
• gp96-secreting tumor vaccines
• Dendritic cell vaccines
• Listeria vector-based vaccines

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Promising vaccination strategies

• DNA vaccines
• Poxvirus vector-based vaccines
• GM-CSF transduced tumor vaccines
• Idiotype GM-CSF
• gp96-secreting tumor vaccines
• Dendritic cell vaccines
• Listeria vector-based vaccines
• NDV-infected autologous tumor vaccines
  (Schirrmacher colleagues)

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Immunological control of cancer

• Where are we going from here

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Promising vaccination strategies

• DNA vaccines
• Poxvirus vector-based vaccines
• GM-CSF transduced tumor vaccines
• Idiotype GM-CSF
• gp96-secreting tumor vaccines
• Dendritic cell vaccines
• Listeria vector-based vaccines
• NDV-infected autologous tumor vaccines
  (Schirrmacher colleagues)

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Specific Active Immunotherapy of CancerCancer
Vaccines
To engender protective immunity in the cancer
patientthat will negatively impact on tumor
progression.
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A multi pronged approach to cancer immunotherapy
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A multi pronged approach to cancer immunotherapy
Immune suppression
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Why is a tumor growing in an immune competent
patient not eliminated by an immune response?
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Why is a tumor growing in an immune competent
patient not eliminated by an immune response?

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Why is a tumor growing in an immune competent
patient not eliminated by an immune response?
Lack of immunogenicity

The immune system is not activatedin response to
the growing tumor - tumor is not sufficiently
distinct from normal tissue
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Why is a tumor growing in an immune competent
patient not eliminated by an immune response?

Immune suppression

Tumors activate mechanisms which suppress the
differentiation and/or function of an otherwise
effective antitumor response
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Tumor-induced immune suppression
Immune suppressive Cell types
Immune suppressive products
Cox-2 generated prostanoids B7H1 TGF? IL-10 Decoy
receptor 3 (DcR3) STAT3 cAMP Adenosine VEGF Indole
amine deoxygenase (IDO) and more
Regulatory T cells IL-13 secreting NKT
cells Immature myeloid cells (ImC) Tolergenic DC
(iDC, pDC) Alternatively activated M?
(AAMs) and other
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...and more evidence

• Immune mediated tumor rejection in the absence of
  vaccination, e.g., blocking TGF? signaling in T
  cells (Gorelik Flavell, Nat. Med. 2001, 71118)
• Tumor induced immune suppression, not lack of
  inherent immunogenicity, the main reason for
  tumor outgowth - in a highly relevant spontaneous
  tumor model where tumors are heterogenous,
  multifocal exhibiting different biologies. (G.
  Willimsky T. Blankenstein. Nature, 2005, 437,
  141-146 )
• Vigorous premalignancy-specific effector T cell
  response in the bone marrow of patients with
  monoclonal gammopathy. Dhodapkar et al., Exp Med,
  2003, 1981753
• Inverse correlation between tumor progression in
  ovarian and colorectal cancer patients and immune
  infiltrate (Zhang et al., N. Engl. J. Med., 2003,
  348203 Galon et al., Science, 2006, 3131960)
• Immune-mediated control of subclinical cancer in
  murine models (Schreiber, Smyth and colleagues,
  CRI meeting, Manhattan, NYC, October 2006)
• Inherent immunogenicity of human cancer
  Frequent induction of immune responses which are
  occasionally associated with better prognosis but
  ultimately fail to reverse disease course
  (reviewed by Hodi Dranoff, Adv. Immunol., 2006,
  90341)

Cancer despite immunosurveillance
immunoselection and immunosubversion. Zitvogel,
L., Tesniere, A. and G. Kroemer. Can. Rev.,
Immunol., 2006, 6715
The seventh hallmark of cancer
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Tumor-induced immune suppression
Immune suppressive Cell types
Immune suppressive products
Regulatory T cells IL-13 secreting NKT
cells Immature myeloid cells (ImC) Tolergenic DC
(iDC, pDC) Alternatively activated M?
(AAMs) and other
Cox-2 generated prostanoids B7H1 TGF? IL-10 Decoy
receptor 3 (DcR3) STAT3 cAMP Adenosine VEGF Indole
amine deoxygenase (IDO) and more
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Naturally occuring CD4CD25 regulatory T cells

• A distinct lineage of thymic origin, comprise
  3-10 of the CD4 T cell population
• Immune suppressive - inhibit CD4 CD8 T cell
  responses
• Function - Preventing autoimmunity by keeping
  autoreactive T cells in check.
• Depletion of Treg in mice with ?CD25 antibodies
• Induces or exacerbates autoimmune pathology
• Potentiates tumor immunity, especially in
  conjunction with vaccination.

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Elimination of Treg using a diphteria toxin-IL-2
conjugate (ONTAK) in RCC patients vaccinated
with tumor RNA transfected DC
Dannull et al., J. Clin. Invest., 2005, 1153623
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Limitation to targeting CD25 for Treg depletion

• CD25, a component of the IL-2 receptor complex,
  is also upregulated on conventional activated
  (vaccine-induced) T cells.
• Treg rebound with time
• The tumor and the vaccination itself can generate
  Treg
• Interfere with an ongoing protective immune
  response against subclinical levels of pathogenic
  infection
• A significant fraction of Treg (10-30),
  especially recently activated Treg, have
  downregulated CD25.
• Depletion is global - risk of autoimmune
  pathology
• CD25 depletion constitutes an additional
  intervention and a reagent not always readily
  available for clinical testing.

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Other Treg-specific markers

• GITR, Lag-3, CTLA-4, CD103 - expressed on the
  cell surface but, like CD25, not specific.
• Foxp3
• Member of the forkhead/wing-helix family of
  transcription factor repressors
• Expression exclusively restricted to Treg
• Master regulator of suppressive phenotype
• CD4CD25foxp3 as well as CD4CD25-foxp3 Treg.

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Foxp3 is a nuclear protein - cannot use
antibodies or ONTAK-like reagents for depletion
of foxp3 expressing cells in vivo
No additional procedure Co-vaccination against
tumor antigen and foxp3
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Immunization (1x) against Foxp3 enhances
antitumor immunity in B16 melanoma tumor-bearing
mice
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Repeated depletion of Treg subsequent to tumor
vaccinationFoxp3 immunotherapy versus ?CD25 Ab
depletion
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Fate of foxp3-expressing cells in mice vaccinated
against foxp3 or treated with ?CD25 Ab Tumor
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Fate of foxp3-expressing cells in mice vaccinated
against foxp3 or treated with ?CD25 Ab Periphery
A. ?CD25
Lymph node
Spleen
B. Foxp3
Nair et al. Can. Res., 2007, 67371
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Why is a tumor growing in an immune competent
patient not eliminated by an immune response?

Immune suppression

Tumors activate mechanisms which suppress the
differentiation and/or function of an otherwise
effective antitumor response
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Tumor-induced immune suppression
Immune suppressive Cell types
Immune suppressive products
Regulatory T cells IL-13 secreting NKT
cells Myeloid derived suppressor cells
(MDSC) Tolergenic DC (iDC, pDC) Alternatively
activated M? (AAMs) and other
Cox-2 generated prostanoids B7H1 TGF? IL-10 Decoy
receptor 3 (DcR3) VEGF STAT3 cAMP Adenosine Indole
amine deoxygenase (IDO) and more
???
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A multi pronged approach to cancer immunotherapy
Persistence of immunity

Immune suppression
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4-1BB
Costimulatory receptors on T cells

• Upregulated on antigen-activated T cells
• Enhances survival and proliferation of activated
  CD8 T cells

• CD28
• CD27
• OX40
• 4-1BB
• CTLA-4
• PD-1
• HVEM
• CD30

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Manipulating costimulation

• Ectopic expression of ligands in APC or tumor
  cells
• Systemic administration of agonistic or blocking
  antibodies
• Clinical trials CTLA-4, PD-1, 4-1BB, OX-40, CD40.

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Agonistic 4-1BB antibodies enhance proliferation
of activated CD8 T cells and potentiate tumor
immunity in mice
Melero, I., W.W. Shuford, S.A. Newby, A. Aruffo,
J.A. Ledbetter, K.E. Hellstrom, R.S. Mittler,
and L. Chen. 1997, Nat Med 3682-685.
Limitations to use of antibodies (or
protein-based ligands) as therapeutic reagents
Antibodies are cell based products

• Complexity cost of development
• Regulatory approval process
• Cost of manufacturing
• Limited uncertain access (companies)

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Antibody combinations - synergistic antitumor
effectsMurine studies
Antibodies
Reference
4-1BB CTLA-4 Kocak, et al., 2006, Cancer Res
6672764 4-1BB OX40 Lee et al., 2004, J.
Immunol., 1733002 4-1BB B7H1 Hirano et al.,
Can. Res., 2005, 651089 4-1BB CD40 DR5 Uno
et al., Nat. Med., 2006, 12693
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Antibody combinations - synergistic antitumor
effectsMurine studies
Antibodies
Reference
4-1BB CTLA-4 Kocak, et al., 2006, Cancer Res
6672764 4-1BB OX40 Lee et al., 2004, J.
Immunol., 1733002 4-1BB B7H1 Hirano et al.,
Can. Res., 2005, 651089 4-1BB CD40 DR5 Uno
et al., Nat. Med., 2006, 12693
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Eradication of established 4T1 breast carcinoma
tumors in mice by combination therapy with
?DR5?CD40?CD137
Uno et al., Nat. Med., 2006, 12693
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Antibody combinations - synergistic antitumor
effectsMurine studies
Antibodies
Reference
4-1BB CTLA-4 Kocak, et al., 2006, Cancer Res
6672764 4-1BB OX40 Lee et al., 2004, J.
Immunol., 1733002 4-1BB B7H1 Hirano et al.,
Can. Res., 2005, 651089 4-1BB CD40 DR5 Uno
et al., Nat. Med., 2006, 12693
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In vitro selection of oligonucleotide aptamers
In vitro selection (SELEX)
Aptamer Library
440 possible sequences
AGGACGAUGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAGAC
GACUCGC
40 nucleotide random region
An Aptamer Library A Vast Shape Library
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Advantages of aptamers vs antibodies as
therapeutics

• Specificity avidity comparable or better than
  antibodies
• Synthesized chemically they are not cell-based
  products.
• Vastly simpler regulatory approval process
• Development manufacturing - cost effective
• Superior pharmacology - tumor penetration
• Lack of immunogenicity
• Amenable to chemical modification

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Aptamers can be made to most any target
Target Protein Affinity(Kd) Function Ref. PDGF
0.1nM Inhibitor Green et al.,
1996 P-Selectin 0.03nM Inhibitor Jenison et
al., 1998 Complement C5 0.03nM Inhibitor Bie
secker et al., 1999 MAb to AChR 60nM
Inhibitor Lee and Sullenger,
1997 Interferon-Gamma 2.7nM Inhibitor Kubi
k et al., 1997 VEGF 0.15 Inhibitor Ruckman
et al., 1998 Factor VIIa 11nM
Inhibitor Rusconi et al., 2000 mCTLA-4
30nM Inhibitor Santulli-Marotto,
et al. 2003
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Isolation of aptamers which bind to murine 4-1BB
in solution
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4-1BB in vitro costimulation assay Enhancement
of proliferation or IFN? release from
suboptimally activated CD8 T cells
Isolate CD8 T cells from BALB/C
mice. Incubate o/n with sub-optimal
concentration of anti-CD3. Add anti-4-1BB
antibody or aptamers coupled to
beads. Proliferation assay 48 hrs later (CFSE
dilution)and/or IFN? release
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4-1BB signaling requires ligand-induced receptor
dimerization
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Sequence and computer-predicted secondary
structure of a 4-1BB binding aptamer
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Generating aptamer dimers using complementary 3
extensions
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4-1BB aptamer dimer binds specifically to
activated CD8 T cells
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Binding of monomeric and dimeric forms of 4-1BB
aptamers to 4-1BB expressed on the cell surface
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4-1BB Aptamer Dimers Costimulate T Cells (CFSE
proliferation assay)
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4-1BB Aptamer Dimers Costimulate T Cells (IFN?
production)
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Rejection of P815 mastocytoma tumors injected
with 4-1BB aptamer dimers
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Summary

• High affinity 4-1BB binding aptamers can be
  isolated
• A subset of which - when multimerized - can
  function as 4-1BB agonists
• A low-tech clinically applicable approach

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The potential of aptamer technology to manipulate
immunity

• Ligands as well as targeting agents for drugs
  (siRNAs)
• Feasibility
• Replace antibodies and expand therapeutic
  applications?

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Concluding thoughts
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or
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Yesterday
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Today
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Mounting evidence that tumors in cancer patients
are capable of stimulating, transiently,
protective immunity

• Frequent induction of immune responses in cancer
  patients. (reviewed by Hodi Dranoff, Adv.
  Immunol., 2006, 90341)
• Correlation between lack of tumor progression in
  cancer patients and immune infiltrates.
• Ovarian cancer Zhang, L., et al. Intratumoral T
  cells, recurrence, and survival in epithelial
  ovarian cancer. N Engl J Med, 2003, 348203
• Colorectal cancer Galon, J., et al.Type,
  density, and location of immune cells within
  human colorectaltumors predict clinical outcome.
  Science, 2006, 3131960

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Tomorrow
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And then...

• Overcoming tumor-induced immune suppression
• Delivering co-stimulatory ligands to the tumor
• Promoting immunogenic death of the tumor
• Enhancing the antigenicity of the tumor

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Duke Cancer Immunotherapy Program
Center for Translational Research, Duke
University Medical Center
Eli Gilboa
Johannes Vieweg
4-1BB aptamers James McNamara Despina
Kolonias Fernando Pastor CollaborationPaloma
Giangrande Bruce Sullenger Lieping Chen Robert
Mittler
Foxp3 vaccination Smita Nair David
Boczkowski Martin Fassnacht
Jenz Dannull Zhen Su Philip Dahm Doris Coleman
Sylvia Sichi Benjamin Yang Melinda Malready Donna
Yancey
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Duke Cancer Immunotherapy Program
Center for Translational Research, Duke
University Medical Center
Eli Gilboa
Johannes Vieweg
4-1BB aptamers James McNamara Despina
Kolonias Fernando Pastor CollaborationPaloma
Giangrande Bruce Sullenger Lieping Chen Robert
Mittler
Foxp3 vaccination Smita Nair David
Boczkowski Martin Fassnacht
Jenz Dannull Zhen Su Philip Dahm Doris Coleman
Sylvia Sichi Benjamin Yang Melinda Malready Donna
Yancey