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Old problems, new directionsEli Gilboa
ESMO International Symposium on ImmunologyNov 15-17, 2007Athens, Greece
Cancer immunotherapy with mRNA transfected dendritic cellsA personalized form of cell therapy
IL-1IL-1, IL-6, TNF-, IL-6, TNF-, PGE, PGE22
Antigen-LoadedAntigen-LoadedMature DCMature DC
DC maturationDC maturation
CryopreservedCryopreservedDC VaccineDC Vaccine
LeukapheresisLeukapheresis
Immature Immature DCDC MonocyteMonocyte
ss
GM-CSF, IL-4GM-CSF, IL-4
Tumor Tumor BiopsyBiopsy
Tumor antigensTumor antigens(mRNA)(mRNA)
Antigen LoadingAntigen Loading(mRNA transfection)(mRNA transfection)
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.
Clinical benefit is minimal, if any.
“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
Preclinical murine studies- Antitumor effects in murine models- No toxicity
Clinical trials- Immune responses- Hints of clinical impact
Clinical benefit is minimal, if any.
“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)
Preclinical murine studies- Antitumor effects in murine models- No toxicity
Clinical trials- Immune responses- Hints of clinical impact
Immunological control of cancer
Where are we going from here
Clinical benefit is minimal, if any.
“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)
Preclinical murine studies- Antitumor effects in murine models- No toxicity
Clinical trials- Immune responses- Hints of clinical impact
Specific Active Immunotherapy of Cancer
Cancer Vaccines
To engender protective immunity in the cancer patientthat will negatively impact on tumor progression.
Induction of immunity
Persistence of immunity
+Immune suppression
Immune escape
A multi pronged approach to cancer immunotherapy
Induction of immunity
Persistence of immunity
+Immune suppression
Immune escape
A multi pronged approach to cancer immunotherapy
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”
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”
Immune suppression
Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response
+
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”
Immune suppression
Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response
+
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”
Immune suppression
Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response
+
Tumor-induced immune suppression
Immune suppressiveCell types
Immune suppressiveproducts
Regulatory T cellsIL-13 secreting NKT cellsImmature myeloid cells (ImC)Tolergenic DC (“iDC”, pDC)“Alternatively activated” M (AAMs)…and other
Cox-2 generated prostanoidsB7H1TGFIL-10Decoy receptor 3 (DcR3)STAT3cAMPAdenosineVEGFIndoleamine deoxygenase (IDO)…and more
...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, 7:1118)
• 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, 198:1753
• Inverse correlation between tumor progression in ovarian and colorectal cancer patients and immune infiltrate (Zhang et al., N. Engl. J. Med., 2003, 348:203; Galon et al., Science, 2006, 313:1960)
• 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, 90:341)
Cancer despite immunosurveillance: immunoselection and immunosubversion. Zitvogel, L., Tesniere, A. and G. Kroemer. Can. Rev., Immunol., 2006, 6:715
“The seventh hallmark of cancer”
Tumor-induced immune suppression
Immune suppressiveCell types
Immune suppressiveproducts
Regulatory T cellsIL-13 secreting NKT cellsImmature myeloid cells (ImC)Tolergenic DC (“iDC”, pDC)“Alternatively activated” M (AAMs)…and other
Cox-2 generated prostanoidsB7H1TGFIL-10Decoy receptor 3 (DcR3)STAT3cAMPAdenosineVEGFIndoleamine deoxygenase (IDO)…and more
Naturally occuring CD4+CD25+ 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.
Elimination of TElimination of Tregreg using a diphteria toxin-IL-2 conjugate (ONTAK using a diphteria toxin-IL-2 conjugate (ONTAK®®) in ) in
RCC patients vaccinated with tumor RNA transfected DCRCC patients vaccinated with tumor RNA transfected DC
Follow-up
Treatment Phase Follow-upPost-Surgery
Week 2 Week 6Week 0Week -4
ONTAKØ +RCC RNA loaded DC
RCC RNA loaded DC
Nephrectomy
EligibilityAssessment
Dosing Schedule: 3 cycles of 1x107 cells i.d per cycle
Leu
kaph
eres
is
InformedConsent
Leu
kaph
eres
is
RA
ND
OM
IZE
750
500
250
0IFN
/105
CD
8+ T
cel
ls
ONTAK® - +n=4 n=6
p=0.019
Dannull et al., J. Clin. Invest., 2005, 115:3623
A. RCC RNA + ONTAK®
B. RCC RNA
3737
RCC PBMCRCC PBMCPBMCRCC PBMCPBMCRCC PBMCPBMC0
250
500
750
1000
RCC RERCC RERERCC RERERCC RERE
0202 --RCCRCC --DABDAB0101 --RCCRCC --DABDAB
597597
7272
900900
64646565
RCC PBMC
0404 --RCCRCC --DABDAB
RCC PBMC
0505 --RCCRCC --DABDAB
RCC PBMC
0606 --RCCRCC --DABDAB
480480 501501
3535
280280
IFN
/10
5 C
D8+
T c
ells
1515
RCCRCC PBMC RCC PBMC RCC PBMC RCC PBMC
0808 --RCCRCC 0909 --RCCRCC 1010 --RCCRCC 1111 --RCCRCC
4040 56569797
3030
286286
38387979
IFN
/10
5 C
D8+
T c
ells
0
250
500
750
1000
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.
1. Treg rebound with time
2. The tumor and the vaccination itself can generate Treg
3. 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.
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• CD4+CD25+foxp3 as well as CD4+CD25-foxp3 Treg.
Stimulate a CD8+ CTL response against foxp3
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
Foxp3 is expressed in the thymus
1. Thymocytes destined to become TregFontenot, J.D et al. 2003. Nat Immunol 4:330-336
2. Thymic stromaChang, X. et al., 2005, J Exp Med 202:1141-1151
Immunization (1x) against Foxp3 enhances antitumor immunity in B16 melanoma tumor-bearing mice
d3d0
Tumor
Treg
Vaccination
(TRP-2)
Impact on tumor growth
ActinFoxp3
TRP-2 + Foxp3TRP-2
Days to tumor onset
Tum
or f
ree
mic
e (%
)
0
10
20
30
40
50
60
70
80
90
100
10 15 20 25 30 35 40
Foxp3 vaccination
Tum
or f
ree
mic
e (%
)
Days to tumor onset
0
10
20
30
40
50
60
70
80
90
100
10 15 20 25 30 35 40
ActinCD25
TRP-2 + CD25TRP-2
CD25 depletion
Repeated depletion of Treg subsequent to tumor vaccinationFoxp3 immunotherapy versus CD25 Ab depletion
Days to tumor onset
0
10
20
30
40
50
60
70
80
90
100
12 16 20 24 28 32 36 40
Actin + CD25TRP-2 + CD25TRP-2 + CD25, CD25
Tu
mor
fre
e m
ice
(%)
0
10
20
30
40
50
60
70
80
90
100
12 16 20 24 28 32 36 40
Actin + Foxp3TRP-2 + Foxp3TRP-2 + Foxp3, Foxp3
Tu
mor
fre
e m
ice
(%)
Days to tumor onset
d10d3d0
Tumor
Treg (1X)
Vaccination
Treg (2X)
Impact on tumor growth
1week
1.32%0.40%
0.46%
0.62% 0.30%
0.12%
TRP-2
TRP-2 +
CD25 Ab
TRP-2 + Foxp3
Sid
e S
catt
er
Foxp3 CD4 CD8
Sid
e S
catt
erS
ide
Sca
tter 0.29%
0.05%
0.07%
TRP-2TRP-2 + CD25 Ab TRP-2 + Foxp3
Rat
io o
f %
pos
itiv
e ce
lls
0
2
4
6
8
10
CD4+Foxp3-/Foxp3+ CD8+/Foxp3+
Treg depletion enhances the ratio of conventionalTcells/Treg in the tumor
Fate of foxp3-expressing cells in mice vaccinated against foxp3 or treated with CD25 Ab
Tumor
CD
25
Side
Sca
tter 2.8% 1.1%
CD
25
Side
Sca
tter 0.2% 0.06%
2.6% 1.1%
0.9%
CD
25
Side
Sca
tter
2.7% 0.5%
1.3%
CD
25
Side
Sca
tter
Foxp3 CD4 Foxp3 CD4
TRP-2
TRP-2 + CD25 Ab
TRP-2
TRP-2 + Foxp3Si
de S
catt
erSi
de S
catt
erSi
de S
catt
erSi
de S
catt
er
CD
25C
D25
CD
25C
D25
Foxp3 CD4 Foxp3 CD4
4.1%
1.8%
3.2%
3.3%
2.5%
1.2%
2.4%
2.2%
A. CD25
B. Foxp3
Lymph node Spleen
Nair et al. Can. Res., 2007, 67:371
Fate of foxp3-expressing cells in mice vaccinated against foxp3 or treated with CD25 Ab
Periphery
Shift emphasis from inducing immunity to developing methods targeting tumor-induced immune suppression
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”
Immune suppression
Tumors activate mechanisms which suppress the differentiation and/or function of an otherwise effective antitumor response
+
Tumor-induced immune suppression
Immune suppressiveCell types
Immune suppressiveproducts
Regulatory T cellsIL-13 secreting NKT cellsMyeloid derived suppressor cells (MDSC)Tolergenic DC (“iDC”, pDC)“Alternatively activated” M (AAMs)…and other
Cox-2 generated prostanoidsB7H1TGFIL-10Decoy receptor 3 (DcR3)VEGFSTAT3cAMPAdenosineIndoleamine deoxygenase (IDO)…and more
???
Induction of immunity
Persistence of immunity
+Immune suppression
Immune escape
A multi pronged approach to cancer immunotherapy
TCR attenuation
Costimulation
4-1BB
• Upregulated on antigen-activated T cells
• Enhances survival and proliferation of activated CD8+ T cells
Costimulatory receptors on T cells
• CD28• CD27• OX40• 4-1BB• CTLA-4• PD-1• HVEM• CD30
Agonistic 4-1BB antibodies enhance proliferation of activated CD8+ T cells and potentiate tumor immunity in mice
1. Complexity & cost of development
2. Regulatory approval process
3. Cost of manufacturing
4. Limited & uncertain access (companies)
Limitations to use of antibodies (or protein-based ligands) as therapeutic reagents
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 3:682-685.
Antibodies are cell based products
Antibody combinations - synergistic antitumor effectsMurine studies
Antibodies Reference
4-1BB + CTLA-4 Kocak, et al., 2006, Cancer Res 66:727644-1BB + OX40 Lee et al., 2004, J. Immunol., 173:30024-1BB + B7H1 Hirano et al., Can. Res., 2005, 65:10894-1BB + CD40 + DR5 Uno et al., Nat. Med., 2006, 12:693
Antibodies Reference
4-1BB + CTLA-4 Kocak, et al., 2006, Cancer Res 66:727644-1BB + OX40 Lee et al., 2004, J. Immunol., 173:30024-1BB + B7H1 Hirano et al., Can. Res., 2005, 65:10894-1BB + CD40 + DR5 Uno et al., Nat. Med., 2006, 12:693
Antibody combinations - synergistic antitumor effectsMurine studies
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Eradication of established 4T1 breast carcinoma tumors in mice by combination therapy with DR5+CD40+CD137
*Uno et al., Nat. Med., 2006, 12:693
Antibodies Reference
4-1BB + CTLA-4 Kocak, et al., 2006, Cancer Res 66:727644-1BB + OX40 Lee et al., 2004, J. Immunol., 173:30024-1BB + B7H1 Hirano et al., Can. Res., 2005, 65:10894-1BB + CD40 + DR5 Uno et al., Nat. Med., 2006, 12:693
Antibody combinations - synergistic antitumor effectsMurine studies
In vitro selection of oligonucleotide aptamers
An Aptamer Library = A Vast Shape Library
Aptamer Library
AGGACGAUGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAGACGACUCGC
440 possible sequences
In vitro selection (SELEX)
40 nucleotide random region
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
Aptamers can be made to most any target
Target Protein Affinity(Kd) Function Ref.
PDGF 0.1nM Inhibitor Green et al., 1996P-Selectin 0.03nM Inhibitor Jenison et al., 1998Complement C5 0.03nM Inhibitor Biesecker et al., 1999MAb to AChR 60nM Inhibitor Lee and Sullenger, 1997Interferon-Gamma 2.7nM Inhibitor Kubik et al., 1997VEGF 0.15 Inhibitor Ruckman et al., 1998Factor VIIa 11nM Inhibitor Rusconi et al., 2000mCTLA-4 30nM Inhibitor Santulli-Marotto, et al. 2003
0.0 0.5 1.0 1.5 2.0 2.5 3.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7Round 12 PoolRNA Library
Fra
ctio
n R
NA
Bou
nd
Log([4-1BB-Fc](nM))
M12-2(722): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGC-3'M12-3(722): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGG-3'M12-5(722): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGG-3'M12-8(722): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGG-3'M12-11(819): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGG-3'
*M12-23(819): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGG-3'M12-2(923): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGA-3'M12-12(923): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGG-3'M12-18(923): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGA-3'M12-33(923): 5'-CGACCGAACGUGCCCUUCAAAGCCGUUCACUAACCAGUGG-3'
M12-17(923): 5'-GAAGUGACAGCUCCCAGCGCUUCAAAGCUCAUCUAUAACU-3'M12-23(923): 5'-CAGAAACUAGACCUCCGAUCGGACACCCGGUCCCUUCGUC-3'M12-24(923): 5'-GAAGCACAAUAGGCCGCAACACUUCAAAACCCAUUCAAUC-3'
*M12-9(722): 5'-CAAGCACUCUUCAGGCUAAGGACUCUCUUGACACCCCGC-3'
*M12-12(722): 5'-GCACAGCAACACCACGACCCCCCCUAGGCUUCCGCCCGCC-3'M12-25(819): 5'-GCACAGCAACACCACGACCCCCCCUAGGCUUCCGCCCGCG-3'M12-5(923): 5'-GCACAGCAACACCACGACCCCCCCUAGGCUUCCGCCCGCA-3'M12-7(923): 5'-GCACAGCAACACCACGACCCCCCCUAGGCUUCCGCCCGCG-3'
*M12-22(819): 5'- GCACAGCAACACCACGACCCCCCCUAGGCUUCCGCCCGCG-3'
M12-1(722): 5'-UAACGGCCCAAUGACUUCGCCUUACUGCCCCCCUAAGCUUC-3'M12-7(722): 5'-AAAGCGACAAUUCUUACUACUCCCCAAGCUCCACGCCUUU-3'M12-15(819): 5'-AAGACGAUACCUAGCCUCAAAAUUCCUCCCCCGACUUCCU-3'
*M12-9.3(819): 5'- CGAGAACCCGCAUCUUCGGAUGCGCCCCCCUAGGACUUAC-3'
*M12-20.1(819): 5'-GACCAAGGGCAGCAUCACCGUUCCCCCCCUAGGAGCUUAC-3'
*M12-20.3(819): 5'-UAACGGCCCAAUGACUUCGCCUUCUGCCCCCCUAAGCUUC-3'
M12-1(923): 5'-CGCUCUCUCACAACCACGACCUCCGAUCUGAUAAUUCGUC-3'M12-16(923): 5'-CGCUCUCUCACAACCACGACCUCCGAUCUGAUAAUUCGUC-3'
*M12-3(819): 5'-GCACCAAACACCGGUUCAGAACCCAUCAUGUAACUCCUUG-3'
*M12-5(819): 5'-AACUACCUCCUCGAACCAUAGUUCAACACCAUCCAGCCAU-3'
Isolation of aptamers which bind to murine 4-1BB in solution
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
4-1BB in vitro costimulation assay
Enhancement of proliferation or IFN release from suboptimally activated CD8+ T cells
4-1BB signaling requires ligand-induced receptor dimerization
+
MAb
+
Sequence and computer-predicted secondary structure of a 4-1BB binding aptamer
M12-23
mutM12-23
mutM12-23-A + mutM12-23-B
M12-23-A + M12-23-B
100b
p L
adde
r
M12
-23-
A +
M12
-23-
B
M12
-23-
A
M12
-23-
B
mut
M12
-23-
A +
mut
M12
-23-
B
mut
M12
-23-
A
mut
M12
-23-
B
Dimer
Monomer
Generating aptamer dimers using complementary 3’ extensions
Stimulated (CD3)
Unstimulated
Competition
4-1BB Ab-AF488
100
80
60
40
0
20
Cou
nts
100 101 102 103 104
a
M12-23 dimer-FAM100 101 102 103 104
100
80
60
40
0
20
Cou
nts
b
mutM12-23 dimer-FAM100 101 102 103 104
100
80
60
40
0
20
Cou
nts
c
M12-23 dimer-FAM +Isotype Ab
100
80
60
40
0
20
Cou
nts
100 101 102 103 104
d
M12-23 dimer-FAM +4-1BB Ab
100
80
60
40
0
20
Cou
nts
100 101 102 103 104
e
4-1BB Ab-AF488100 101 102 103 104
100
80
60
40
0
20
Cou
nts
f
M12-23 dimer-FAM100 101 102 103 104
100
80
60
40
0
20
Cou
nts
g
4-1BB aptamer dimer binds specifically to activated CD8+ T cells
Binding of monomeric and dimeric forms of 4-1BB aptamers to 4-1BB expressed on the cell surface
100
101
102
103
104
4 nM
40
100
80
60
0
20
Cou
nts 2.5
Cou
nts
20 nM10
010
110
210
310
4
40
100
80
60
0
20
3.0
Cou
nts
100
101
102
103
104
100 nM
40
100
80
60
0
20
7.3
Cou
nts
100
101
102
103
104
500 nM
40
100
80
60
0
20
97.4
100
101
102
103
104
4 nM
40
100
80
60
0
20
Cou
nts 9.9
100
101
102
103
104
20 nM
40
100
80
60
0
20
Cou
nts 27.4
100
101
102
103
104
100 nM
40
100
80
60
0
20
Cou
nts 77.7
100
101
102
103
104
500 nM
40
100
80
60
0
20
Cou
nts
98.3
Monomer
Dimer
AptamersAntibodies
Untreateda
IgG + CD3b
4-1BB + CD3c
mutM12-23 dimer + CD3
d
M12-23 dimer + CD3e
M12-23 monomer + CD3f
M12-23 dimer + IgGg
0.02%
0
20
40
60
80
100
Cou
nts
100 101 102 103 104
CFSE
0
20
40
60
80
100
Cou
nts 13.5% 13.7%
0
20
40
60
80
100
Cou
nts
100 101 102 103 104
CFSE
40.2%
0
20
40
60
80
100
Cou
nts
100 101 102 103 104
CFSE
11.3%
100 101 102 103 104
CFSE
0
20
40
60
80
100
Cou
nts
32.3%
100 101 102 103 104
CFSE
0
20
40
60
80
100
Cou
nts
100 101 102 103 104
CFSE
0
20
40
60
80
100
Cou
nts
0.08%
4-1BB Aptamer Dimers Costimulate T Cells(CFSE proliferation assay)
0
2
4
6
8
10
12
14
16
IgG
4-
1BB
Ab
mut
M12
-23
dim
er
M12
-23
dim
er
Unt
reat
ed
IFN
re
leas
e (f
old
incr
ease
)
4-1BB Aptamer Dimers Costimulate T Cells(IFN production)
0
200
400
600
800
1000
2 3 4 52 3 4 5
Mea
n tu
mor
vol
ume
(mm3 )
Day 4
2 3 4 52 3 4 5
Day 6
2 3 4 52 3 4 5
Day 8
0 2 4 6 8 10 12
Mea
n t
um
or
vo
lum
e (m
m3 )
Days after injection
800
600
400
200
0
PBSIsotype Ab4-1BB AbMutM12-23 dimerM12-23 dimer
A
B C
0 2 4 6 8 10 12 14 160
2
4
6
8
10
PBSIsotype Ab4-1BB AbMutM12-23 dimerM12-23 dimer
Days after injection
Num
ber
of M
ice
Rem
aini
ng
D
0
100
200
300
400
500
Mea
n t
um
or
Vo
lum
e (m
m3 )
Rejection of P815 mastocytoma tumors injected with 4-1BB aptamer dimers
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
Next:1. Improve aptamer potency by post selection modifications2. Antitumor potential - stringent murine immunotherapy models3. Do monomers block 4-1BB signaling?4. Development of human 4-1BB agonists and clinical studies.
The potential of aptamer technology to manipulate immunity
• Ligands as well as targeting agents for drugs (siRNAs)
• Feasibility
• Replace antibodies and expand therapeutic applications?
Concluding thoughts
Vaccination: Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens
orPotentiating the ability of the disseminated tumor to stimulate immune responses on its own
Vaccination: Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens
Yesterday
Vaccination: Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens
Potentiating the ability of the disseminated tumor to stimulate immune responses on its own
Today
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, 90:341)
• 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, 348:203
– Colorectal cancer: Galon, J., et al.Type, density, and location of immune cells within human colorectaltumors predict clinical outcome. Science, 2006, 313:1960
Vaccination: Inducing de novo, or expanding preexisting, immune responses against tumor-associated antigens
Tomorrow
Potentiating the ability of the disseminated tumor to stimulate immune responses on its own
And then...
Potentiating the ability of the disseminated tumor to stimulate immune responses on its own
Overcoming tumor-induced immune suppression
Delivering co-stimulatory ligands to the tumor
Promoting “immunogenic” death of the tumor
Enhancing the antigenicity of the tumor
Concept development Translation 2-Arm clinical trials
• Murine studies• Human in vitro studies
Development andoptimization ofclinical reagents andprotocols
1o Immunological2o Clinical
End points:
4-1BB aptamers
James McNamaraDespina KoloniasFernando Pastor
CollaborationPaloma GiangrandeBruce Sullenger
Lieping ChenRobert Mittler
Jenz DannullZhen SuPhilip DahmDoris Coleman
Center for Translational Research, Duke University Medical Center
Foxp3 vaccination
Smita NairDavid BoczkowskiMartin Fassnacht
Sylvia SichiBenjamin YangMelinda MalreadyDonna Yancey
Eli Gilboa Johannes Vieweg
Duke Cancer Immunotherapy Program
Concept development Translation 2-Arm clinical trials
• Murine studies• Human in vitro studies
Development andoptimization ofclinical reagents andprotocols
1o Immunological2o Clinical
End points:
4-1BB aptamers
James McNamaraDespina KoloniasFernando Pastor
CollaborationPaloma GiangrandeBruce Sullenger
Lieping ChenRobert Mittler
Jenz DannullZhen SuPhilip DahmDoris Coleman
Center for Translational Research, Duke University Medical Center
Foxp3 vaccination
Smita NairDavid BoczkowskiMartin Fassnacht
Sylvia SichiBenjamin YangMelinda MalreadyDonna Yancey
Eli Gilboa Johannes Vieweg
Duke Cancer Immunotherapy Program