tim-3 as a target for tumor immunotherapy...responses and ensuing immunopathology •ctla-4, pd-1,...
TRANSCRIPT
Tim-3 as a target for tumor immunotherapy
Ana Carrizosa Anderson
Harvard Medical SchoolBrigham and Women’s Hospital
Disclosures
A portion of the work has been performed as part of asponsored research agreement with MedImmune LLC.
Outline•Immune checkpoint molecules:
•Tim-3 vs PD-1
•Role of Tim-3 and PD-1 in anti-tumor immunity:-T cell exhaustion-Regulatory T cells
Immune checkpoint molecules•Definition- negative regulatory molecules upregulated onactivated T cells that serve to contract ongoing T cell responses thereby preventing uncontrolled immuneresponses and ensuing immunopathology
•CTLA-4, PD-1, Tim-3, Lag-3
•Cancer - Dysregulated expression of immune checkpoint molecules on T cells
•Clinical relevance: immunotherapies that target immunecheckpoint molecules have been shown to improve immunity in cancer (mice and human)
T cell exhaustion•Exhaustion- state of T cell dysfunction- failure to proliferate andexert effector function in response to TCR stimulation
•Hierarchy of exhaustion- loss of proliferation/CTL function and IL-2, then loss of TNF, then loss of IFN
•Express PD-1 and blockade of PD-1/PD-L1 interactions partiallyrestores T cell function
•Exhausted T cells also express Tim-3 (HIV, HCV) and blockade of Tim-3 signaling restores T cell function
Expression:•PD-1 is upregulated on all T cells 24-72 hrs after activation•Tim-3 is selectively expressed on IFN--secreting CD4+ and CD8+ T cells
Signaling:•PD-1 has ITIM and ITSM motifs in cytoplasmic tail•Tim-3- 6 tyrosine residues in cytoplasmic tail- no ITIM or ITSM
Ligands:•For PD-1: PD-L1 (widely expressed) and PD-L2•For Tim-3: galectin-9- widely expressed
•Both Tim-3 and PD-1 ligands are upregulated by IFNs andexpressed on tumors
Tim-3 vs PD-1
10 0 10 1 10 2 10 3 10 4
FL2-H
0
20
40
60
80
100
% o
f Max
10 0 10 1 10 2 10 3 10 4
FL2-H
0
20
40
60
80
100
% o
f Max
10 0 10 1 10 2 10 3 10 4
FL2-H
0
20
40
60
80
100
% o
f Max
10 0 10 1 10 2 10 3 10 4
FL2-H
0
20
40
60
80
100
% o
f Max
CT26
0
500
1000
1500
2000
2500
3000
3500
Expression of Tim-3/PD-1 and their ligands on tumor cell lines
10 0 10 1 10 2 10 3 10 4
FL2-H: FL2-Height
0
20
40
60
80
100
% o
f Max
10 0 10 1 10 2 10 3 10 4
FL2-H: FL2-Height
0
20
40
60
80
100
% o
f Max
PDL-1 Tim-3 Gal-9 Gal-9 (IC)
B16F10
0
5000
10000
15000
20000
25000
30000
35000
PDL-1 Tim-3 Gal-9 Gal-9 (IC)
10 0 10 1 10 2 10 3 10 4
FL2-H
0
20
40
60
80
100
% o
f Max
10 0 10 1 10 2 10 3 10 4
FL2-H
0
20
40
60
80
100
% o
f Max
CD8 CD4
FMO (Tim‐3)
PD‐1Tim‐3
CD4
CD8
Tim‐3
‐ PD‐1
+Tim
‐3+ PD‐1
+
Tim‐3
‐ PD‐1
‐
Tim‐3
‐ PD‐1
+Tim
‐3+ PD‐1
+
Tim‐3
‐ PD‐1
‐
Tim‐3
‐ PD‐1
+Tim
‐3+ PD‐1
+
Tim‐3
‐ PD‐1
‐
A BCT26 4T1 B16
One‐Way ANOVA, Tukey’s multiple comparison test
*p<0.001, **p<0.05
High frequency of Tim‐3+ PD‐1+ cells in CD8 positiveTumor Infiltrating Lymphocytes (TILs)
0 10 2 103 104 105
0
102
10 3
10 4
105
2
2.72
0 10 2 103 104 105
0
102
10 3
10 4
105 19.7
31.4
45.8
0 10 2 103 104 105
0
102
10 3
10 4
105 73.3
12.4
10.1
0 10 2 103 104 105
0
10 2
10 3
10 4
10 5 14.1
85.2
0.25
0 10 2 103 104 105
0
10 2
10 3
10 4
10 5 67.8
29.7
0
CD8 TILs
A
B
LAG3
CTLA‐4
LAG3 and CTLA-4 expression on Tim-3 and PD-1 expressing TILs
0 102 103 104 1050
20
40
60
80
100
86.4
0 102 103 104 1050
20
40
60
80
100
58.70 102 103 104 105
0
20
40
60
80
100
14
0 102 103 104 1050
20
40
60
80
100
13.8
0 102 103 104 1050
20
40
60
80
100
42.4
0 102 103 104 1050
20
40
60
80
100
36.7
Tim‐3‐PD‐1‐
Tim‐3‐PD‐1+
Tim‐3+PD‐1+
Tim‐3‐PD‐1‐
Tim‐3‐PD‐1+
Tim‐3+PD‐1+
LAG3
CTLA4
0
25
50
75
100
0
25
50
75
100
TIM-3 -PD-1 -
TIM-3 -PD-1 +
TIM-3 + PD-1 +
CD8 + TILS in CT26
IFN
TNFTim
-3
PD-1
Cyt
okin
e
CD8
IL-2
Tim-3+PD-1+ CD8 TILS are more impaired in cytokineproduction relative to Tim-3-PD-1-TILS
0 102
103
104
105
0
102
10 3
10 4
10 5
81.9
17.6
0 102
103
104
105
0
102
10 3
10 4
10 5
90.9
9.14
0 102
103
104
105
0
102
10 3
10 4
10 5
98.8
1.24
0 102
103
104
105
0
102
10 3
10 4
10 5
79.8
20
0 102
103
104
105
0
102
10 3
10 4
10 5
60.2
39.8
0 102
103
104
105
0
102
10 3
10 4
10 5
89
10.9
0 102
103
104
105
0
102
10 3
10 4
10 5
63.7
36.3
0 102
103
104
105
0
102
10 3
10 4
10 5
73.2
26.7
0 102
103
104
105
0
102
10 3
10 4
10 5
94.6
5.31
0 102 103 104 105
0
102
103
104
105 12.8
32.2
48.7
IL-2 - TILS
0
10
20
30
40
50
60
70 *TNF - TILS
0
10
20
30
40
50
60
70 *IFN - TILS
0
10
20
30
40
50
60
70 *
*p<0.0001
Tim-3 and PD-1 expression in cytokine non-producing TILS
0 1000 2000 3000 4000 5000
0
102
103
104
105
G0
G1S->M
Tim-3 and PD-1 expression in CD8+TILS entering cell cycle
TO-PRO-3
Ki-6
7
G0 G1 S->M0
1
2
3
4
5
6
7 *
P<0.05, One-Way ANOVA, Tukey’s MultipleComparison Test
Effect of targeting the Tim-3 and PD-1 signaling pathwayson CT-26 tumor growth
0 5 10 15 20 250
100
200
300
400
Time (Days)0 5 10 15 20 25
0
50
100
150
200
250 Control
Anti-Tim3 Ab
Anti-PD-L1 Ab
Anti-Tim-3 + Anti-PD-L1
Time (days)
Tum
or s
ize
(mm
2 )
Combined targeting of Tim-3 and PDL-1 in melanoma
B16F10 Melanoma
0 10 200
25
50
75
100
125ControlAnti-Tim-3Anti-PD-L1Anti-Tim-3 +Anti-PD-L1
Time (days)
Anti-Tim-3 and Anti-P-L1 Antibody Treatment of established Tumors
CT26
2/5 completeregression
Anti-Tim3 AbAnti-PD-L1 Ab
D
Tim-3 and PD-1 marks exhausted cells in AML
Zhou et al. Blood 2011
Combined therapy increases survival in AML
Zhou et al. Blood 2011
Pre-clinical models of cancer
•Tim-3 and PD-1 are uniquely co-expressed in TILs
•CD4 FoxP3+ Tregs
•CD8 exhausted phenotype
•In both solid and non-solid cancers combined targeting of the Tim-3 and PD-1 pathways is a highly effective therapy
•Human cancer (melanoma mets)
•CD8+ TILN vs PBL: exhausted phenotype only in TILN,
Expression of inhibitory receptors (Tim-3,PD-1) enriched in TILN (Baitsch et al JCI 2011)
•Blockade of Tim-3 and PD-1 (in vitro) synergizes to
increase cytokine production in melanoma specific
T cells (Fourcade et al JEM 2010)
Why target both Tim-3 and PD-1?
Additional reasons to target Tim-3 in cancer
•Tim-3 has a role in promotion of myeloid-derived suppressorcells (MDSC) (Dardalhon et al, JI 2010)
•Tim-3 is expressed on cancer stem cells- AML (human)
AcknowledgementsVijay K. Kuchroo
Kaori SakuishiLionel ApetohJenna M. Sullivan
University of MinnesotaQing ZhouBruce Blazar