adu-s100 combines with checkpoint blockade to elicit an ......•approaches to stimulate priming of...
TRANSCRIPT
STimulator of INterferon Genes (STING) is a critical component of an innate immune pathway that activates robust anti-viral and anti-tumor responses in mouse models. Activation of the STING pathway by intratumoral (IT) injection of synthetic cyclic dinucleotides (CDNs) is being explored as a cancer therapy and has shown potent anti-tumor activity in preclinical models. Here we assessed the benefit of combining immune checkpoint blockade with ADU-S100 (MIW815), a CDN under clinical evaluation, in different syngeneic mouse tumor models. In mice bearing dual flank 4T1 mammary carcinoma tumors, adding a single dose of ADU-S100 with αPD-1 induced eradication of both injected and non-injected tumors, leading to near complete responses, demonstrating that ADU-S100 potentiates the activity of checkpoint blockade. Tumor control was CD8+ T cell-dependent and correlated with an enhanced CD8+ T cell effector profile in both the periphery and non-injected tumors. Remarkably, the resistance of CT26 Pten-/- tumor to αPD-1 was overcome by combining with ADU-S100. In addition, combining a single injection of ADU-S100 with αPD-1 elicited enhanced tumor control in the dual flank MC-38 colon carcinoma model compared to ADU-S100 or αPD-1 treatment alone. Those mice which cleared tumor by combination treatment were also protected from tumor re-challenge. Moreover, in the poorly immunogenic B16.F10 model, adding ADU-S100 to the combination therapy of αPD-1 and αCTLA4 induced tumor-specific CD8+ T cell responses and tumor control, leading to multiple complete responses and durable immunity in surviving animals. Together, these results highlight the immune correlates of STING-mediated anti-tumor efficacy and illustrate the potential of combining ADU-S100 with checkpoint inhibitors for the treatment of human cancer. Clinical trials of ADU-S100 in combination with αPD-1 or with αCTLA4 are ongoing and could further elucidate the immunological mechanism of action and therapeutic effect in humans.
MC38 DF tumorbearing mice
S100 1x± αPD-1 Tumor
outgrowth
0 10 20 300
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a-PD-1 comboprimary tumor
Days after implantation
Tum
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S100, isotype
Vehicle, a-PD1
S100, a-PD1
Tumor free
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a-PD-1 combodistal tumor
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*Vehicle + isotype (0/8)Vehicle + αPD-1 (0/8)S100 + isotype (0/8)S100 + αPD-1 (2/8)
Presented at the 2018 Tumor Immunology and Immunotherapy, November 27 - 30, 2018, Miami, FL
Statistical Significance: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 byANOVA (exceptions: 8B, log-rank (Mantel-Cox) test; 8C, Fisher’s exact test);Statistics are versus Naïve or Vehicle (V) controls unless indicated otherwise.Lines (except 8C) or bars and error represent the mean ± SEM and eachsymbol in bar graphs represents an individual animal. In legends, (x/x) notationindicates (number of cures/total mice in experimental group). Arrow on tumorgrowth curves indicates day of IT injection.
Abbreviations: IT, intratumoral; CDN, cyclic dinucleotide; IFN, type I interferon; TDLN, tumor-draining lymph node; CPI, checkpoint inhibitor; V, vehicle; ns, non-significant; SF, single flank; DF, dual flank; S100, ADU-S100/MIW815; 1x, single IT injection; 2x, two IT injections; 3x three IT injections; GrzB, Granzyme B; SFC, spot-forming cells; (x/x), (cures/total n of mice)
References:1) Diamond MS et al., The Journal of Experimental Medicine. 2011.2) Fuertes MB et al., The Journal of Experimental Medicine. 2011.3) Ishikawa H et al., Nature. 2008.4) Woo S-R et al., Immunity. 2014.5) Corrales L et al., Cell Reports. 2015.6) Francica BJ et al., Cancer Immunology Research. 2018.7) Corrales L et al., Journal of Clinical Investigation. 2016.
Ethics Approval: All animals were used according toprotocols approved by Institutional Animal Use Committee ofAduro Biotech, Inc. and maintained in specific pathogen-freeconditions in a barrier facility.
ADU-S100 Combines with Checkpoint Blockade to Elicit an Anti-Tumor CD8+ T Cell Response to Control Non-Injected Tumors in Preclinical Models
Weiwen Deng1, Anthony L. Desbien1, Kelsey Sivick Gauthier1, Gabrielle Reiner1, Leticia Corrales1, Tamara Schroeder1, Natalie H. Surh1, Brian Francica1, Justin J. Leong1, Ken Metchette1, Lianxing Zheng2,
Charles Cho3, Yan Feng2, Jeffrey M. McKenna2, Steven L. Bender3, Chudi Ndubaku1, Meredith L. Leong1, Andrea van Elsas1, and Sarah M. McWhirter1
1Aduro Biotech, Inc., Berkeley, CA, 2Novartis Institutes for BioMedical Research, Cambridge MA; 3Genomics Institute of the Novartis Research Foundation, San Diego, CA
A,B) Mice bearing a single flank 4T1 tumor received one IT injection of vehicle alone or 1, 10, 100, or 500 μg ADU-S100, A) ADU-S100 cleared injected tumor in a dose dependent manner. B) The frequency of replicating tumor-specific T cells, as measured by H-2Ld-AH1 (AH1) tetramer staining, exhibited a bell-shaped curve in a single flank setting.
C,D) Mice bearing dual flanks 4T1 tumor received one IT injection of vehicle alone or 1, 10, 100, or 500 μg ADU-S100, C) Both immunogenic and ablative doses of ADU-S100 elicited a robust tumor-specific CD8+ T cell response in the dual flank setting. D) Significant amounts of S100 were found in non-injected tumors at the 500 μg dose (134.2 ng/ml ± 29.62), reaching a concentration on the same order of magnitude as tumors injected with 10 μg (923.8 ng/ml ± 371.4).
• The magnitude of tumor-specific CD8+ T cell responses is dependent on the dose of ADU-S100.
• Lower immunogenic dosing regimens result in local STING activation and durable adaptive immune responses.
• More aggressive ablative dosing regimens, while effective in clearing injected tumors, result in systemic drug distribution and compromised T cell immunity.
• Immunogenic doses of ADU-S100 combine effectively with checkpoint inhibitors αPD-1 and/or αCTLA4 in multiple tumor models including CPIs resistant models.
• Together, these results identify immune correlates of STING-mediated anti-tumor efficacy in mice and illustrate the potential of combining ADU-S100 with checkpoint inhibitors for the treatment of human cancer.
CONCLUSIONS
A)Treating MC38 tumor-bearing mice with an immunogenic dose of S100 (10 μg) combined with αPD-1 induced eradication of the non-injected tumors, while either agent alone was insufficient for tumor control (right: green squares vs. blue squares or red circles).
B)Surviving mice demonstrated durable immunity.
Figure 5. Combination of ADU-S100 and αPD-1 Enhances Non-Injected Tumor Control in a CD8+ T Cell-Dependent Manner in 4T1 Mammary Carcinoma Tumor-Bearing Mice
A)Treating 4T1 tumor bearing mice with an immunogenic dose of S100 (10 μg) combined with αPD-1 induced eradication of both injected and non-injected tumors in a CD8+ T cell-dependent manner (green inverted triangles vs. yellow diamonds).
B)An immunogenic dose of S100 (10 μg) activated and expanded tumor-specific effector T cells, while αPD-1 potentiated the functionality of these cells in the non-injected tumor microenvironment.
BACKGROUND• T cell inflamed tumors in humans are
correlated with an interferon-β (IFNβ) transcriptional signature in the tumor microenvironment.
• Approaches to stimulate priming of tumor-specific CD8+ T cells for any individual and/or initiate productive immune responses in “cold” tumors have potential as cancer immunotherapies.
• Different mechanisms, including the loss of function mutations, were involved in relapse to checkpoint inhibitors (CPIs).
• Intratumoral (IT) injection of cyclic dinucleotide (CDN) STING agonists induces IFNβ, and activates tumor-resident dendritic cells capable of priming tumor-specific CD8+ T cells in mice.
• Here, the benefit of combining IT CDN therapy optimized for CD8+ T cell induction with immune checkpoint blockade was explored using ADU-S100, a CDN under clinical evaluation, in different syngeneic mouse flank tumor models.
Figure 1. STING-Activating CDNs Drive T Cell Priming
Figure 4A
± αPD-1, ± αCD8 Tumoroutgrowth
S100 1x
4T1 DF tumorbearing mice
Figure 4B
DistalTumorFlow
Cytometry4T1 DF tumorbearing mice
12days
± αPD-1S100 1x
4T1 DF tumorbearing mice
S100 1x± αPD-1, ± αCD8 Tumor
outgrowth
• Phosphorothioate substitution increases resistance to phosphodiesterase cleavage.
• Mixed-linkage configuration facilitates broad activation of human STING alleles.• ADU-S100 has enhanced potency over natural CDN ligands in humans cells
and mouse models.• ADU-S100 was selected based on balance of efficacy and tolerability in pre-
clinical studies.
• X-ray crystal structure (stick model) of ADU-S100• Structure contains two adenine bases covalently bonded
by a 2’-5’ and a 3’-5’ linkage (aka “mixed” linkage) and phosphorothioate substitutions in the R,R configuration
• X-ray crystal structure of ADU-S100 bound tothe C-terminal domain of human STING
Figure 2. Development of Clinical Compound ADU-S100 (S100)
Carbon (white)PhosphorousNitrogen OxygenSulfur
ABSTRACT
BFigure 2A
Figure 2B
Figure 2C
TDLNFlow
Cytometry4T1 DF tumorbearing mice
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Figure 7. Combination of ADU-S100 and αPD-1 Enhances Non-Injected Tumor Control and Induced Durable Immunity
Figure 3. IT STING Activation Can Be Modulated to Induce Local Versus Systemic Immune Activation
Day Post Implantation
VehicleS100 10 µgS100 500 µg
+/− αCD8 Tumoroutgrowth
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CD8+ T cells were necessary for control of injected tumors afforded by an immunogenic dose of S100 (1x 10 μg, top: gray triangles) and for control of non-injected tumors afforded by an ablative dose of S100 (1x 500 μg, BOTTOM: black squares). In contrast, CD8+ T cells were dispensable for control of injected tumors afforded by an ablative dose of S100 (1x 500 μg, TOP: black squares).
4T1 DF tumorbearing mice
S100 1x± αCD8 Tumor
outgrowth
Figure 4. CD8+ T Cells Are Necessary for Anti-Tumor Immunity Elicited by Immunogenic, but Not Ablative, Doses of ADU-S100
Tum
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RESULTS
A)CT26 tumor partially responded to αPD-1 while CT26 Pten-/- tumor was resistant to the treatment.
B)Treating CT26 Pten-/-
tumor bearing mice with an immunogenic dose of S100 (10 μg) combined with αPD-1 induced eradication of non-injected tumors, while S100 was insufficient for tumor control (right: green squares vs red circles).
CT26 Pten-/-
DF tumorbearing mice
S100 1x± αPD-1
Tumoroutgrowth
± αPD-1D7
Figure 6. The Resistance of CT26 Pten-/- Tumors to αPD-1 Was Overcome by Combining with ADU-S100
0 10 20 300
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Figure 2A
Figure 2B
Figure 2C
TDLNFlow
Cytometry4T1 DF tumorbearing mice
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TumorLC/MS/MS4T1 DF tumor
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Tumoroutgrowth
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B
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A) In the poorly immunogenic B16.F10 model, combining an immunogenic dose of S100 (10 μg) to the αPD-1 + αCTLA4 CPIs combination therapy induced tumor-specific CD8+ T cell responses B)and tumor clearance, resulting in a significant survival benefit. C) Surviving mice demonstrated durable immunity as a significant number of animals were resistant to autologous rechallenge.
B
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Figure 8. Combination of ADU-S100, αPD-1, and αCTLA4 Enhance Anti-Tumor Immunity in B16.F10 Melanoma Tumor-Bearing Mice
B16 SF tumorbearing mice
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daysPBMC
ELISpot Tumorrechallenge
Cure mice
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CFigure 2A
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Figure 2C
TDLNFlow
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DFigure 2A
Figure 2B
Figure 2C
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100
µg S100:100
Injected TumorDistal Tumor
101
102
103
104
105
10 500 10 500
**
InjectedNon-Injected
ng/m
lADU
-S10
0
µg S100:100
Injected TumorDistal Tumor
101
102
103
104
105
10 500 10 500
**Tu
mor
Vol
ume
(mm
3 )
Day Post Implantation