Discovery of Small Molecule Aryl Hydrocarbon Receptor (AhR)
Antagonists for Cancer Immunotherapy
Immuno-Oncology: Emerging Targets and TherapeuticsNew ways to Refocus the Immune System to Attack Cancer
Thomas Hoffmann
Boston, Sept. 17, 2019
Discovery on Target, Boston, Sept. 17, 2019 1
o Heidelberg based, private small molecule drug discovery company
o Focus on Liver / Metabolic Diseases and Cancer
o Multi-disciplinary project teams span Translational Biology and MedicinalChemistry
o Partnered projects:
• 2012 – Contract with Janssen/J&J: Licensing of Psoriasis Program on
RORgt, up to 135 Mio. US$; in Phase 1
• 2014 – Contract with Gilead: Sale of NASH Program on FXR, volume
up to 470 Mio. US$; in Phase 2b in NASH, in Phase 3 in PSC
o Pipeline of metabolic projects (i.e. LXR inverse agonists for NASH) andcancer projects targeting the metabolic microenvironment of tumors
Phenex at a glance
Discovery on Target, Boston, Sept. 17, 2019 2
Immune checkpoint inhibitors do not always overcome the suppressive metabolic alterationsin solid tumors
Anti-PD1 / PD-L1Anti-CTLA4
Stephen M. Hatfield et al., Sci Transl Med 2015;7:277ra30
Hypoxic coreCD8
There is a strong need for safe, controllableand affordable small molecule based TMEmodulating therapies to increase the efficacyof existing treatments!
Checkpoint inhibitors do not workin many solid tumors
i.e. immune therapy combinationscould have deleterious pro-inflammatory side effects
Discovery on Target, Boston, Sept. 17, 2019 3
Phenex aims at significantly improving response rates:
o in combination with standard of care – chemotherapy, and / or
o in combination with checkpoint inhibitor therapies, with
• AhR Program
• further Tumor Metabolism Programs in the pipeline
Phenex interested in targets that break the `metabolic shield` of solid tumors
Discovery on Target, Boston, Sept. 17, 2019 4
Crystal structure of AhR/ARNT complexbound to its target DNA
Schulte, Daumke et al, Structure 2017, 25, 1-9
O
OCl
Cl
Cl
Cl
TCDD (Dioxin)
NH
HN
O
6-formylindolo[3,2b]-carbazole (FICZ)
Cyp1a1, Aldh1a3, Aldh3a1, ugt1a1, Mdr1→ xenometabolism, chemoresistance
IL-17, IL-22,…→ immune regulation
adapted from:Zhou, Trends Immunol 2016, 37, 17-29
AhR - multifaceted receptor
AhR is a ligand controlled transcription factor (basic helix-loop-helix, bHLH family)AhR is expressed in most cells, in particular hepatocytes and certain immune cells
Discovery on Target, Boston, Sept. 17, 2019 5
AhR - Sensor with Multiple Ligands & Functions
Microbiota derived Metabolitesof skin, gut, lung
Environmental Ligands: Dioxin, Biphenyls,Polyaromatic Hydrocarbons (PAHs)
Kinases (MEK, c-Src etc)
Xenometabolism / Chemoprevention: CYP1A1, UGT1A1
Toxicity: Dioxin
Endogenous MetabolitesTryptophan derived via IDO1/TDO2
− Agonist → Immune suppression:increased Treg, MDSC and M2
− Antagonist → Immune stimulation:increased CD8+ T cells and M1,reduced Treg; Reduced IL-22
Chemoresistance:ALDH1A3, ALDH3A1, MDR1
Increase of Immune barrier protection:via IL-22
Dietary Metabolites:Flavonoids, Indoles
AhR
Discovery on Target, Boston, Sept. 17, 2019 6
Counterregulation of systemic immune response:
Variety of activating ligands Multiple biological functions
CD8+ T cells
Treg cellsMDSCs
AhR
Activation with an Agonist ligand ----> Immune suppression
Discovery on Target, Boston, Sept. 17, 2019 7
Deactivation with an antagonist ----> Immune stimulation
CD8+ T cells
Treg cellsMDSCs
AhR
Discovery on Target, Boston, Sept. 17, 2019 8
AhR as a target for cancer immunotherapy in the literature
Selected publications on the role of AhR in the immune system and harnessing its potential as therapeutic target:
o Quintanta et al., Nature 2008, 453 (7191), 65-71: Treg and TH17 differentiation is controlled by AhR
o Veldhoen et al., Nature 2008, 453 (7191), 106-9: AhR links TH17-cell-mediated autoimmunity to environmental toxins
o Opitz et al., Nature 2011, 478 (7368),197-203: AhR agonism of Kynurenine, formed by IDO-1/TDO promotes tumor survival
o Bessede et al., Nature 2014, 511 (7508), 184-190: Aryl hydrocarbon receptor control of a disease tolerance defense pathway
o Takenaka et al., 2019 Nat Neurosci., 22(5):729-740: Control of tumor-associated macrophages and T cells in glioblastoma via AHR and CD39
Discovery on Target, Boston, Sept. 17, 2019 9
Cancer type References
Pancreatic cancer Koliopanos et al. Oncogene 2002
Breast cancer-Triple Negative Breast cancer
Powell et al. J Cancer Ther. 2013; Li et al. Int J Clin Exp Pathol 2014; D’Amato et al. Cancer Res 2015; Romagnolo et al. BMC Cancer. 2015; Vacher et al. PLoS One 2018
Non-Small Cell Lung Cancer Lin et al. Toxicologic Pathology 2003;Ye at al. Clin Cancer Res 2018
Prostate cancer Richmond et al. PLoS One 2014; Ghotbaddini et al. PLoS One 2017; Yu et al. DNA Cell Biology 2017
Glioblastoma Opitz et al. Nature 2011; Gabriely et al. Trends in Endocrinology & Metabolism 2017
AhR in tumor indications
ESCA - Esophageal carcinomaGBM - GlioblastomaLAML - Acute Myeloid LeukemiaPAAD - Pancreatic adenocarcinomaSTAD - Stomach adenocarcinoma
TCGA/GTEx data – GEPIA2
Discovery on Target, Boston, Sept. 17, 2019 10
The AhR project @ Phenex - Assay Path
Chemical synthesis
Series 1
PK/PD/ADME Syngenic models
Microsomal stabilityPlasma protein binding
Cyp inhibitionSelectivity panel
hERG
Efficacy evaluation usingPhenex immuno-oncology research platform
Series 2 Series 3
PX-A446Series 2
PX-A548Series 3
PX-A590Series 1
Series 1 1/30
Series 3 1/29
Series 2 1/69
PX-A590
PX-A446
PX-A548
Series 1,2 and 3 : patents filed Feb. 2017Series 4 : patent filed July 2018
Series 4
Assay format
Direct reporter assay
Cellular target gene regulation assay
Mouse splenocyte and human PBMC assay
Discovery on Target, Boston, Sept. 17, 2019 11
Are Phenex AhR antagonists on target in vitro?
In HepG2 cells – AhR translocation upon ligand occupancy
Discovery on Target, Boston, Sept. 17, 2019 12
Are Phenex AhR antagonists on target in vivo?
PK/PD study
Discovery on Target, Boston, Sept. 17, 2019 13
Effects on AhR on the immune-tumor cell axis
Aryl hydrocarbon receptor
Direct effects on the tumor Effects on immune system / tumor microenvironment
Combination with standard of care chemotherapy
Tumor growth inhibition via IL-22 suppression
Inhibition ofIDO/TDO-Kyn-AhR axis
Effects on tumor-infiltrating immune cells
Checkpoint inhibitors
Discovery on Target, Boston, Sept. 17, 2019 14
Effects on AhR on the immune-tumor cell axis
Aryl hydrocarbon receptor
Direct effects on the tumor Effects on immune system / tumor microenvironment
Combination with standard of care chemotherapy
Tumor growth inhibition via IL-22 suppression
Inhibition ofIDO/TDO-Kyn-AhR axis
Effects on tumor-infiltrating immune cells
Checkpoint inhibitors
Discovery on Target, Boston, Sept. 17, 2019 15
AhR antagonists enhanced the efficacy of chemostatic drugs
0
5 0 0
1 0 0 0
1 5 0 0R
TV
(%
) o
n d
ay
25
P X -A 2 7 5
G e m c ita b in e
V e h ic le
G e m c ita b in e+ P X -A 2 7 5
ns * ****
*
days after cell inoculationC57BL/6 mice0 8 15 22 29
Gemcitabine 50mg/kg i.p.
vehicle / PX-A275 20mg/kg/d p.o.
1
52
26
randomization
Panc02-HA tumors(tumor vol = 50-250mm3)
Discovery on Target, Boston, Sept. 17, 2019 16
Effects on AhR on the immune-tumor cell axis
Aryl hydrocarbon receptor
Direct effects on the tumor Effects on immune system / tumor microenvironment
Combination with standard of care chemotherapy
Tumor growth inhibition via IL-22 suppression
Inhibition ofIDO/TDO-Kyn-AhR axis
Effects on tumor-infiltrating immune cells
Checkpoint inhibitors
Discovery on Target, Boston, Sept. 17, 2019 17
o IL22 produced ----> ILCs, Th22 cells ----> effects on homeostasis, protection & regeneration of epithelial tissues (gut, skin, brain, pancreas,liver etc.)
o It directly participates in inflammatory diseases in the gut (IBD) and skin (Psoriasis)
o IL-22 binds to IL22RA1 to drive tumor growth and metastasis via Stat3 signaling
Modified from Hernandez 2018, EJI, Vol. 48: 15-31
Bridging the immune-tumor axis via Ahr controlled IL22
Discovery on Target, Boston, Sept. 17, 2019 18
Wu et al. Clin Immunol 2014Kirchberger et al. JEM 2013
o IL-22 and Th22 cells are increased in the TME of various tumors (e.g. lung, gliomas, colorectal carcinoma and pancreatic ductaladenocarcinoma)
IL22 and IL22+ lymphocytes negatively correlate with survival of colon cancer patients
Survival in Colon Cancer Patients
Discovery on Target, Boston, Sept. 17, 2019 19
PX-A548
PX-A275
PX-A758
PX-A446
IL22 as a PD marker of AhR Antagonism
Th17/Th22 cells
AhR antagonism
AhR agonism Increase in IL22
Decrease in IL22
Facilitatestumor progression
Restrictstumor progressionTh17/Th22 cells
PX-A548
PX-A275PX-A758PX-A446
Discovery on Target, Boston, Sept. 17, 2019 20
Screening syngeneic models for AhR & IL22/IL22RA1 expression
Discovery on Target, Boston, Sept. 17, 2019 21
Increase in intra-tumoral CD8+ T cells and M1 macrophages (day 35)
AhR antagonist shows single agent anti-tumor activity in syngeneic pancreatic tumor model
C57BL/6 mice (n=10)
0 16 35
Panc02-HA 1x106 cells implanted
Randomization
Days after tumor cellinoculation
daily p.o. treatment
PX-A590
31
v e h icle
1 0mg /k
g
3 0mg /k
g
1 0 0mg /k
g0
2 0 0
4 0 0
6 0 0
8 0 0
d a y 2 8
Tu
mo
r V
olu
me
[m
m3
] * * **
P X -A 2 4 5 9 0PX-A590
Ve h ic le P X -A2 4 5 9 00
1 0
2 0
3 0
% C
D8
+ o
f li
ve
ce
lls
*
PX-A590
PX-A590
Discovery on Target, Boston, Sept. 17, 2019 22
Aryl hydrocarbon receptor
Direct effects on the tumor Effects on immune system / tumor microenvironment
Combination with standard of care chemotherapy
Tumor growth inhibition via IL-22 suppression
Inhibition ofIDO/TDO-Kyn-AhR axis
Effects on tumor-infiltrating immune cells
Checkpoint inhibitors
Effects on AhR on the (metabolic) immune-tumor cell axis
Discovery on Target, Boston, Sept. 17, 2019 23
AhR antagonist
---> CD8+ T cell increase---> Treg reduction---> MDSC reduction
Inhibiting the IDO-Kyn-AhR pathway
Opitz et al. Nature 2011Murray et al. Nat Rev Cancer 2014Platten et al. Frontiers in Immunology 2015Lui et al. Nat Commun. 2017Cheong et al. Trends Pharmacol Sci. 2018Xue et al. Frontiers in Immunology 2018
Discovery on Target, Boston, Sept. 17, 2019 24
IDO/TDO impact ---> Kynurenine are present across a spectrum of tumor
Li et al. Nature Med. 2019
Discovery on Target, Boston, Sept. 17, 2019 25
AhR antagonists enhance anti-PD-L1 efficacy in MC38 tumor model
randomizationMC38 cell
inoculation
6 9 12 25
a-PD-L1
vehicle / compounds 2x10mg/kg/d
C57BL/6 mice15 days after cell inoculation
0 1 0 2 0 3 00
1 0 0
2 0 0
3 0 0
4 0 04 0 05 0 06 0 07 0 08 0 0
d a y s a fte r M C 3 8 c e ll in o c u la tio n
Tu
mo
r V
olu
me
[m
m3
] v e h ic le
a -P D -L 1
a -P D -L 1 + P X -A 2 5 5 4 8
a -P D -L 1 + P X -A 2 5 7 5 8
***
****
PX-A548
PX-A758
Discovery on Target, Boston, Sept. 17, 2019 26
Effects on AhR on the immune-tumor cell axis
Aryl hydrocarbon receptor
Direct effects on the tumor Effects on immune system / tumor microenvironment
Combination with standard of care chemotherapy
Tumor growth inhibition via IL-22 suppression
Inhibition ofIDO/TDO-Kyn-AhR axis
Effects on tumor-infiltrating immune cells
Checkpoint inhibitors
Discovery on Target, Boston, Sept. 17, 2019 27
D M S O P X -A2 7 50
1 0 0
2 0 0
3 0 0
CD
8+
T c
ell
s/u
l
P B M C IL -2
P B M C IL -2 + T G F b
C D 8 + T C e ll n u m b e rs(a fte r re s tim u la tio n )
Direct AhR antagonist activity on immune cell subsets
P X-A7 5 8 P X-A5 4 80
1 0 0 0 0 0
2 0 0 0 0 0
3 0 0 0 0 0
4 0 0 0 0 0
A h R c o m p o u n d s
IFN
g p
g/m
l
H u m a n P B M C s0 .2 2 2 µM
0 .0 7 4 µM
0 .0 2 5 µM
0 .0 0 8 µM
0 .0 0 3 µM
D M S O
P X-A7 5 8 C H 2 2 3 1 9 11 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
A h R c o m p o u n d s
IFN
g p
g/m
l
M o u s e N K c e lls5 µ M
1 .2 5 µ M
0 .3 1 3 µ M
0 .0 7 8 µ M
0 .0 2 0 µ M
0 .0 0 5 µ M
D M S O
P X-A7 5 8 P XA5 4 8
5 0 0 0
1 0 0 0 0
1 5 0 0 0
A h R c o m p o u n d s
IL2
pg
/ml
H u m a n P B M C s0 .2 2 2 µM
0 .0 7 4 µM
0 .0 2 5 µM
0 .0 0 8 µM
0 .0 0 3 µM
D M S O
Immune cellsAhR antagonism Myeloid cells T cells
NK cells Increase in:
IFNg production
Increase in :
CD8 numbersIFNg productionIL2 production
Discovery on Target, Boston, Sept. 17, 2019 28
o AhR is an attractive, small-molecule cancer target
o AhR (as a transcription factor) contributes to a variety of cancer relevant pathways
o Combining direct effects on tumor tissue with direct effects on the immune system
o A series of proprietary, selective and potent AhR antagonists have been identified by Phenex
o In syngeneic tumor models AhR antagonists have:
o Single agent activityo Synergy with standard-of-care chemotherapyo Synergy with checkpoint inhibitors
o Preclinical development activities for a development candidate has been started
29
Summary
Discovery on Target, Boston, Sept. 17, 2019
Acknowledgements
Chemistry/Medicinal Chemistry
Christoph Steeneck
Aurelie Mallinger
Olaf Kinzel
Marta Czekanska
Biology
Sheena Pinto
Ulrich Deuschle
Larisa Buselic-Wölfel
Michael Albers
Helen Krol
Floriane Braun
Ibrahim Sektioglu
Gisela Eisenhardt
Preclinical & Clinical Development
Manfred Birkel
Sabine Eigenbrod-Giese
Our AhR Team
Discovery on Target, Boston, Sept. 17, 2019 30
