This document outlines the history and mechanisms of the indoleamine-2,3-dioxygenase (IDO) pathway and its role in cancer immunotherapy. It discusses key findings such as: 1) Munn's 1999 discovery that IDO suppresses T-cell mediated rejection of tumors and fetal allografts by depleting tryptophan. 2) Mechanistic studies showing IDO induces T-cell arrest and inhibits proliferation through tryptophan depletion and kynurenine production, leading to Treg differentiation and CTL inhibition. 3) Preclinical studies combining IDO inhibitors with chemotherapy or vaccines, showing enhanced anti-tumor effects. 4) Ongoing clinical

1. Indoleamine-2,3 Dioxygenase
One Key from Innate to Adaptive
IDO Pathway from Bench to
Clinic
Houssein Abdul Sater
12/11/15
It’s difficult but not impossible to summarize great men work in one hour!

2. Outline
• Background and History
• Primary Bench Work On IDO and its Inhibitors
• Key Immunology Concepts Learned over years
• Case Presentation
• Rationale of Combination Immuno therapy
• Rationale of combination with Chemotherapy
• Ongoing Clinical studies: Different Molecules, Efficacy, and Toxicity
• Back to case
• IDO In HNSCC project

3. History
• 1863 Rudolph Virchow: potential role of Immune system in cancer
• Early 20th Century: Paul Ehrlich Theory of Immunosurveillance
• 1950s: Macfarlane Barnet Revitalized
Lewis Thomas
• 1960s Studies on partially immunodef “Nude” mice provided
• 1970s arguments against immunosurveillance
• Late 1990s-Early 2000s: Transgenic Mice with defined defects in
innate or adaptive immunity used in carcinogenesis studies
• 2004: Robert Schreiber Immuno editing Theory

4. Notions behind Immunoediting
• Immunodeficient Mice develop more spontaneous and carcinogen
induced tumors
• ‘Darwanian’ concept where Immunity exert pressure to select tumor
cells that eludes it. Analogy with Abx resistance
• All happen in tumor microenvironment or “battle field”

5. Immunosuppressed Patients at Increased risk
of cancer
• Transplant pts, AIDS, Immunodef Sds
• Virally Induced: NHL, Kaposi, GU and anogenital tumors ( HIV,
HHV8,EBV, HPV, HCV etc…)
• Just another evidence of Immunosurveillance
• What about non virally induced tumors?
• Knock out mice for key immunoregulatory genes as INF𝛾, Stat 1 and
Rag2 had increased tumor incidence
• Chronic Inflammation and Autoimmune disease have increased risk of
developing cancer!
• Induced Inflammation by surgery, XRT, chemo etc help cancer ?

7. Steps in Stimulating Cancer Specific Immunity
Melanoma
“Danger”
e.g. HSP
IFN-α
MAGE I
MARTI
(TAAs)
Immature DC
Activated mature DC
Migration to lymph node
Lymph node
Migration from
lymph node
CD8
CD4
DC
CD4
TH
CD8
CTL
APC
CD40
CD40
B7
TCR
MHC I
CD28 CD40
CD40L
TCR
MHC II
CD40L
CD40
B7
CD28
Help
e.g.
IL-2

8. Tumor induces Immunosuppression through
various mediators

9. Why Do Most Tumors Evade
Immune Recognition?
Murphy et al, 2008.

10. Foxp3 = forkhead box P3. Fridman et al, 2011; Disis, 2010a.
Multiple Factors Impact the
Tumor Immune Microenvironment
Pro-Tumorigenic
Inflamation
Anticancer
Immunosurveillance
Cell Types M2 macrophages
Myeloid-derived suppressor
cells
Neutrophils
Foxp3+ T reg. Th17 cells
Dendritic cells
M1 macrophages
cytotoxic CD8+ T cells with a
memory effector phenotype
Cytokine
Profiles
Th2, Th17 Th1
CX3CL1
CXCL9, CXCL10
Distribution Peritumoral Intratumoral, close to cancer
cells, as well as in the
invasive front
Associated
Features
Stat3 phosphorylation High endothelial venules
Functional
Impact
Negative prognostic impact Positive prognostic and
predictive impact

11. Tryptophan

12. Tryptophan Observations
• 1 of the 9 essential AA and 22 AA Total
• Precursor for Neurotransmitter Serotonin
• Present in lots of food and “Turkey”
• 1936: 1st paper on its metabolism in Germany
• 1967: Increased in urine from Breast cancer pts, and decreased after
tumor resection. Rose et.al
• Increased in different cancers: Hodgkin, Leukemia, Breast, Prostate
etc

13. What is IDO
• One of Tryptophan Catalyzing enzymes- Essential
• Originally called D Tryptophan Pyrrolase in 1963
• First isolated from Rabbit intestine by Hayaishi in Berlin
• 2 genes INDO 1 and INDO2 on chromosome 8
• IDO is ubiquitous and may catalyze other indols: Serotonin,..
• 2 domain structure of alfa helical domains with central heme
• TDO another enzyme in Liver that catabolizes Tryptophan
• Both IDO and TDO lead to L Kynurenine

15. IDO Regulation
• IDO induced by viruses, LPS, INF
• Inflammatory signals INF gamma , JAK-STAT Increased IDO
• JAK inhibition Inhibit IDO pathway
• Increase in INF gamma lead to cells in cell cycle arrest reversed by IDO
inhibition: due to increased caspase 3
• TNF alfa and INF gamma synergize to increase Kynurenines
• TDO induced by Tyrosine, histidine, glucocorticoids, kynurenine

16. IDO pathway Roadmap: Anti to Pro Cancer
Preclinical Observations
Prognostic significance and expression
IDO Pro (immunosuppressive) or Anti Cancer (part of immune response)
Ground breaking discoveries by Dr Munn 1999
Mechanistic studies: T cells, DCs, monocytes, macrophages, and microglia
GCN2 pathway: Munn et al., Metz et al.
Treg activation and CTL inhibition(proliferation and differentiation)
AHR : Treg Differentiation
mTOR
Animal Studies
IDOI + Vaccine
IDOI + Chemo: Muller et al.
Clinical Studies: Phase I & Phase II studies

19. IDO expression and prognosis
• Mostly Sporadic, but could also be focal, or diffuse (diffuse worse prognosis
in study of Ov Ca by Okamoto et al. 2005; 11m:17m:41m)
• High versus low ( high a/w increased mets to liver from colon ca,
Brandacher et al. 2006; 70% vs 50%; no survival diff)
• Good prognosis
HL
HCC
• Bad prognosis
 Ovarian: a/w paclitaxel resistance
 Endometrial
 Colon
 Melanoma
 Lung : Eos +IDO

20. Groundbreaking Studies
• Pregnant female Mice treated with
1-methyl tryptophan (1-MT) lose tolerog-
enic state that protects fetus from mater-
Nal immune system
• T cells activation inhibited by G1 arrest
by catabolism of Tryptophan in APCs sec
To IFN-γ

21. Placenta

22. Conclusion – a new paradigm for T-cell
suppression?
• IDO creates immune tolerance zone where Tryptophan lacks and T
cells can’t attack fetal cells to maintain allogeneic conceptus safe
• New mechanism to suppress T Cell Function/proliferation inVivo
• What is the mechanism?
Tryprtophan depletion
Tryptophan Metabolites, any role?
How can the fetus grow without tryptophan?

23. Figure 8. Overexpression of IDO before transplantation suppresses allograft rejection. Upper panel: Gross anatomy of transplanted lungs (L), and
native lungs (R) in untreated, empty vector-, or IDO vector-treated allograft recipients. Untreated and empty vector-treated allografts were shrunken,
hard, and disfigured. In contrast, IDO vector-treated allografts were soft and exhibited nearly normal gross anatomy. Lower panel: Histology of
transplant and native lungs from each group. Untreated and empty vector–treated allografts showed severe (Grade 4) rejection with extensive
mononuclear cell infiltrates. In contrast, few cellular infiltrates were observed in IDO-vector–treated allografts consistent with mild (Grade 1) rejection
(×20 magnification). Data are representative of three individual transplants per group.
Am J Respir Cell Mol Biol,
http://www.atsjournals.org/doi/abs/10.1165/rcmb.2003-0268OC

24. Figure 4. Lung iAPC-derived IDO actively suppresses allogeneic T cell proliferation. Freshly isolated irradiated C57BL/6 lung iAPCs (circles), or liver
(squares) or splenic (triangles) APCs, were co-cultured with allogeneic (A) lung or (B) splenic T cells (3 × 105) at the indicated ratios. Proliferative
responses were determined as mean ± counts per minute (CPM) of thymidine incorporated into the T cells in triplicate cultures (#P < 0.01 compared
with DC:T cultures from liver and spleen at same ratios; *P < 0.02 and **P < 0.001 comparing DC:T cultures from liver and spleen at the same ratios).
(C) APCs and allogeneic lung T cells were cultured at 0.5: 1 (APC:T) in the presence of increasing concentrations of the IDO inhibitor, 1-MT. As a
control, 1-MT did not affect spontaneous proliferation in DCs or T cells cultured alone (*P < 0.01comparing lung DC: lung T co-cultures with 250 and
500 mg/ml 1-MT to untreated cultures).

25. IDO Expressed by Dendritic Cells decreases MLR
• Lee et al. 2003 IDO + APC exist even before overt mets
• Munn et al. 2004
• Hold Poor Prognosis

26. J Clin Invest. 2007 Sep 4; 117(9): 2570–2582. Sharma et al.

27. IL = Interleukin; TNF = tumor necrosis factor; TGF = transforming growth factor.
DeNardo et al, 2010.
The Immune System Is All About “Checks and
Balances”
NK/NKT TH1 TH17 TH17 B cell TH2 TREG
Tumor Regression Tumor Progression
Angiostatic Pro-
angiogenic
Direct
cytotoxicity
Tissue
remodeling
Immune
surveillance
Immune
suppression
Myeloid Phenotype
Regulatory loops
IL-12, IL-2, IFN-g, TNF-a IL-4, IL-5, IL-10, TGF-b
N2
M2
DC2DC1
M1
N1

29. IDO pathway Roadmap: Anti to Pro Cancer
Preclinical Observations
Prognostic significance and expression
IDO Pro (immunosuppressive) or Anti Cancer (part of immune response)
Ground breaking discoveries by Dr Munn 1999
Mechanistic studies: T cells, DCs, monocytes, macrophages, and microglia
GCN2 pathway: Munn et al., Metz et al.
AHR : Treg Differentiation
mTOR
Treg activation and CTL inhibition (proliferation and differentiation)
Animal Studies
IDOI + Vaccine
IDOI + Chemo: Muller et al.
Clinical Studies: Phase I & Phase II studies

34. IDOTGF-βTregs

36. • Hongbo Chi 2012
• Nature Reviews
Immunology

37. AHR- Aryl Hydrocarbon Receptor

38. Munn et al. 2013- Indoleamine 2,3 dioxygenase
and metabolic control of immune responses

39. IDO-activated Tregs block alloreactive T cell responses in vivo.
Babak Baban et al. J Immunol 2009;183:2475-2483Copyright © 2009 by The American Association of Immunologists, Inc.

40. Blocking STAT3 activation inhibits MDSC development and tumor metastasis in the 4T1 mouse
mammary tumor model. 4T1 mouse mammary carcinoma cells (3 × 106/mouse) were injected
into mammary fat pads of BALB/c mice.
Jinpu Yu et al. J Immunol 2014;193:2574-2586
Copyright © 2014 by The American Association of Immunologists, Inc.

42. IDO functions or potentials
• Induced by inflammation
• Apoptosis
• Immune suppression
• New Epitope Tolerance
• Treg prolipheration
• MDSC proliferation
• Neurotixic
• Diabetic retinopathy (endothelial cells)
• Etc

43. IDO functions or potentials
• Anti-inflammatory: suppress innate immune inflammation
• Tolerogenic: creates antigen-specific unresponsiveness in T cells, seen
as a continous extent of thymic function in periphery
• IDO inhibition proves evidence for role in allogeneic tolerance in mice
placenta and mucosal tolerance
• IDO inhibition led to autoimmunity and increased inflammation, but
not in CTLA4/Treg deficient mice
• Lethal lupus like autoimmunity in mice IDO inhibited treated with
apoptotic cells

44. Cancer use of IDO pathway
• Acquired tolerance to apoptotic cells. Apoptotic cells induce IDO in
CD169+ macrophages in the spleen
• Although IDO sounds to be local, and expressed in low % in tumor,
APC and immune cells so how can tumors use it systemically?
• IDO drives CD4+Tcells into FoxP3 inducible Tregs (PD1 Dependant)
• Tregs are either thymically derived or newly produced in periphery
• They will have increased CTLA4 and PD-1 and ready to suppress
• IDO stabilizes Tregs by maintaining corepressor Eos (1kzf4) and
prevent IL-6 into Thelper like prominflammatory cells

45. Cancer use of IDO pathway
• Cell death free DNA, ATP, HMGB1, phosphatidylserineTLR, TGF
• Human Tumor mileu highly immunosuprressive
High TGF, IL-10, sup Tregs
M2-like macros
High MDSC
High IDO APC in tumor and TDLN
• Also: chronically inflamed, may express antigens not normally
expressed in normal tissues(oncofetal), may present normal self Ags
in aberrant way(immunogenic?), continuous cell death
• Would chemo or radiation help unveil tumors or their antigens and
how does it affect IDO expression

46. IDO Inhibitors
• 1-methyl-dl-tryptophan, β-(3-benzofuranyl)-dl-alanine and β-[3-
benzo(b)thienyl]-dl-alanine are competitive inhibitors for indoleamine
2,3-dioxygenase. By S Cady et al. 1991
• Newlinks : 1MT or Indoximod , and GDC-0919 and many
others(IDO/TDO inhibitors)
• Incyte Corp. : Epacadostat
• Bristol-Myers Squibb: F001287
• Curadev Pharma: IDO/TDO-I: PCT/US14/24920
• IOmet Pharma: IDO/TDO-I:

47. Indoximod (1-Methyl-D-tryptophan, Phase I
NSC-721782; IND # 78189, IND#78060)
• Prioritized by NCI’s RAID program for clinical development as an IDO
pathway inhibitor
• Good oral bioavailability with favorable pharmacokinetics with once
daily dosing.
• No significant toxicity was observed in animals.
• 65 patients with a variety of solid malignancies (breast, colon,
melanoma, sarcoma, pancreatic, lung).
• The maximally administered dose of indoximod was 2000mg PO BID
given in continuous 28 day cycles
• . The conclusion was the drug was well tolerated, biologically active,
and had modest activity as monotherapy.

48. Rationale for Combination of Indoximod and
Chemotherapy
• 2005: Muller et al. studied MMTV-Neu mice with autochthonous tumors on the
interaction between indoximod and various chemotherapeutic agents.
• Mice with 5-10mm tumors were enrolled into control and treatment groups.
1. Indoximod alone
2. Chemotherapy alone (paclitaxel, doxorubicin, cyclophosphamide, and others)
3. Combination of indoximod and chemotherapy
• The combination of indoximod plus paclitaxel caused 30% tumor regression and
histologically there was significantly enhanced tumor cell death with the
combination versus either agent alone.
• This synergism was abrogated when the mice underwent CD4+ T cell depletion
prior to treatment with the combination, suggesting the immune response played
a role in the observed effect.

49. IDO inhibition enhances the efficacy of certain commonly used cancer chemotherapeutic agents

50. Potential Combinations
• IDO Blockage +Checkpoint inhibitors  better results
• CD19 Car Tcells treatment was strongly inhibited by IDO exp and this
was reversed by IDO Inhibition
• Vaccine therapy + IDO blockage
• Potential roles for key tyrosine kinase pathways as PI3K, MTOR and
additional effect on MDSC and Tregs
• Car T cells +IDO blockage

51. Vaccine Therapy and 1-MT in Treating Patients With Metastatic Breast Cancer
Participants receive adenovirus-p53 transduced dendritic cell
(Ad.p53-DC) vaccine ID in weeks 1, 3, 5, and 10, and then every
3 weeks for 6 total doses. Participants also receive
1-methyl-d-tryptophan (indoximod) orally (PO) daily (QD)
on days 1-21. Treatment with 1-methyl-d-tryptophan repeats
every 28 days (patients with stable disease) for up to 12 courses
in the absence of disease progression or unacceptable toxicity.
Treatment (Vaccine Therapy,
1-methyl-d-tryptophan)
Number of Participants
[units: participants] 44
Age
[units: participants]
<=18 years 0
Between 18 and 65 years 36
>=65 years 8
Age
[units: years]
Mean (Full Range)
52.68 (27 to 73)
Gender
[units: participants]
Female 40
Male 4
Region of Enrollment
[units: participants]
United States 44
ClinicalTrials.gov Identifier:
NCT01042535
First received: January 4, 2010
Last updated: September 21, 2015
Last verified: September 2015

52. Vaccine Therapy and 1-MT in Treating
Patients With Metastatic Breast Cancer

53. [TITLE]

54. Phase I INDOXIMOD+TMZ
• 12 Patients: 10 GBM, 1ODG, 1 AAstro
• 3 received Ind 600mg BID, 3 1000mg BID, 6mg 1200 BID
• 4 remain on study and 9 still alive
• MTD 1200 BID
• AE: Mainly Fatigue in 2 pts related to Indoximod and GI upset
treatment related

55. [TITLE]

56. A first in man phase I trial of the oral immunomodulator,
indoximod, combined with docetaxel in patients with
metastatic solid tumors.
• Soleman et al. 2014
• Phase I
• Docetaxel at 60 or 75mg/m2 IV Q3weeks plus indoximod at 300,
600,1000, 2000, and 1200 mg PO twice daily continuously
• Well tolerated with no increase in expected toxicities or
pharmacokinetic interactions.
• 18% (4/22) had partial response (2 breast, 1 lung, 1 thymic)