Indoleamine-2,3 DioxygenaseOne Key from Innate to Adaptive

IDO Pathway from Bench to ClinicHoussein Abdul Sater

12/11/15

It’s difficult but not impossible to summarize great men work in one hour!

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

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

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”

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 ?

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

CD8CD4

DC

CD4

TH

CD8

CTLAPC

CD40

CD40

B7

TCR

MHC I

CD28 CD40

CD40L

TCR

MHC II

CD40L

CD40

B7CD28

Help

e.g. IL-2

Tumor induces Immunosuppression through various mediators

Why Do Most Tumors Evade Immune Recognition?

Murphy et al, 2008.

Foxp3 = forkhead box P3. Fridman et al, 2011; Disis, 2010a.

Multiple Factors Impact the Tumor Immune Microenvironment

Pro-Tumorigenic Inflamation

AnticancerImmunosurveillance

Cell Types M2 macrophagesMyeloid-derived suppressor cells NeutrophilsFoxp3+ T reg. Th17 cells

Dendritic cellsM1 macrophagescytotoxic CD8+ T cells with a memory effector phenotype

Cytokine Profiles

Th2, Th17 Th1CX3CL1CXCL9, 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

Tryptophan

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

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

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

IDO pathway Roadmap: Anti to Pro CancerPreclinical ObservationsPrognostic significance and expression IDO Pro (immunosuppressive) or Anti Cancer (part of immune response)Ground breaking discoveries by Dr Munn 1999Mechanistic studies: T cells, DCs, monocytes, macrophages, and microgliaGCN2 pathway: Munn et al., Metz et al.Treg activation and CTL inhibition(proliferation and differentiation)AHR : Treg DifferentiationmTORAnimal StudiesIDOI + VaccineIDOI + Chemo: Muller et al.Clinical Studies: Phase I & Phase II studies

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 prognosisHLHCC• Bad prognosis Ovarian: a/w paclitaxel resistance Endometrial Colon Melanoma Lung : Eos +IDO

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 arrestby catabolism of Tryptophan in APCs sec To IFN-γ

Placenta

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 depletionTryptophan Metabolites, any role?How can the fetus grow without tryptophan?

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

Published in: Kena A. Swanson; Yan Zheng; Kathleen M. Heidler; Teruaki Mizobuchi; David S. Wilkes; Am J Respir Cell Mol Biol  2004, 30, 311-318.DOI: 10.1165/rcmb.2003-0268OC© 2004 The American Thoracic Society

One PowerPoint slide of each figure may be downloaded and used for educational not promotional purposes by an author for slide presentations only. The ATS citation line must appear in at least 10-point type on all figures in all presentations. Pharmaceutical and Medical Education companies must request permission to download and use slides, and authors and/or publishing companies using the slides for new article creations for books or journals must apply for permission. For permission requests, please contact the Publisher at dgern@thoracic.org or 212-315-6441.

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).

Am J Respir Cell Mol Biol, http://www.atsjournals.org/doi/abs/10.1165/rcmb.2003-0268OC

Published in: Kena A. Swanson; Yan Zheng; Kathleen M. Heidler; Teruaki Mizobuchi; David S. Wilkes; Am J Respir Cell Mol Biol  2004, 30, 311-318.DOI: 10.1165/rcmb.2003-0268OC© 2004 The American Thoracic Society

One PowerPoint slide of each figure may be downloaded and used for educational not promotional purposes by an author for slide presentations only. The ATS citation line must appear in at least 10-point type on all figures in all presentations. Pharmaceutical and Medical Education companies must request permission to download and use slides, and authors and/or publishing companies using the slides for new article creations for books or journals must apply for permission. For permission requests, please contact the Publisher at dgern@thoracic.org or 212-315-6441.

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

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

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

AngiostaticPro-

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

IDO pathway Roadmap: Anti to Pro CancerPreclinical ObservationsPrognostic significance and expression IDO Pro (immunosuppressive) or Anti Cancer (part of immune response)Ground breaking discoveries by Dr Munn 1999Mechanistic studies: T cells, DCs, monocytes, macrophages, and microgliaGCN2 pathway: Munn et al., Metz et al.AHR : Treg DifferentiationmTORTreg activation and CTL inhibition (proliferation and differentiation)Animal StudiesIDOI + VaccineIDOI + Chemo: Muller et al.Clinical Studies: Phase I & Phase II studies

GCN2

IDOTGF-βTregs

• Hongbo Chi 2012• Nature Reviews Immunology

AHR- Aryl Hydrocarbon Receptor

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

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.

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.

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

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

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

Cancer use of IDO pathway• Cell death free DNA, ATP, HMGB1, phosphatidylserineTLR, TGF• Human Tumor mileu highly immunosuprressiveHigh TGF, IL-10, sup TregsM2-like macrosHigh MDSCHigh 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

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:

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.

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 alone2. 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.

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

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

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:NCT01042535First received: January 4, 2010Last updated: September 21, 2015Last verified: September 2015

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

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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

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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)