The Liver as Target of Autoimmunity in Immunotherapeutic Approaches to Complex

Diseases

Amy S. Rosenberg, M.D.Supervisory Medical Officer, Office of Biotechnology

Products, CDER, FDA

2

The Immune System on a Knife’s Edge: Tipping the Balance for Therapy of

Serious Diseases

CancerChronic Infection

AutoimmunityGraft Rejection

Treg

Teff

Treg

Teff

Treg

Teff

Autoimmunity Cancer/Chronic Infection

Immunological Tolerance• Definition:

– unresponsiveness to an antigen induced by exposure of lymphocytes to that antigen; antigen-specific (unlike “immunesuppression”)

• Significance:– All individuals are tolerant of their own antigens to a greater or lesser

extent (self-tolerance); breakdown of self-tolerance results in autoimmunity

– Therapeutic potential: Inducing tolerance may be exploited to prevent graft rejection, treat autoimmune and allergic diseases, and prevent immune responses in gene therapy and stem cell transplantation; breaking tolerance has led to impressive tumor responses in some patients

3

FOREIGN SELF

ExpectImmunogenicity

No toleranceNeutralize Product

Hypersensitivity

PotentialImmunogenicity

Incomplete toleranceAltered structure/Antigen Present

Epitope spreading

RareImmunogenicity

Robust toleranceNovel Route of Administration AdjuvantsHLA Haplotype Specific

• Low abundance self-protein• Aggregates of self proteins• Chemical degradation/modification of self proteins• Adjuvants

Immune Tolerance to Self Proteins is not Absolute

5

Autoimmunity is Suppressed by both Thymic and Peripheral Tregs

Local immune suppressionOral toleranceFetal toleranceMucosal tolerance

Nrp-1

APC

ThymustTreg

Tnaive

Teff

pTreg

TeffSite ofInflammation

specializedAPC

Immune homeostasisAutoimmune responses

Yadav M et al Frontiers in Immunology 2013

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Tregs Express Constitutively High Levels of CD25 (IL-2Ra) and CTLA-4 Essential for their Function

Tang and Bluestone, Nat Immunol 9:239, 2008 6

Strip CD28 ligands

Mechanisms by which Tregs Suppress Immune Responses

7

Consequences of Self Antigen Recognition in Thymus

Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 7th edition, 2011 c Elsevier8

The Hallmark of Regulatory T cells is the Transcription Factor FoxP3: Autoimmunity is Consequence of FOXP3

Mutations(Sakaguchi et al 2008)

Mother of IPEXpatient

IPEXpatient

NormalPolyendocrinopathy

Enteropathy

Immune Dysreg

X

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Liver is Relatively Spared as Focus of Autoimmunity in IPEX

• Hallmarks of IPEX are enteropathy, type 1DM and eczema: early onset of one or more in all patients

• Only 12/128 IPEX cases reported AIH, Hepatitis, Cholangitis, or Cholestatic Hepatitis (Barzaghi F et al. Frontiers in Immunology 2012)

• Immunoregulatory circuits in liver not solely dependent on Tregs

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Immunosuppressive Circuits in Liver Mediated Mainly Through Liver Resident Myeloid Cells

IL-10

TGF-β1

IDO

Cox2->PGE2

FasL

HEP

DeadHEP

“MDSC”

LSEC

pDC

mDC

HSC Kupffer

PD-L1

LSECtin

IL-10

IL-27

PD-L1

IDOPD-L1

TGF-β1 +retinol

PD-L1

kynurenine

IL-10TGFβ1other

IFN-γ

HGF

Treg

Treg

8+

4+

Crispe, IN 2014. Hepatology

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What Self-Antigens are Expressed in the Thymus?

• Ubiquitous cell-associated and circulating proteins

• Peripheral tissue antigens: expression mediated by Autoimmune Regulator (AIRE) transcription factor in thymic medullary epithelial cells and extrathymic bone marrow derived APC (eTACs)• signal self-reactive thymocytes for death or lineage deviation to

Tregs

AIRE Promotes Expression of Peripheral Tissue Antigens in the Thymus, Deleting or Deviating High Affinity Autoreactive T cells

(Mathis and Benoist 2007)

Tregs

AIRE Mutations are Associated with Autoimmune PolyendocrineSyndrome Type I: Autoimmune Polyendocrinopathy-Candidiasis-

Ectodermal Dystrophy(Kampe et al 2008)

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AIRE Mediated Expression of of Liver Proteins in the Thymus

Cyp1a2

Prg4Apoa1FggTdo2AmbpGys2FgbAlbItih4

Apoa2Apoc2Mat1aOrm1Itih3HpIgfbp1RelnHalCyp26a1

Hamp

Cytochrome P450, family 1, subfamily A, polypeptide 2

Proteoglycan 4Apolipoprotein A-IFibrinogen gamma chainTryptophan 2,3-dioxygenaseAlpha-1-microglobulin/bikunin precursorGlycogen synthase 2 (liver)Fibrinogen beta chainAlbuminInter-alpha-trypsin inhibitor heavy chain

family, member 4Apolipoprotein A-IIApolipoprotein C-IIMethionine adenosyltransferase I, alphaOrosomucoid 1Inter-alpha-trypsin inhibitor heavy chain 3Haptoglobininsulin-like growth factor binding protein 1reelinHistidine ammonia-lyasecytochrome P450, family 26, subfamily a,

polypeptide 1Hepcidin antimicrobial peptide

227.2

263.1626.8174.9138.4139.292.2153.5146.5191

334.1345.699.5298.3144.8108.315.212.010327.3

337

20.6

31.795.92722.526.619.536.743.958

111.8121.637.81165849.68.322.32278.671.4

946.1

Gene ID Gene Annotation WT Aire KO

11

866654433

22222210.50.50.4

0.4

FoldChange

223

9480435906279364152664373775681

11107249424565130015456416152814

163

Liver ExpressionProtein atlas*

The list is the combination of SI from PNAS 2012 by Matthieu Giraud and 02670 Table2 (Microarray data from Affymetrix MgU74Av2 gene chip by using 2004.04.29 Affymetrix annotation *FPKM values or 'number of Fragments Per Kilobase gene model and Million reads', were calculated as the sum of all its protein-coding transcripts, and the average FPKM value for replicate samples were used as abundance scores) from RNA-seq analysis

16

Organ Specific Non-Endocrine Disease at Higher Freqencyin American APECED Patients

Ferre EMN et al JCI insight 2016)

16

100

80

60

40

20

0

% o

f pat

ient

s

Expression in thymicmedullary epithelial cells in AIRE WT

Expression in thymicmedullary epithelial cells in AIRE KO

Expression of cyp1a2

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Autoantibodies to CYP1A2 and Amino Acid Decarboxylase in APECED Patients

(Gebre-Medhin G et al FEBS Letters 1997)

Organ-Specific Autoimmunity Requires “Two Hits”

Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 7th edition, 2011 c Elsevier

Failure of control mechanisms is the underlying cause of most autoimmune diseases

19 Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 7th edition, 2011

20

Immune Suppression Mediated by Tregs and Immune Activation of T effector cells Modulated Orthogonally by “Checkpoint” MoleculesA B

Wolchok J et al 2016 NATURE REVIEWS CLINICAL ONCOLOGY

HLA is the Strongest Genetic Factor for Susceptibility to Autoimmune Disease

21

22

HLA Association with AIH: Implicated DRB1 Alleles Vary in Distinct Populations(Czaja A and Donaldson PT 2000 Imm Rev)

DRB1DRB1DRB1DRB1DRB1DRB1DRB1

Locus Allele Population

0301040104050404040513011301

Caucasian (NA&Europe)CaucasianJapaneseMexicanArgentina (adults)Argentina (children)Brazil

Non-HLA Genes Implicated in Autoimmunity

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Lymphadenopathy, enteropathy, eczema, infection

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The CTLA4 and PD-1 Genes are Linked to Self-Tolerance and are Involved in Autoimmune Diseases

Thyroiditis, Graves’ disease CTLA-4 European Ueda, Nature 2003 [8]and Hashimoto’s disease Vaidya, Rheumatology 2002 [72]

Diabetes mellitus CTLA-4 European Ueda, Nature 2003 [8]Asian Zhernakova, Hum Genet, 2005 [73]

Zalloua PA, Hum Immunol 2004 [10]Jin, P of Endocrinol Investig, 2014 [74]

Celiac disease CTLA-4 European Zhernakova, Hum Genet, 2005 [73]Song, Hum Immunol, 2013 [75]

Myasthenia gravis CTLA-4 South American Fernandez-Mestre, Hum. Immunol. 2009 [76]

Systemic lupus erythematosus CTLA-4 Asian Hudson, Hum Genet, 2002 [77]

PD-1 European and Mexicans Prokunina, Nat Gene, 2002 [9]Bertsias, Arthritis Rheum. 2009 [78]

Rheumatoid arthritis CTLA-4 European Vaidya, rheumatology 2002 [72]

PD-1 European and Asian Lee, Z. Rheumatol. 2015 [79]

Addison’s disease CTLA-4 European Blomhoff, J Clin Endocrinol Metabol 2004 [8]

Autoimmune disease Polymorphism Ethnic group Referred studies

CTLA-4: cytotoxic T-lymphocyte-associated antigen 4; PD-1: programmed cell death protein.

25

The Immune System on a Knife’s Edge: Tipping the Balance for Therapy of

Serious Diseases

CancerChronic Infection

AutoimmunityGraft Rejection

Checkpoint Inhibitor

Antagonists

Checkpoint Inhibitor Agonists

Treg

Teff

Treg

Teff

Treg

Teff

Autoimmunity Cancer/Chronic Infection

Organ-Specific Autoimmunity Requires “Two Hits”

Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 7th edition, 2011 c Elsevier

Failure of control mechanisms is the underlying cause of most autoimmune diseases

26 Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 7th edition, 2011

Two Hit Model for Autoimmunity in Cancer Therapeutic Setting

• Tissue Damage (chemo, RT) PLUS• Checkpoint Inhibition

• Multiple check point inhibitors• Affecting multiple cell types

• HLA haplotype associated with AIH in context of CP inhibitor therapy may not follow that for primary AIHs: further investigation warranted

27

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Deliberately or Incidentally Breaking Self Tolerance in Immunotherapeutic Approaches

• Monoclonal antibodies to checkpoint inhibitors (PD-1, PDL-1, CTLA-4)

• Monoclonal antibodies to T reg expressed and requisite molecules (eg CTLA-4, IL-2Ra (CD25))

• Chimeric Antigen Receptor T cells (CAR-T)• Inhibitors of Indoleamine Diooxygenase (IDO)

www.fda.gov

The Clinical Spectrum of Immune-Related Adverse Events Pertaining to Checkpoint Inhibition

Michot JM et al 2016

30

Immunotherapy-related Adverse Events for IpilimumabImmunotherapy-related

adverse eventsN (%)

Ipilimumab N = 234 Pembrolizumab N = 10 Nivolumab N = 7

Gastrointestinal 93 (39.7)aColitis/enterocolitis 68 (29.1)Colitis complicated by intestinal perforation 12 (5.1)Hepatitis 17 (7.3)Pancreatitis 2 (0.9)

Endocrine 79 (33.7) 2 (20.0) 3 (42.9)Hypophysitis (manifestedaspanhypopitutarism)

68 (29.1)

Thyrotoxicosis 4 (1.7) 1 (14.3)Hypothyroid 4 (1.7) 1 (10.0) 2 (28.6)Syndrome of inappropriate secretion ofantidiuretic hormone

1 (0.4)

Central adrenal insufficiency 1 (0.4)Primary adrenal insufficiency 1 (0.4)Diabetes Mellitus 1 (10.0)

Dermatologic 60 (25.6)b 5 (50.0) 2 (28.6)Rash 26 (11.1) 1 (14.3)Pruritus 15 (6.4)Vitiligo 8 (3.4) 1 (10.0)Dermatitis 7 (3.0) 3 (30.0)aSweet syndrome 3 (1.3)Drug eruption 2 (0.9)Poliosis 1 (0.4)Delayed hypersensitivity reaction 1 (0.4)Alopecia universalis 1 (0.4)Groverdisease 1 (0.4)Pyodermagangrenosum 1 (0.4)Toxic epidermal necrolysis 1 (0.4)Chronic non-caseationgranuloma 1 (0.4)Bullous pemphigoid 1 (10.0)Psoriasis 1 (14.3)

Abdel-Wahab N et al PLOS One 2016

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Comparison of Immune Related Toxicities Between PD-1/PD-L1 and CTLA-4 Inhibitors

De Velasco G et al Cancer Imm Res 2017

Two Hit Model for Autoimmunity in Cancer Therapeutic Setting

• Tissue Damage (chemo, RT) PLUS• Checkpoint Inhibition

• Multiple check point inhibitors• Affecting multiple cell types: Treg (loss of

function) and Teff (gain of function)

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Checkpoint Inhibitors Acting on Orthogonal Targets Have Greater Incidence and Severity of Immune Related AEs

(Larkin J et al NEJM 2015)

34

The Two-Faced Cytokine IL-2

• Interleukin-2 is the prototypic T cell growth factor, required for initiating clonal expansion of T cells in response to antigen

• BUT: knockout of IL-2 or the α or β chain of the IL-2R results not in immune deficiency but in systemic autoimmunity and lymphoproliferation

35

IL-2 Receptor-α Deficiency and Features of Primary Biliary Cirrhosis

Aoki CA et al J. Autoimmunity (2006)

36www.fda.gov

What is the second hit? HLA? Autoimmune context?

ZINBRYTA (daclizumab,anti-CD25 mAb) treatment of Multiple Sclerosis: Risk of Autoimmune Hepatitis and Other Autoimmune Phenomena

Other Immune-Mediated DisordersIn addition to autoimmune hepatitis, immune-mediated disorders such as skin reactions, lymphadenopathy, and non-infectious colitis can occur in patients treated with ZINBRYTA. Overall, serious immune-mediated conditions were observed in 5% of patients treated with ZINBRYTA

Two Hit Model for Autoimmunity in Cancer Therapeutic Setting

• Tissue Damage (chemo, RT) PLUS• Checkpoint Inhibition

• Multiple check point inhibitors• Affecting multiple cell types

37

IPI plus Dacarbazine Caucasian Population: The most common grade 3 or 4 immune-mediated AE: hepatitis

(Robert C et al NEJM 2011)

Increased Susceptibility to to Auotimmune Hepatitis in Japanese Patients Treated with Ipi plus

Dacarbazine: HLA Related? (N. Yamazaki et al Cancer Chemother Pharmacol (2015)

Treatment of Immune Mediated Hepatitis following Ipilimumab and Dacarbazine

(Wolchok J NEJM 2011)

• 80% of patients with high grade immune mediated received glucocorticoids and/or MMF in the Ipi + Dac group– LFTs normalized in 68% of patients-median time to normalization 9.9

weeks (1-56 weeks)

• 33% of patients in Dac alone group with grade 3-4 elevations in LFTs treated with steroids

• No patient deaths

Acknowledgements

• Jeff Bluestone, UCSF• Abul Abbas, UCSF• Steven Kozlowski, OBP, FDA• Lourdes Villalba, OND, FDA• Mark Avigan, OSE, FDA

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Expression in thymicmedullary epithelial cells in AIRE WT

Expression in thymicmedullary epithelial cells in AIRE KO

Expression of AADC

43

Autoantibodies against L-amino acid decarboxylase in patients with APECED

(Husebye E et al J. Clin. Endo.&Metab. 1997)