New treatments for Type 1 diabetesPrinciples of Pediatric Clinical Pharmacology

February 21, 2018Kevan C. Herold, MD

Departments of Immunobiology and Internal MedicineYale University

Disclosures

• I have a patent application for an assay to measure beta cell death

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Worldwide Incidence Trends

Vehik & Dabelea. DMRR 27: 3-13, 2011

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Results from the DCCT: 1993

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There remains an unmet clinical need for treatment of type 1 diabetes: clinical practice in the 21st century Country Median A1c (%) n

Age < 15

England 8.4 15959

Denmark 8.0 1499

US 8.4 10,870

Age 15-24

England 9.1 20939

Denmark 8.5 2575

US 8.4 7189

Age > 24

England 8.3 144840

Denmark 7.9 18648

US 7.5 7461(McKnight et al, Diabet Med, 2014)

(Miller et al, Diabetes Care, 2015)

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Outline

• Beta cells are involved in diabetes!• Successes and failures of immune therapy for Type

1 diabetes• Next steps:

• Prevention• individualized approaches• Mechanistically directed combinations

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General concepts of the pathogenesis of Type 1 of relevance to treatment• T and B lymphocytes are involved in the pathogenesis of the

disease. In addition, the limited human anatomic data indicates that innate immune cells are also present in human insulitis.

• Autoantibodies are not responsible for destruction of insulin producing cells but B cells are required in the early stages for disease pathogenesis. T cell alone can mediate beta cell destruction.

• The T cell target(s) and receptor(s) that are involved in human disease have not been identified. However, important antigens include insulin, GAD65, IGRP, and others. The multitude of antigens suggest a polyclonal response is involved although there appears to be a hierarchy of antigens.

• Re-establishment of immune regulation prevents and even reverses diabetes.

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Updated natural history of Type 1 diabetes

(Herold, Vignali, Cooke, Bluestone, NRI, 2013)

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Progression to Diabetes vs Number of Autoantibodies(GAD, ICA512, Insulin)*

Ziegler et al JAMA 2013

*3 large observational studies (DIPP, Baby DIAB/DIET, DAISY)

Rationale for an assay to detect beta cell death in vivo• Beta cell function is may be affected by environmental factors

such as glucose, fatty acids, etc.• Beta cell death is a silent event. We only know it has happened

after it has happened.• Methylation is one of the epigenetic control mechanisms that can

affect gene transcription.• When cells die, they release their DNA into the bloodstream.• The only source of demethylated insulin DNA should be dead

beta cells.• The amount of demethylated insulin DNA, however, is likely to be

below limits of detection even by PCR.• We first used a nested PCR (Akirav PNAS 2011) and then droplet

digital PCR (Usmani-Brown Endocrinology 2014) to detect beta cell-specific DNA amidst methylated DNA from other cells.

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TN-01 “Pathway to prevention”

• Subjects are enrolled if they have a first or 2nd

degree relative with T1D and are at least 1 biochemical autoantibody+

• Subjects undergo oral glucose tolerance testing and PBMC and serum are collected. The samples are collected on a ~q6 mo basis.

• Some individuals progress to T1D (progressors) and others do not (non-progressors) over the period of observation. The general understanding about progression of disease is:

Relative, 0 autoabs→ 1 autoantibody→2 or more autoabs→2 or more autoabs+dysglycemia→diabetes

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TrialNet Natural History subjects:

Subject groups:1) “at risk”: autoantibody+ relative of a patient, followed for up to 4

years. Some developed T1D (“progressors”), others did not (“non-progressors”).

2) “high risk”: 2+ autoantibody+, dysglycemic relatives. The vast majority will develop diabetes within 5-10 yrs (Wherrett et al Diabetes Care, 2015)

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High risk individuals have high levels of unmeth INS DNA

(Herold et al JCI 2015)

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Beta cell death in at-risk participants followed for up to 4 yrs

Average levels All data points

(Herold et al JCI 2015)

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Modified model of T1D

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STAGE 4

GeneticRisk

ImmuneActivation

ImmuneResponse

STAGE 1 STAGE 2 STAGE 3

T1D Disease Progression

Normal Blood Sugar≥ 2 autoantibodies START OF T1D

Abnormal Blood Sugar≥ 2 autoantibodies

Clinical Diagnosis≥ 2 autoantibodies

Immune ResponseDevelopment of single

autoantibody

Starting PointIf you have a relative:

15x greater risk of developing T1D

GeneticRisk

ImmuneActivation

ImmuneResponse

STAGE 1 STAGE 2 STAGE 3

Long-standingT1D

Immune ActivationBeta cells are attacked

The Stages to Type 1 Diabetes

STAGE 4

67.4% are in the detectable range (> 0.03 nmol/L)

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Non α, non β cells in islets of 9 wkold NOD mice

NOD B6

Glucagon

Insu

lin

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A new subpopulation of beta cells develops during progression of diabetes in NOD mice

Bottom

Top

Rui et al, Cell Metabolism (in press)Herold | Slide #19

RNA-seq analysis of gene expression in Btm/Top β cells

Rui et al, Cell Metabolism (2017)

- 1 5 - 1 0 - 5 0 5 1 0 1 5

1 0 - 1 5

1 0 - 1 0

1 0 - 5

1 0 0

L o g f o ld c h a n g e

p-v

alu

e

T 1 D a n t i g e n s

m e t a b o l i s m

r e p l i c a t io n

d e a t h / s u r v i v a l

i m m u n e m e d ia t o r s

The new β cell subpopulation shows reduced expression of β cell identity genes.

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I ns 1

I ns 2

S l c 2 a 2G c g

S s tM

a f a

F o x o 1

N k x 6 - 1P d x 1

C h g a

N e u r o g 3

1 2

1 6

2 0

2 4

2 8

3 2

∆C

t+2

0

T o p β c e l l s B tm β c e l l s

* * * *

* * *

* * *

* * *

*

** * * *

* * * *

p = 0 . 0 5

* * *

The new subpopulation has reduce expression of diabetes antigens and increased expression of immune inhibitory ligands

Rui et al, Cell Metabolism (in press)Herold | Slide #22

CycloSaline Ctl

SS

C

FSC

The new subpopulation survives immune attack

Rui et al, Cell Metabolism (in press)Herold | Slide #23

To

pβ

(%

of

tota

lβ

ce

lls

)S a l in e C t r l C y c lo

0

2 0

4 0

6 0

8 0

* * *

Human beta cells also change with immune attack

Rui et al (in press)

SS

C

FSC

Media PBMC-HC PBMC-T1D

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M e d ia + P B M C -H C + P B M C -T 1 D0

2 0

4 0

6 0

Btm

β

(% o

f to

talβ

cells

)

********

Immune factors: Pro-inflammatory cytokinesAuto-reactive T cellsInflammatory mediators

Mature β cells Stressed β cells

Top (mature) β cells

Btm β cells

β cell features

PD-L1, Qa-2Stemness

Summary: Changes in β cells during progression of autoimmune diabetes

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Th1

Insulin

T Cell activationEffector T cells

B CellActivation

B Cell

Beta cell

CapillaryAntigen Specific

+

Treg

T cell receptor/CD3

AutoantibiodiesCD28

CD4+or8APC activationHLA

CD80/86

TLR

IL-1 TNFα

Immune therapy of Type 1 diabetes

CD2LFA3

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Outcomes of human trials

“Worked”• aCD3 mAb (x6)• Abatacept (CTLA4Ig)• Rituximab (aCD20)• Alefacept (LFA3Ig)• ATG/G-CSF

“Didn’t work”• GAD65• Anakinra (IL-1RA), canakinumab

(anti-IL-1β)• Mycophenolate mofetil+ daclizumab• Diapep277??• Rapa/IL-2• Sitagliptin+lansoprazole• Oral insulin (?)(prevention)• Parental insulin (prevention)• Nicotinamide (prevention)• Thymoglobulin

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Current Anti-CD3 mAbs

• There are 3 non-FcRbinding anti-human CD3 mAbs at the present time.

• Preventing FcR binding is important for minimizing cytokine release that occurs with OKT3.

• Otelixizumab and teplizumab are humanized.

• NI-0401 (foralumab) is fully human

Chatenoud et al

teplizumab

otelixizumab

foralumab

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Rituximab (anti-CD20)(Pescovitz, 2009)

Abatacept (CTLA4Ig)(Orban, 2011)

FcR non-binding anti-CD3 mAbs have shown efficacy in 6 clinical trials

Study 1 (Herold et al, NEJM 2002)

Protégé (Sherry et al, Lancet 2011)

(Keymeulen et al, NEJM,2005)

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From the AbATE trial (ITN027AI)

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0 10 20 300.00.10.20.30.40.50.60.70.8

Drug Control

Month

C-p

ep(ln

AUC

(nm

ol/L

)+1)

15.9 mos

FcR non-binding anti-CD3 mAbs have shown efficacy in 6 clinical trials (Protégé)

5% of subjects in the full dose group were off of insulin at 1 year vs 0% in placebo (p<0.05)

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Abatacept (CTLA4Ig- binds to CD80/86) in Type 1 diabetes

(Orban, 2011)

Alefacept (LFA3Ig) (binds to CD2 on T cells)

(Rigby et al JCI 2015)

The problem is that none of the interventions have had lasting effects on beta cell function

Teplizumab (FcR non-binding anti-CD3 mAb) Otelixizumab (FcR non-binding anti-CD3 mAb)

Abatacept (CTLA4Ig) Rituximab (anti-CD20 mAb)

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Months

C-p

epti

de

AU

C (

pm

ol/m

l)

0 10 20 300.0

0.2

0.4

0.6

0.8DrugControl

** *** *** **

Can we personalize therapy: ieidentify responders, is prevention possible?

This may also tell us how the drug works!

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*Delay=Phase II RPCT of teplizumab in patients with T1D 4-12 mos duration^AbATE=ITN Phase II open label trial of teplizumab in pts with < 3 mos T1D

Analysis of CD8CM T cells in responders in Delay* and AbATE^

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Baseline predictors of C-peptide @ 24 m

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Children show a more robust response to teplizumab (anti-CD3 mAb) than adults (Protégé)

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Placebo14-day Teplizumab

Children show a more robust response to teplizumab than adults (Protégé)

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Proposed actions of FcR non-binding anti-CD3 mAb

(Esplunges, Flavell, Nature 2011Waldron-Lynch, Herold, Sci Trans Med, 2012) (Bluestone et al)

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Fetal liver Isolate hCD34+ cells Inject them in to pupsWith in 48 hours

NOD/SCID/IL-2Rγnull mice

Week 12

Changing the microflora with antibiotics in humanized mice: What are the personal factors that may modify

the drug effects?

Test for Reconstitution

Adult mice

Treat with Teplizumab (single

dose)

Follow for graft

rejection, isolate organs

Spleen

Liver

Lymph nodes

Adrenal glands

Blood

Treat with antibiotics x 2 wks

Place a skin xenograft

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Efficacy of anti-CD3 mAb in preventing xenograft rejection is reduced with antibiotics

Anti-CD3 mAb vs Ctl Ig no antibiotics

Anti-CD3 mAb vs Ctl Ig with antibiotics

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Anti-CD3 mAb (teplizumab) prevention trial (NIDDK/TrialNet)

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Goal of the Study: Delay progression from Normal to Abnormal Glucose tolerance

Population: 2 or more antibodies (not mIAA), relatives, “dysglycemia” or abnormal glucose toleranceAge 8 and aboveN: 75

Exendin-4 enhances the reversal of diabetes by anti-CD3 mAb in NOD mice

Insu

lin fr

ee (%

)

aCD3 aCD3 +Ex-4 Ins+IgG Ex-40

20

40

60

80

All mice

Glu<350 mg/dl*

(*p<0.05; Sherry et al, Endocrinology 2007)

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Examples of mechanistically driven combinations:• Selection of responders• Anti-CD3 mAb with:

• GLP-1 receptor agonist of DPP-IV inhibitor• Anti-IL-6r mAb• Anti-IL-7r mAb

• Anti-CD20 (rituximab) followed by CTLA4Ig (abatacept)

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ActivationEffector T cells

Normal: reactions against pathogensInflammatorydisease, e.g. reactions against self

ToleranceRegulatory T cells

Controlled response to pathogensNo response to self

Can we develop new therapies for spontaneous diabetes by understanding other forms of autoimmune diabetes? The

immunological equilibrium: balancing lymphocyte activation and control

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Age/sex Primary diagnosis Past hx Other /previous chemotherapy

Diabetes presentation Random C-peptide/glucose*

Timeafter anti-PD-1 mAb

Pt 1 55/f Melanoma with metastatic disease

Autoimmune thyroid disease

Ipilimumab, prednisone DKA, glucose=532mg/dl;HbA1c=6.9%

< 0.1 ng/ml/52 mg/dl

5 mo

Pt 2 83/f Non-small cell lung Ca

Remote smoker

None DKA; Glucose=350 mg/dl; HbA1c=7.7%

< 0.1/336 mg/dl < 1 mo

Pt 3 63/m Renal cell carcinoma

Hypertension Proleukin, bevacizumab, interferon

Random glucoses of 247, 340; HbA1c=8.2%

1.3/79 mg/dl 4 mo

Pt 4 58/m Small cell lungcancer

Type 2 diabetes

Carboplatin/etoposide, paclitaxel

Hx of T2D; DK, glucose=749; HbA1c 9.7% (from 8.5%

< 0.1/2840.6/523 mg/dl

1 wk

Pt 5 64/f Melanoma Autoimmune thyroiddisease, psoriasis

None Glucose=703 mg/dl; +ketonuria; HbA1c=7.4%

0.5 ng/ml/268 mg/dl < 1 mo

*Nl: 1.1-4.4 ng/ml

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Features of diabetes induced with check point inhibitors

• New onset of diabetes in elderly or dramatic increase in insulin requirements in a patient with known Type 2 diabetes

• May present with diabetic ketoacidosis• May or may not have autoantibodies (30-50% are +)• Increased amylase or lipase in about ½ (6/10 in our series)• Often associated with thyroid dysfunction. • No FH of autoimmune diabetes but frequently a family history of

autoimmune diseases• Rapidly progresses to undetectable levels of C-peptide • It is not clear whether steroids will prevent complete loss of beta cell

function• Recovery is very uncommon• Glucose lability is consistent with absolute deficiency of insulin• Does not occur with anti-CTLA-4 alone

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Expression of PD-L1 on β cells in NOD mice

r= 0.91, p<0.0001

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Conclusions• The immune assault on beta cells reaches a crescendo in the peri-

diagnosis period. • There are both functional and anatomical effects of insulitis.

Preclinical data suggests that beta cells may acquire stem-like features that may allow them to resist immune destruction.

• A number of immunologics have been able to delay progression of T1D but none have been able to do so permanently.

• Activation of T cells with non-FcR binding anti-CD3 mAb appears to diminish responsiveness of CD8+ T cells: i.e. partial exhaustion

• “Responders” can be identified. They tend to be children very soon after diagnosis with residual C-peptide. There may be other personal features that can identify them (e.g. microbiome).

• Prevention remains an important goal.

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Acknowledgements:• Joyce Rui• Songyan Deng• Elke Gulden• Sahar Usmani-Brown• Eitan Akirav• Frank Waldron-Lynch• Jasmin Lebastchi• Paula Preston-Hurlburt• Pam Clark

• Funding• ITN• TrialNet• NIDDK/HIRN• NIAID• JDRF• Brehm Coalition• Howalt family

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