Hematology winter symposium in Kaohsiung Veterans General hospital

Dec. 17th, 2016 in Kaohsiung

Clinical Application of Anti-CML immunity in the era of TKI therapy

Hiroshi Fujiwara, MD,PhD

Department of Hematology, Clinical Immunology and Infectious Diseases,Graduate School of Medicine, Ehime University

COI Disclosure Information

Hiroshi Fujiwara, MD, Ph.D.

Honoraria ; BMSTravel accommodation ; BMS

Interphase Nuclear FISH

ABL1 (9q); red

BCR (22q); yellow

White arrow; BCR/ABL or ABL1/BCR fusionFusion protein

Ph1 chromosome and BCR/ABL fusion gene; driver gene alteration for CML

Tom Strachan, Judith Goodship, Patrick Chinnery. Genetics and Genomics in Medicine.

1st ed. 2015, published by Garland Science, part of Taylor & Francis Group LLC.

(A)

(B)

(C) Fusion signal

Nowell P, Hungerford D. Science (1960).

Rowley JD. Nature (1973).

Douglas Hanahan , Robert A. Weinberg. Hallmarks of Cancer: The Next Generation. Cell, Volume 144, Issue 5, 2011, 646 - 674

Tactics of anticancer therapy based on characteristics of cancer cells

Selective TKI therapy

Allogeneic HSCT; the most powerful treatment option, however enforcing

pts to live with substantial Treatment-related Toxicity.

Target specific, i.e. less toxic systemically.

Kantarjian H, et al. Harrison’s Principles of Internal Medicine 2014. Slide credit: clinicaloptions.com

68%

92%

83%

43%

35%

8%

0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4 5

Yrs

Surv

ival P

robabili

ty

6 7 8 9 10 11 12 13 14

Treatment Era

TKI 2001-today

(CML-related deaths)

TKI 2001-today

(all deaths)

1996-2000

1991-1995

1983-1990

≤ 1982

CML Survival by Era

Inness AJ et al. Nat Rev Clin Oncol. 2016.

The number of HSCTs carried out for CML-CP remarkably decreased.

CML advanced(> CP2, AP, BC)

HS

CT

(n

um

be

r o

f c

as

es

)

Year

Since the first trial of imatinib opened

CML early

(CP1)

Steep decline in transplants for CML early

Remained stable for

CML advanced

Data from EBMT

Ponatinib vs SCT for CML With T315I Mutation

Ponatinib vs SCT

Median OS, Mos

Disease

Group Ponatinib SCT

P

Value

CP NR 103 .013

AP NR 56 .889

BP 7 11 .026

Ph+ ALL 7 32 .136

CP

AP

BP

Nicolini FE, et al. Blood. 2015;126. Abstract 480. Slide credit: clinicaloptions.com

0

20

40

60

80

100

OS

(%

)

0 12 24 36 48

Ponatinib

SCT

Mos

0

20

40

60

80

100

0 12 24 36 48

Mos

0

20

40

60

80

100

0 12 24 36 48

Mos

OS

(%

)O

S (

%)

Ponatinib

SCT

Ponatinib

SCT

Overall response rates to TKIs in pts with CML-CP

IM 1st line Tx.60% durable

response

40% eligible for

IM treatment

withdrawal

Up to 50%

achieve

Operational

cure

40% require

2nd TKI

50% durable

response

50% require

3rd TKI

50% durable

response

50% require

alternative

treatment

Around 10-15% of

initial group might

benefit from HSCT

2nd TKI

1st line Tx.

70-80% durable

response

20-30% require

3rd TKI

50% durable

response

50% require

alternative

treatment

Around 10-15% of

initial group might

benefit from HSCT

(A)

(B)

Inness AJ et al. Nat Rev Clin Oncol. 2016. modified.

Treatment

withdrawal

under evaluation

Clinical questions in the treatment of CML in the era of TKI

Withdrawal of TKI ?

MRD ; minimal or measurable? residual disease

How to manage after TKI withdrawal

/ Concept of “Treatment-Free Remission” “Operational Cure”

CML stem cell

Required alternative strategy ?

To pts with refractory to TKI or advanced disease w/o T315I mutation

Interfere the disease control after TKIs withdrawal

allo-HSCT Immunotherapy

Immunotherapy

> 3rd TKIs

NCCN(National Comprehensive Cancer Network)

Clinical Practice Guidelines in Oncology. 2012

European LeukemiaNet recommendations

for the management of chronic myeloid leukemia.

Baccarani M et al. Blood, 2013

“ Propose continuation of TKI treatment indefinitely

in all responders”

A challenge;

To establish a strategy to avoid the life-long dependency on TKIs.

….Secondary Malignancies, financial issues,

recognized and/or unrecognized AEs (for young pts),

impairment of QoL (pregnancy issue), …

e.g. For whom ?

Secondary malignancis in Imatinib-treated CML patients

Mirand MB et al. Leukemia,2016

Tx.before Requisite TFR% discontinuation for discontinuation (m edian follow -up tim e)

IM discont. trialsSTIM 1 100 IFN then IM for>3y C M R for > 2y 39% (55 m o.)STIM 2 200 IM > 3y As for STIM prelim inary 46% @ 2yEU RO -SKI 809 IM , N IL, D AS M R4 for > 1y in progress

TKI for > 3y prelim inary 61% @ 0.5yIM /N IL/D AS discont. trialsSTO P 2G -TKI pilot 50 N IL or D AS C M R for m edian 29m o. in progress

prelim inary 61.1%EN ESTFreedom 175 N IL front-line M R4.5 for > 1y in progressEN ESTop 117 N IL 2nd-line >3y M R4.5 for > 1y in progress

EN ESTPath 1058 IM >2 and N IL M R4.5 for > 1y vs in progressM R4.5 for >2y RC T

Japanese trial* 88 D AS 1y as consoli. D M R, unclearly defined 49% @ 6m o.(63 discont.)

Study Pt. N o.

“Operational Cure”

; Prolonged survival in molecular remission without therapy

by Goldman JM et al. J Natl Compr Canc Netw. 2012

What does MRD in leukemia really mean?

Goldman JM, Gale RP. Leukemia 2014

Saußele S et al, Leukemia 2016, modified.

Levels of molecular response and corresponding log-reduction

and BCR-ABL transcript levels on the International Scale.

Michele Baccarani, and Simona Soverini Blood

2014;124:469-471

© 2014 by American Society of Hematology

NCCN Guidelines Version 1.2017

Chronic Myeloid Leukemia

Discontinuation of TKI therapy

Criteria for TKI Discontinuation

1) Age > 18 y. 2) CP with no prior history of AP or BC, 3) TKI Tx. > 3 y.4) Prior evidence of quantifiable BCR-ABL transcript, 5) Stable MR4; < 0.01% IS for > 2y.6) No history of resistance to any TKI, 7) Access to a reliable QPCR test with a sensitivity, of detection of > 4.5 logs that reports

results on the IS and provides results within 2w, monthly molecular monitoring for the first 6 mo. following discontinuation, bimonthly during months 7-24, and quarterly thereafter (indefinitely) for pts who remain in MMR (MR3; <0.1% IS),

8) Consultation with a CML Specialty Center to review the appropriateness for TKIdiscontinuation, including TKI withdrawal syndrome

9) Prompt resumption of TKI, with a monthly molecular monitoring for the first 6 mo. following resumption of TKI and every 3 mo. thereafter is recommended indefinitely for pts with a loss of MMR. For those who fail to achieve MMR after 6 mo. of TKI resumption, BCR-ABL1 kinase domain mutation testing should be performed, andmonthly molecular monitoring should be continued for another 6 mo..

Low risk (sokal score) CML at diagnosis

Timely available and highly sensitive (> 4.5 log sensitivity

below the standard baseline) MRD measurement

Optimal response achieved to prior TKI cessation.

Achievement of deep molecular remission > 2y

with prior TKI treatment >3y before the discontinuation

Discontinuation should be as a “clinical trial”

with Higher NK cell number

Bhalla S et al. Clin Lymphoma, Myeloma & Leukemia, 2016, modified

Although the depth of molecular response seems

associated with TFR (Treatment Free Remission) in

fact, the correlation between TFR and the “long-term

outcome” still remains to be elucidated.

A primitive subset of CD34+ CML cells survives complete Bcr-Abl inhibition in growth factor–free medium for 12 days.

Ashley Hamilton et al. Blood 2012;119:1501-1510

Chronic myeloid leukemia stem cells are not dependent on Bcr-Able kinase activity for their survival

NC

DAS 150nM

Untreated

Abraham SA et al. Nature, 2016

In CML stem cells ; c-MYC & p53 level

c-MYC & p53 ; novel therapeutic targets for CML-LSC

e.g.

(based on the transcriptome analysis)

Douglas Hanahan , Robert A. Weinberg. Hallmarks of Cancer: The Next Generation. Cell, Volume 144, Issue 5, 2011, 646 - 674

Tactics of anticancer therapy based on characteristics of cancer cells

Selective TKI therapy

Immunological intervention

CTL: non-labeled moving cells, Leukemia cell : Green labeled

Dead cells: red labeled, Fibroblasts: by-stander cells

Biostation IM, NIKON Inc.Asai H. & Fujiwara H.

Leukemia-specific CD8+ T cells (CTLs) selectively killed leukemia cells.

CTL

CD8

CD3

α β

Garcia KC, et al. Science, 1996

TCR

HLA class I β2MG

Time-lapse study

Perforin/Granzyme

IFN-g/TNF-a/Fas-L

Being target-specific might reflect the less systemic toxicity

(on-target/off-tumor AE).

Immunotherapeutic strategy

Hantschel O, et al. Leuk Lymphoma 2008; 49: 615

Imatinib Nilotinib Dasatinib

ABL ABL ABL BMX ILK

ARG ARG ARG TXK LIMK1

BCR-ABL BCR-ABL BCR-ABL DDR1 LIMK2

KIT KIT KIT DDR2 MYT1

PDGFR PDGFR PDGFR ACK NLK

DDR1 DDR1 SRC ACTR2B PTK6

NQO2 NQO2 YES ACVR2 QIK

FYN BRAF QSK

LYN EGFR/ERBB RAF1

HCK EPHA2 RET

Src family LCK EPHA3 RIPK2

FGR EPHA4 SLK

BLK EPHA5 SLK36/ULK

FRK FAK SYK

B-cell development

CSK GAK TAO3

Haematopoietic stem cell Differentiation/proliferation

BTK GCK TESK2

TEC HH498 TYK2

ZAK

Colorectal cancerCell differentiation / Cancer metastasis

Molecular targets of imatinib, nilotinib and dasatinib

Lo

g10

(BC

R-A

BL)

Time course (mo.)

Mathematical mode; Autologous immune responsemight interfere the variation of BCR-ABL transcripts

- 3.5 log reduction- 3.5 log reduction

- 3.5 log reduction

Clapp GD et al. Oncoimmunol. 2016. modified.

Based on data from IM first-line Tx.

Immune

window

drug

induced

Immune response-mediated disease suppression

Host immune responses are involved in the clinical course of CML pts treated with TKIs

Lymphocytosis

Schiffer CA et al. Cancer, 2016

NK cell increase

Christiansson L et al. Mol Cancer Ther., 2015

Christiansson L et al. Mol Cancer Ther., 2015

MDSCA

rg1

(ng/m

L )

IL-6

(lin

ear

NP

X )

IL-1

2 (

linear

NP

X )

IL-8

(pg/m

L )

Naka K and Ichinohe T. Stem Cell Investig 2016

Natural Killer cells in the clinical efficacy of TKIs

A phase I/II clinical trial of autologous cytokine-induced killer cells as adjuvant immunotherapy

for acute and chronic myeloid leukemia in clinical remission. Linn YC, et al. Cytother. 2012

Yeung DT, et al, Blood, 2015

Nievergall E, et al, Blood, 2014

NK cells seem involved in anti-CML efficacy mediated by TKIs

Adoptively transferred NK cells seemed less effective

ADCC mediated by NK cells might be able to kill CML stem cells

Dasatinib IC50 value <25 nM in 14 of 15 imatinib-resistant mutations tested

Ba/F3

Bcr-Abl

E255K

T315I

M351T

M244V

G250E

Q252H

Q252R

Y253F

Y253H

E255V

F317L

E355G

F359V

H396R

F486S

Concentration of Dasatinib (nM)

1.2

5025

0

0.2

0.4

0.6

0.8

1.0

52.50.50

T315I

Rela

tive G

row

th A

fter

48 H

ours

of

Dra

g E

xp

osu

re

Shah NP, et al. Science 2004, 305:399-401

O’Hare T, et al. Cancer Res 2005, 65:4500-4505

Dasatinib inhibits the majority of imatinib-resistant BCR-ABL mutations, but not T315I mutation, tested in vitro.

0

200

400

600

800

+3M +6M +9M +12M +15M +18M +21M +24M +27M +30M +33M +36M +39M

Nilotinib800mg/day

T315I(+)

Interferon α600 x10^4 U/day

CHR

Ph chr.in BMG-banding

20/20 0/20 0/20 1/20 0/20 2/20 4/20 1/20

CCyR Loss of CCyR MR3.0(MMR)

Dasatinib100mg/day

Dasatinib140mg/day

0% 0.8% 1.5% 0.1% 2.3% 5.0% 2.4% 2.1%

0.0854% 0.0664% 0.0783% 0.0549%

Major bcr-abl mRNA(IS)

Registration to JMDPMSD; N/A 6/6 MUD; N/A

Ineligible for the Ponatinib clinical trial

Clinical Course

Copy /assay

Major bcr-abl mRNA(TMA)

FISH (PB)

T315I(-)

46 y.o male ; CML-CP+ myeloid sarcoma (rt.hip)

MRI

Science 2013 Trends in Immunology 2016Nature 2014

Blood 2015;125:4017

CD19-CAR-T

Journal of clinical pharmacology, 2015,doi10.1002/jcph.591

Anti-PD1 mAb

Induction or

Adoptive transfer of

High quality effector cell

Removal of

immuno-regulatory elements

Tactics of immunotherapy for CML

Drawn by author

MDSC

Tactics of immunotherapy for CML-2

BCR-ABL1 derived antigens

Driver mutation = BCR-ABL1

Overexpressed

protein

LAA

CML cell

HLA1

A gene

B gene

PGPs;

PR3 (PR1)

NE (PR1)

Cath-G (CG-1)

WT1

AURKA

p210 BCR-ABL

CML specific AgC

ML

rela

ted

Ag

s

Treg.

HLA1

B gene

CML stem cell

anti-CML

-CAR-T

CD123

Primary Granular Proteins as therapeutic targets of cellular immunotherapy for CM-CP: lineage specific Ags

1 2 3 4 5 6

CG

PR3

GAPDH

HNE

Lane 1, 2; Normal G-CSF

mobilized PB

CD34+ cells

Lane 3, 4; CD34+ cells

from CML patient

Lane 6; HL60 as positive

control

Lane 6; dH2O as negetive

control

(PGP mRNAs detected by RT-PCR)

Fujiwara H et al, Clin Cancer Res, 2005

Molldrem J.J. et. al., Nat Med,2000

PR1 peptide-specific CD8 killed CML cells

Fujiwara H. et. al., Blood,2004

NE-specific CD8 killed CML progenitor cells

*

CD3+/ HNE

0

50

100

150

200

50:125:112.5:16.25:10:1

*

Untreated CD3+

CD3+/HNE with anti-class I

E:T ratio

Nu

mb

er

of

co

lon

ies

/10

e3

ce

lls

CTLs specific for PR1 peptide which derived from both PR3 and NE display tumoricidal activity against CML cells (CML-BM-CD34+ cells).

HL60 RNAs to cDNAs / PR3, NE, CG pcDNA3/ PGP

CD40L-B

electroporation

PGPs producing CD40L-B

GFP, IE1-pp65

(as control)

Target gene

Fujiwara H et al, Clin Cancer Res, 2005

Generation of PGPs-specific T cells

PGPs

Artificial APC

By PR3 expressing CD40B

By HNE expressing CD40B

By CG expressing CD40B

By mock transfected CD40B

Cyto

lyti

c a

cti

vit

y (

%)

E:T ratio

50:1 12.5:125:1

E:T ratio

CML cells Normal BM cells

0

10

20

30

40

50

60

70

50:1 12.5:125:1

- CML-CP (case# 5)-

Responder T-cells

Fujiwara H et al, Clin Cancer Res, 2005

Successful generation of PGPs-specific T cells derived from CML patient’s CD3+T cells at day 90 after allo-HSCT

using PCP gene-modified autologous CD40LBs

CML CML

Zhang M, et al, Clin Cancer Res, 2012, Alatrash G. Oncoimmunology, 2013,

Sergeeva A, et al, Leukemia, 2016, Qing M, et al, Cytother. 2016,

Ataie N, et al. J Mol Biol 2016, modified,

Pep.vaccine

+

adjuvant

Multiple pep.vaccine

+

adjuvant

SS

Va

Ca

Vb

Cb

Single target

Multiple targets

Adoptive transfer

VL VH

or

or

T

T

T

8F4-CAR

8F4-Bite

C

C

C

8F4 mAb

Up-dated strategy to target PGPs

ESK1-CAR

CML CML

Immunogenic determinant epitopederived from AURKA

1 51 132 383 402

H2N COOHKinase domain

KEN motif D-box activating motif D-box (PxxRxxL)

PNILRLYGYF HDATRVYLILEYAPLGTVYR ELQKLSKFDE191 230207 215

Ochi T, et al. Blood. 2009

ALLBMMCs(n=7)

AMLBMMCs(n=17)

NormalPBMCs(n=4)

PHA-blasts(n=3)

CMLBMMCs(n=10)

0

10

20

3040

120

Re

lative

exp

ressio

n o

f A

UR

KA

m

RN

A AURKAmRNA

%Specific lysis

Pt#1 (CML)

Pt#2 (CML)

Pt#4 (AMLM2)

Pt#5 (AMLM0)

Leukemia cells

Pt#6 (ALL L2)

0 20 40 60

7.0

19.1

9.4

9.5

27.3

HLA-A*0201

+

-

-

-

+

Pt#3 (CML) +

10.5

Chr. 20q

Vα Vβ

εδ

Cα Cβ

ε γz z

Vα Vβ

εδ

CD3

Cα Cβ

ε γz z

HLA class I

ITAM

TCR gene-modified (CD8+) T cells

Introduced Therapeutic

TCR

Endogenous TCR*

Immunogenic epitope

Cancer Cell

CD3ITAM

Development of AURKA-specific siTCR-T cells

Casey N, et al, PLoSONE, 2016

AURKA-siTCR retroviral vector

CML (BMMCs) Cord blood (CBMC)

34+38low

34+38low

CD34

CD

38

CML

CD34＋

38low

Normal

PBMCs

(n=4)

0

5

10

15

CML

PBMCs

CML

whole

BMMCs

10.6

10.9

4.4

1.1 1

CBMC

CD34＋

38low

Normal

PBMCs

(n=4)

Rela

tiv

e a

mo

un

t o

f

Au

rora

-Am

RN

A

1. e.g. CML stem cells subset overexpressing AURKA mRNA Ochi T, et al. Blood. 2009

Hoechst Red-A

Hoe

ch

st B

lue

-A

2. e.g. Side population (SP) of GANMO-1

Targeting leukemia stem cells by AURKA-specific CTLs

AURKA mRNA expressionCD107a positive CD8 cells (%)

Whole SP Non-SP

SP

Non -SP

GANMO-1

NGM-T siTCR-T conTCR-T

AURKA specific effector T cells

Casey N, et al, PLoSONE, 2016

Against SP

PD-1 Pathway Overview

PD-1

Type

• Co-inhibitory receptor

• Can be induced through T-cell

activation1

Ligands • PD-L1 and PD-L22

Expression

• Receptor expression: On a range of

immune cells, including activated

T cells3

• Ligand expression: On tumors and

immune cells and low levels in normal

tissues3

• Amplification of 9p24 results in

increased ligand expression4

Activation• Through interaction of tumor and

T cell at tumor site3

Blockade• Blockade results in target cell killing

and inhibition of Akt signaling and

PI3K activity3,5

1. Keir ME et al. Annu Rev Immunol. 2008;26:677-704. 2. Korman AJ et al. Adv Immunol. 2006;90:297-339. 3. Hamid O, Carvajal RD. Exp Opin Biol Ther. 2013;13(6):847-861.4. Ansell SM et al. N Engl J Med. 2015;372(4):311-319. 5. Parry RV et al. Mol Cell Biol. 2005;25(21):9543-9553.

APC, antigen-presenting cell; Akt, protein kinase B; PD-1, programmed death-1; PD-L1, PD ligand-1; PD-L2, PD ligand-2; PI3K, phosphoinositide 3-kinase.

PD-L1 PD-L2

PD-1

Immune Cell

Tumor cell/APC

↓ T-cell proliferation↓ Cytokine production↓ CD8+ T-cell cytolytic

function↓ T-cell survival

Adapted from Hamid O, Carvajal RD 2013.[3]

PD-1 is up-regulated on CD8+ T cells of CML patients in the chronic phase of the disease.

Sabine Mumprecht et al. Blood 2009;114:1528-1536

© 2009 by American Society of Hematology

DMSO

DMSO + IFN-g

CD274-PE(PD-L1)

CD274-PE(PD-L1)

MFI: 360 vs. 399

MFI: 393 vs. 1641

MOLM-13 (AML-M5a) LCL

DMSO

DMSO + IFN-g

MFI: 360 vs. 4689

MFI: 393 vs. 5250

Via IFN-g stimulation Constitutive expression

Maruta M, Fujiwara H, Unpublished data

Constitutiveexpression

PD-L1 expression in human leukemia cells

PD-L1 expression by CML-SCs induced by CTL-IFN-g Increasing PD-1 expression on CTL

Combined inhibition of PD-1/PD-L1 binding by mAb or siRNA improved the survival of CML mouse. During this process, CML-CSs were preferentially suppressed..

Combined therapy using inhibitor of PD-1/PD-L1 binding andadoptive transfer of CML-specific CTLs. - Murine model -

Riether C et al. Leukemia, 2015

(a) (b)

(c) (d)

Future Direction

CR/MRDOvert leukemia Relapse/ Progression

Prolonged MRD

Cure

To generate the long-lasting anti-leukemia memory immunity in patients

LSC targeting Molecular target Tx.

LSC targeting Vaccine ( Th1 helper) Tx.

Immune Checkpoint Inhibition

TCR-T /CAR cells targeting LCS

Functional Cure

: LSC

: non-LSC

Robust oligoclonalTCR/CAR-T cells

bearing different targets

LSC

Minimum required chemo-radio Tx

Fujiwara H. IJH, 2014, modified

Take-home messages

In the era of TKIs, the prognosis of pts with CML-CP has beautifully improved.

Studies of clinical responses and underlying mechanisms again emphasized the

importance of host immune responses.

Immunological strategies would be beneficial not only for the enhancement

of efficacy of TKI therapy, e.g., maintenance after withdrawal of TKIs via

suppression of CML-SCs, but also for the breakthrough strategy of

TKI-resistant and aggressive CML, particularly for pts ineligible for allo-HSCT.

Furthermore, immunotherapy could be applied for disease relapse after allo-

HSCT.

Accumulated knowledge regarding anti-CML immunity during TKI treatment

will have to provide a scientific rationale not only for the treatment of leukemias,

but also for solid tumors.

Acknowledgement

Toshiki Ochi Kozo NagaiFumihiro OchiNicholas CaseyKazushi TanimotoTaichi AzumaMasaki Yasukawa

Kiyotaka Kuzushima

Sachiko OkamotoJunichi Mineno

Ehime University

Aichi Cancer Center

Takara Bio Inc.

Mie University

Hiroshi Shiku

Thank you very much for your attention !

A. John Barrett

NHBLI/NIH

Katayoun Rezvani

MDACC

Dogo Hotspring in Matsuyama, Ehime