Giacomo Oliveira...Toffalori )et al., in preparation Genomic loss of the mismatched HLA haplotype targeted by donor T cells is a frequent mechanism of Acute Myeloid Leukemia (AML)

The TK approach: engineered donor T cells enabling safe and effective haplo-HSCT - ISSCR 7th Annual Meeting, July 8-10, 2009

Giacomo Oliveira San Raffaele Scientific Institute, Milano

SIES Annual Meeting,

Rimini, October 17, 2014


Schreiber, Science, 2011

Cancer Immunoediting


After targeted immunotherapy with CD19 Chimeric Antigen Receptor-modified T Cells, Acute Lymphoblastic Leukemia relapsed with a CD19-negative tumor variant

Grupp et al., New Engl J Med, 2013

Ex vivo evidences in human leukemia

HLA C l a s s I I s e l ec t i ve t ran sc r i p t i ona l dowregulation frequently occurs in AML relpases after allogeneic HSCT

Toffalori et al., in preparation

Genomic loss of the mismatched HLA haplotype targeted by donor T cells is a frequent mechanism of Acute Myeloid Leukemia (AML) relapse after HSCT from partially incompatible donors Vago et al., New Engl J Med, 2009

Toffalori et al., Blood, 2012 Vago et al., Semin Oncol, 2012 Vago et al, in preparation


Evidences from mouse tumor models

Unedited Progressor Unedited Regressor Edited progressor

Parental! sarcoma!

!

Parental sarcoma

Edited progressor

Schreiber et al., Nature, 2012

In murine models of spontaneous or induced solid tumors, emergence of edited tumor variants through loss of immunogenic epitopes

Affinity Score


Why a Mouse-Human Chimeric Model?

Ex vivo human studies

•  High clinical relevance and translational potential •  High disease variability, reports often anecdotic

•  Enable investigation of new therapeutic approaches

Primarily murine tumor models

•  High reproducibility, allow study of mechanisms •  Species-specificity of tumor and immune system

Mouse-human chimeric models

•  High clinical relevance and translational potential •  High reproducibility, allow study of mechanisms


Setting up the model

Primary leukemia

Leukemia Engrafted in

NSG mice

Leukemia engrafted in mice displays a stable gene expression profile

UPN#1

Primary human AML

Primary recipient

NSG mouse

25 primary leukemia tested Engratment rate: 44% (11/25)

Disease related-variables P value Total=14 Total=11

FAB M4 type 1/8 (12.5%) 4/8 (50%) 0,129

Abnormal karyotype 4/12 (33.3%) 4/10 (40%) 0,746

FLT3-ITD positivity 5/13 (38.5%) 6/10 (60%) 0,309

NPM1 mut A positivity 5/13 (38.5%) 6/11 (54.5%) 0,433

Primary induction failure 3/13 (23.1%) 5/11 (45.5%)

0,253

Relapse post Allo-HSCT 2/11 (18.2%) 8/9 (88.9%) 0,007

No Engraftment Engraftment

Primary AML tested in NSG mice


Setting up the model

Primary AML blasts stably engraft and expand in non irradiated 4-10 weeks old NOD/SCID y-chainnull (NSG) mice.

1st recipients

2nd recipients

3rd recipients

4th recipients

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UPN#12

0 25 50 75 100 125 1500

250

500

750

1000

2000

4000

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Days after infusion of leukemic cells

leukem

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UPN#16

Leukemic cells stably engrafted in mice display a more aggressive kinetic of outgrowth.

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Primary human AML

Primary recipient

Experimental design

vs

vs

vs

vs

vs

Haploidentical T cells

Predicted alloreactivity

Fully mismatched T cells

HLA Identical T cells

Autologous T cells

no T cells

In vivo Immune pressure

NSG mouse

Secondary recipient


Immune pressure with HLA-matched DLIs

Immune pressure with HLA-mismatched DLIs

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Donor-Host HLA disparity


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T-cell antileukemic effect is proportional to HLA disparity

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Evidences of relapse

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Development of extramedullary

Sarcoma


Primary human AML

Primary recipient

What is the gene signature of immuno-edited leukemias?

vs

vs

vs

vs

vs


Predicted alloreactivity



Autologous T cells

no T cells

In vivo Immune pressure

NSG mouse

Puri

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Gene expression profile of leukemia upon immune pressure

• Immune response: IFN alfa/beta signaling pathway

• Development: transcrip5onal regula5on of

granulocytes development

• Immune response: anAgen presentaAon by MHC class I

• Cytoskeleton remodeling

• Immune response: OncostaAn M signaling via MAPK in human cells

Top deregulated processes

Comparative Enrichment analyis between T cell-treated vs untreated leukemia harvested from mice

Dia

gnos

is

Dia

gnos

is 2p 2p 2p

HLA

_ID

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HLA

_ID

_IE

HLA

_ID

_IE

Aut

o_IE

Aut

o_IE

Aut

o_IE

MPOELANEPSMB10HLA−BFOSBCFL1EIF4A1JUNMYL6ACTBHLA−AB2MROCK2JUNDFOSACTG1LYZSTAT1TAP1IRF1OSMPRTN3PIK3R2NCLPSMD6CALRPSMB4SEC61BEGR1TYK2ARPC5PSMC5PSME2TIMP1MYBHSP90B1SEC61GMRLC2PSMB1HSP90AB1CEBPAEIF4A2HSP90AA1CEBPBCSNK2BEIF4HSEC61A1MYL12APSMA4PSMA5MYH9PSME1ARPC3PSMB3PSMB6EIF4G2RHOAPSMB7RPN1PSMA6ACTR2PSMC1PMLLIMK2PTPRCFOSL1TRIOE2F1SERPINE1PSMA7PXNMAPK13MAPK14ZNF225PKD1PSMD9MAPK12MYH3CTNNB1WASLDOCK1PRKCHNCF2PSMD11MPNDEIF4EBP1SOS2SOS1MAP2K4PRKCIMYL5PRKD2PSMD13MAXJAK2SOCS3GATA1SERPING1LAMB2STAT5BITGAVPIK3R1PSMD5ATF2PRKCBFOSL2PAK1PPP1R12AMYH10CSNK2A1TCF4CSNK2A2MAPK9PSMA2PIK3CBTCF3AVPIFNAR1MLC1CRKSMAD3PSMD3FLNAPIP5K1CVAV1PLAULRG1MAP3K11MAPK1TRAP1MAP3K7JAK1LIFSOCS1IFIT2TAP2CYBBNCF1CEBPEPSMB9STAT2PSMC2PSMB2IRF9PSMB8ISG15IFI6PTPN6FESARPC1BACTN4ZYXLDLRCDKN1AJUNBTAPBPPSMA3PTPN1PSMD2PSMC3CSF3RMYCACTN1CANXSPI1STAT5AMAP3K1MYL6BPSMB5ARPC1APRMT1PSMD4PSMD7PLAURPRKCB1TLN1TGFBR2MXD1PSMD14FHSTAT3MAP2K2PSMD8PSMD1PPP1CBPKD2PTENCOL4A5RUNX1PTPN11PIK3CDRXRAANPEPEIF4G3PRKCQPSMD10PSME3RAF1DSTNPSMC4RPN2GSK3BACTR3MAP2K1GRB2MAPK3

Immune genes

−2 0 2Row Z−Score

Color Key

Prim

ary

le

ukem

ia

Unt

reat

ed

Trea

ted

wit

h H

LA id

enti

cal

T ce

lls

Trea

ted

wit

h A

utol

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s

T ce

lls

Leukemia engrafted in mice


Pathway Analysis: DeregulaAon of HLA Class I PresentaAon

Proteasome and immuno-‐proteasome subunits

Classical and non classical MHC class I molecules


•  We set up a mouse-human chimeric model to dissect in vivo the mechanisms of leukemia immunoediting

•  The infusion human T cells is able to impair the in vivo growth of leukemic cells, and genetic disparity between T cells and leukemia is directly proportional to the observed antileukemic effect

•  Leukemic cells harvested upon immune pressure display a specific immune signature, comprising a significant deregulation of most of the genes involved in antigen processing and presentation

•  The outgrow of AML upon removal of T lymphocytes suggests the existence of an immune equilibrium between minimal residual disease and antileukemic immunity, and provides a model to investigate its biological bases

Conclusions


Acknowledgements Carolina Caserta

Cristina Toffalori Laura Zito

Luca Vago Unit of Molecular and Functional Immunogenetics Barbara Camisa Attilio Bondanza

Chiara Bonini Experimental Hematology Unit

Lara Crucitti Massimo Bernardi

Matteo Carrabba Jacopo Peccatori

Fabio Ciceri Hematology and BMT Unit

Cristina Tresoldi OSR Leukemia Biobank

Jose Manuel Garcia-Manteiga Gabriele Bucci

Dejan Lazarevic Elia Stupka Centre for Translational Genomics and Bioinformatics

Documents

Giacomo Oliveira...Toffalori )et al., in preparation Genomic loss of the mismatched HLA haplotype targeted by donor T cells is a frequent mechanism of Acute Myeloid Leukemia (AML)