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Argument for combination immunotherapies in therapeutic cancer vaccine development Thomas F. Gajewski, M.D., Ph.D. Associate Professor, Departments of Pathology and Medicine Program Leader, Immunology and Cancer Program of the University of Chicago Cancer Research Center


Results of clinical studies of immunization with melanoma antigen peptides and IL-12 • Clinical response rate 10% (+ ~20% stable/mixed) – Better than zero – Not as high as hoped based on preclinical studies

• T cell responses (usually modest magnitude) induced in the majority of patients • Some patients had a high T cell response to melanoma antigens even pre-treatment (up to 1% of CD8s) spontaneous immunity • Pivotal question: why does high T cell response not always lead to tumor regression? – Quantitative deficiency (magnitude still not high enough) – Qualitative limitations (TCR avidity, phenotypic subtleties) – Downstream resistance mechanisms at the level of tumor microenvironment Int. J. Can. 1999 Clin. Can. Res. 2001 J. Clin. Oncol. 2003


Anti-tumor immune responses: Taking into account the effector phase Vaccine Endogenous Lymphatic

APC

nCD8

APC

Inhibitory mechanisms

Granzymes perforin

eCD8 IL-2

Blood Chemokines

IFNIFN-Îł

eCD8

eCD8

Lymph node (Priming phase)

Tumor microenvironment (Effector phase)


Can we profile tumor microenvironment and identify correlates with clinical outcome? Phase II study with 4 peptide vaccine + IL-12 • 19 HLA-A2+ patients with metastatic melanoma • All vaccinated with 4 peptides (MelanA, NA17, gp100, MAGE3) pulsed onto PBMC + rhIL12 q 3 weeks • Patients had pre-treatment biopsy to prepare RNA for gene array analysis • Clinically, 1 patient had a CR, 1 PR, and 4 had prolonged disease stabilization (>6 months) • Affymetrix gene array on pretreatment samples: – U133A chips utilized, data were normalized – Supervised hierarchical clustering done comparing patients with SD or better versus patients with PD – Looking for genes differentially expressed 2-fold or greater


Affymetrix gene array analysis of pre-treatment biopsies from patients on melanoma vaccine sorted by clinical outcome

Represents only 7 genes: • 4 upregulated • 3 downregulated

6 mos SD or better


Tumors from favorable clinical outcome patients express higher levels of TCRÎą Îą, CXCL9, and CCL21


Expression of a subset of chemokine genes is associated with presence of CD8 transcripts

CD8β CCL2 CCL4 CCL5 CXCL9 CXCL10 CCL19 CCL21


Superior recruitment of human CD8+ effector T cells in NOD/scid mice bearing “chemokine-high” M537 melanomas Blood

Spleen

Tumor

(Chemokine “high”)

(Chemokine “low”)

Harlin et al, Cancer Research, 2009


Why are melanomas that do attract CD8+ T cell not rejected spontaneously? CD8 IHC A

• Lack of migration

B

• Presence of immune inhibitory mechanisms: – – – –

IDO PD-L1/PD-1 Tregs T cell anergy


Co-expression of IDO, PD-L1, and FoxP3 transcripts in individual tumors

Note: these are more abundant in metastases that contain CD8+ T cells

Immunol. Rev. 2006 Clin. Can. Res. 2007


Strategies to uncouple immune inhibitory mechanisms for clinical translation 1. Promote increased migration into tumor sites – Chemokines, innate immune factors (e.g. type I IFNs), TLR ligands – LIGHT (Yu et al. J. Immunol. 2007)

2. Uncouple negative regulation • IDO inhibition (Uyttenhove et al, Nat Med, 2003) – 1-methyltryptophan (RAID program); other inhibitors

• Blockade of PD-L1/PD-1 interactions (Blank et al, Can Res 2004) – Anti-PD-1 or PD-L1 mAb (Medarex)

• Depletion of CD4+CD25+FoxP3+ Tregs (Kline et al, CCR 2008) – Ontak (denileukin diftitox: IL-2/DT fusion; Daclizumab) – Ex vivo bead depletion of CD25+ cells from T cell product

• Anergy reversal (Brown et al, JI 2006; Zha et al, Nat. Immunol. 2006) – Homeostatic cytokine-driven proliferation (lymphopenic recipient) – Inhibition of anergy factors (e.g. DGK-α)

• Combinations of negative regulatory pathway blockade – Synergy between Treg depletion and anergy reversal with homeostatic proliferation (Kline et al, CCR 2008)


Intratumoral LIGHT adenovirus in B16 melanoma: Promotes chemokine production, CD8+ T cell recruitment, primary tumor control, and rejection of non-injected distant metastases CD8+ T cell infiltrate

Tumor rejection

1st tumor Ad-LIGHT

3000

2nd tumor

17.2%

14.9%

Ad-control

Tumor volume (mm3)

4000

4.56%

3.52%

2000 Ad-LIGHT

Ad-control 1000 0 0

10

20

30

10

Days after tumor challenge

20

30 CD8

Yu et al, J. Immunol. 2007


1-methyltryptophan reverses immunosuppression by IDO and improves tumor control in vivo

Uyttenhove et al, Nature Med. 9:1269, 2003


PD-1-/- TCR Tg T cells are superior at tumor rejection in vivo

Blank et al, Cancer Research, 2004


DGK as a drugable inhibitor of T cell activation in the anergic state

PIP2

Ras

X

DAG GRP1

IP3 PLC-γγ1 DGK

XX ERK

JNK

Zha et al., Nature Immunol. 2006


A pharmacologic inhibitor of DGK recovers IL-2 production by anergic T cells

Implies that it may be possible to develop small molecule immunopotentiating drugs to improve T cell function in the context of cancer and chronic infections


Anergic 2C TCR Tg T cells reject tumors after homeostatic proliferation in RAG2-/- hosts

No homeostatic proliferation

With homeostatic proliferation

Brown et al., J. Immunol., 2006


Uncoupling multiple immune suppressive mechanisms: Combined Treg depletion and anergy reversal supports rejection of B16 melanoma and leads to vitiligo

Kline et al., Clin. Can. Res. 2008


Comprehensive view of levels at which a spontaneous anti-tumor T cell response can fail Lymph node 3. APC maturation/ costimulation

2. Antigens/ 1. innate immune Ag processing awareness

Lymphatic

APC

nCD8

Tumor microenvironment

APC

8. Target cell apoptosis Granzymes perforin

eCD8 IL-2

Blood Chemokines

IFNIFN-Îł

eCD8 4. T cell repertoire/ eCD8 activation 6. Effector T cell 5. T cell differentiation/ trafficking 7. T cell effector function expansion/persistence (negative regulation)


Candidate approaches to overcome these immunologic checkpoints 1.

Innate immune awareness/Ag presentation/APC maturation –

2.

Innate immune cells and cytokines, TLR agonists, CD40 ligands, vaccination—novel Ag sources

T cell repertoire/initial activation – –

3.

B7 and other costimulatory ligands Interference with lymph node-based or systemic negative regulators (CTLA4, IDO, arginase, anergy, Tregs, IL-10)

T cell differentiation/expansion/persistence – –

4.

Differentiation cytokines (IL-12, IL-18) Expansion, survival factors (IL-2, IL-7, IL-15, anti-41BB; homeostatic proliferation)

T cell trafficking into tumor sites – –

5.

Intratumoral chemokines, LIGHT Pro-inflammatory treatments (XRT, TLR agonists, innate cytokines)

Executing effector function in tumor microenvironment – –

6.

Blockade of tumor microenvironment-based negative regulators (IDO, PD-1/PD-L1, Tregs, anergy, TGF-β, IL-10, iNOS) Promote effector cell proliferation (regenerate cytotoxic granules)

Tumor cell susceptibility to recognition and killing – –

Blockade of key anti-apoptotic molecules (Bcl2 and Spi inhibitors) Inhibit oncogenic pathways that create resistant phenotype and/or resistant microenvironment (Stat3; MEK? Notch? Wnt?)


Anti-CTLA-4 mAb + GM-CSF-transduced B16 vaccine induces tumor rejection and leads to vitiligo

van Elsas, Allison et al. JEM 1999


Anti-4-1BB + anti-PD-L1 Combination induces induces rejection of PD-L1expressing tumors in vivo

Hirano, Chen et al. Cancer Res. 2005


Vaccine + CpG + Treg depletion: Control of mammary tumors in Neu Tg mice

Nava-Parada, Celis et al. Cancer Res. 2007


Conclusions and implications • The spontaneous natural host immune response against melanoma is heterogenous; mechanism unclear: – Somatic differences between tumors? – Germline polymorphisms in immunoregulatory genes? • Implies that dominant barrier to T cell-mediated tumor rejection may be distinct in different subsets of patients, e.g.: – Failed T cell priming – Defective T cell recruitment to tumor sites – Immune suppressive factors blocking T cell effector phase • Additional immunotherapeutic interventions beyond (or in place of) vaccination may be needed to maximize tumor rejection by antitumor T cells • Importantly, clinical grade reagents and methodologies have been developed, enabling clinical testing of these concepts • Many of these principles also likely apply to chronic infections • Final point: in the meantime, can we better select patients for vaccine trials based on immunologic features of tumor microenvironment?


Gene expression profiling in context of Erlangen dendritic cell-based vaccine in melanoma Survival based on clinical response

Survival groups

Survival probability (%)

8 2 0 G

7 9 5 G

6 5 6 G

6 2 4

6 3 7

8 8 6 G

6 0 4 G

8 0 6 G

7 1 2 G

6 3 0 G

7 5 0 G

7 6 1 G

Long

8 7 8 G

7 5 9 G

8 6 3 G

8 5 4 G

100

8 0 5 G

7 3 1 G

Short

C O L 1 1 A 1 : c o lla g e n , ty p ... FL T 1 : Fm s -re la te d ty ro s ... C O L 1 1 A 1 : c o lla g e n , ty p ... C O L 1 1 A 1 : c o lla g e n , ty p ... S T C 1 : s ta n n io c a lc in 1 D S G 2 : d e s m o g le in 2 D S G 2 : D e s m o g le in 2 P E G 1 0 : p a te rn a lly e x p re ... S T C 1 : S ta n n io c a lc in 1 L D L R : lo w d e n s ity lip o p r... FL T 1 : Fm s -re la te d ty ro s ... S E R P IN H 1 : s e rp in p e p ti... M A L A T1 : m e ta s ta s is a s ... P T P 4 A 1 : p ro te in ty ro s in ... S N T B 1 : s y n tro p h in , b e ta ... D H C R 2 4 : 2 4 -d e h y d ro c h ... S Q L E : s q u a le n e e p o x id ... S E R P IN E 1 : s e rp in p e p ti... IG F 2 B P 3 : in s u lin -lik e g r... E E D : e m b ry o n ic e c to d e r... P D K 1 : p y ru v a te d e h y d ro ... R A P G E F6 : R a p g u a n in e ... R N F4 3 : R in g fin g e r p ro t... S L C 1 6 A 1 : s o lu te c a rrie r ... S T C 1 : s ta n n io c a lc in 1 Fu ll le n g th in s e rt c D N A c ... IL 1 R 1 : in te rle u k in 1 re c e ... A K R 1 C 1 : a ld o -k e to re d u ... U C P 2 : u n c o u p lin g p ro te i... FL J 2 2 0 2 8 : h y p o th e tic a l ... S T A U 2 : S ta u fe n , R N A b i... R P S 1 1 : R ib o s o m a l p ro te ... U S P 4 0 : u b iq u itin s p e c ific ... M R N A ; c D N A D K F Zp 5 8 ... Fu ll le n g th in s e rt c D N A c ... K B TB D 1 1 : k e lc h re p e a t ... FM O 2 : fla v in c o n ta in in g ... M S R B 2 : m e th io n in e s u lf... H C S T : h e m a to p o ie tic c e ... H o m o s a p ie n s , c lo n e IM ... FA M 1 0 7 B : fa m ily w ith s e ... P L A C 9 : p la c e n ta -s p e c ifi... M R N A ; c D N A D K F Zp 4 3 ... M P S T: m e rc a p to p y ru v a t... C 1 8 o rf4 5 : c h ro m o s o m e ... P D E 3 B : P h o s p h o d ie s te r... R A S E F : R A S a n d E F-h a ... K L F 1 2 : K ru p p e l-lik e fa c t... Tra n s c rib e d lo c u s H O X C 1 0 : h o m e o b o x C 1 0 TS P A N 3 3 : te tra s p a n in 3 3 H O X C 4 : h o m e o b o x C 4 TS T: th io s u lfa te s u lfu rtra ... W F D C 9 : W A P fo u r-d is u lf... C 1 8 o rf3 7 : c h ro m o s o m e ... C D 2 8 : C D 2 8 m o le c u le R A S E F : R A S a n d E F-h a ... IQ C E : IQ m o tif c o n ta in in ... TR IM 4 : trip a rtite m o tif-c ... TH Y 1 : Th y -1 c e ll s u rfa c ... L O C 3 9 2 6 1 7 /// L O C 6 4 1 ... g b :N M _ 0 1 6 9 4 7 .1 /D B _ X ... S N O R D 5 0 A /// S N H G 5 //... C D N A c lo n e IM A G E :4 8 2 ... C D N A c lo n e IM A G E :5 2 6 ... K IA A 0 9 4 7 : K IA A 0 9 4 7 p r... C Y P 1 B 1 : c y to c h ro m e P 4 ... C TS G : c a th e p s in G R E C K : re v e rs io n -in d u c in ... C D N A c lo n e IM A G E :5 2 6 ... A K R 1 C 2 : a ld o -k e to re d u ... L O C 2 8 4 7 0 1 /// FL J 1 4 1 8 ... P R R 1 4 : p ro lin e ric h 1 4 M A D 1 L 1 : M A D 1 m ito tic ... C D R 2 : C e re b e lla r d e g e n ... G A TA 3 : G A T A b in d in g p ... G IM A P 7 : G T P a s e , IM A P ... g b :B C 0 4 3 6 0 1 .1 /D B _ X R ... g b :A I7 6 1 5 7 3 /D B _ X R E F ... C 8 o rf1 3 : C h ro m o s o m e ... C D 3 3 L 3 : C D 3 3 m o le c u l... ZN F 6 7 7 : Z in c fin g e r p ro t... M S X 1 : m s h h o m e o b o x h ... E T V 5 : E ts v a ria n t g e n e ... TH Y 1 : Th y -1 c e ll s u rfa c ... M TA C 2 D 1 : m e m b ra n e t... IL 2 7 R A : in te rle u k in 2 7 r... P T E R : p h o s p h o trie s te ra ... E M C N : e n d o m u c in B T G 1 : B -c e ll tra n s lo c a ti... A P L P 2 : A m y lo id b e ta (A ... E M L 1 : e c h in o d e rm m ic r... R A M P 3 : re c e p to r (c a lc it... E M L 1 : e c h in o d e rm m ic r... g b :N M _ 0 1 8 0 4 1 .1 /D B _ X ... Fu ll le n g th in s e rt c D N A ... FB L N 1 : fib u lin 1 L O C 6 4 3 7 4 9 : H y p o th e tic ... Tra n s c rib e d lo c u s L P L : lip o p ro te in lip a s e C C D C 6 9 : c o ile d -c o il d o ... D N A H 3 : D y n e in , a x o n e ... C D N A : F L J 2 3 5 6 6 fis , c lo ... P O L D IP 3 : P o ly m e ra s e (... M R G P R F : M A S -re la te d ... L R R C 1 5 : le u c in e ric h re ... H o m o s a p ie n s , c lo n e IM ... D C N : d e c o rin FK S G 4 9 : F K S G 4 9 C D N A F L J 3 6 5 8 2 fis , c lo ... L H P P : p h o s p h o ly s in e p h ... L O C 6 4 4 4 5 0 : H y p o th e tic ... A K R 1 C 3 : a ld o -k e to re d u ... M R N A , c lo n e IC R Fp 5 0 7 ... C D N A F L J 3 0 6 3 8 fis , c lo ... R U N X 3 : ru n t-re la te d tra ... TIM M 8 A : tra n s lo c a s e o f ... C D N A F L J 1 2 9 3 2 fis , c lo ... IG F 1 : in s u lin -lik e g ro w th ... P C N X : p e c a n e x h o m o lo ... S E P T 1 : s e p tin 1 C P A 3 : c a rb o x y p e p tid a s e ... P R K C B 1 : P ro te in k in a s e ... g b :B F 4 3 3 2 1 9 /D B _ X R E ... ZF H X 1 B : Z in c fin g e r h o ... C X C L 1 2 : c h e m o k in e (C -... H o m o s a p ie n s , S im ila r t... g b :N M _ 0 1 4 1 3 6 .1 /D B _ X ... IS L R : im m u n o g lo b u lin s ... C D N A c lo n e IM A G E :5 2 6 ... A R P C 5 : A c tin re la te d p r... H O X A 7 : h o m e o b o x A 7 L O C 2 3 1 1 7 : K IA A 0 2 2 0 -li... g b :B F 5 1 0 5 0 6 /D B _ X R E ... Fu ll le n g th in s e rt c D N A ... L R P 1 1 : L o w d e n s ity lip o ... TR 2 IT 1 : T h io re d o x in re d ... Fu ll le n g th in s e rt c D N A c ... g b :A I6 8 4 8 3 1 /D B _ X R E F ... C L U : c lu s te rin M Y O 1 D : M y o s in ID TT L L 5 : tu b u lin ty ro s in e li... FL J 2 0 5 8 1 : h y p o th e tic a l ... A K R 1 C 2 : a ld o -k e to re d u ... C D N A : F L J 2 3 5 7 2 fis , c lo ... O IP 5 : O p a in te ra c tin g p r... A G TR L 1 : a n g io te n s in II ... R B P 7 : re tin o l b in d in g p r... R U N X 1 : R u n t-re la te d tra ... O G N : o s te o g ly c in (o s te o ... H TR A 3 : H trA s e rin e p e p ... P R K C B 1 : P ro te in k in a s e ... FO X O 1 A : Fo rk h e a d b o x ... W B S C R 1 9 : W illia m s B e u ... B C L 1 1 B : B -c e ll C L L /ly m ... L O C 3 8 9 6 3 4 : H y p o th e tic ... C L U : c lu s te rin IK Z F1 : IK A R O S fa m ily z i... C L U : c lu s te rin C X C L 1 4 : c h e m o k in e (C -... TP S T 1 : T y ro s y lp ro te in s ... A P C : a d e n o m a to s is p o ly ... W T A P /// L O C 6 5 3 1 5 0 : ... R A R R E S 2 : re tin o ic a c id ... N A R G 2 : N M D A re c e p to r... M G C 7 2 1 0 4 : S im ila r to F ... B C L 1 1 B : B -c e ll C L L /ly m ... C D N A c lo n e IM A G E :5 2 6 ... C C D C 3 : c o ile d -c o il d o m ... g b :B G 3 8 9 7 8 9 /D B _ X R E ... K IA A 0 2 5 1 : K IA A 0 2 5 1 p r... C C L 1 9 : c h e m o k in e (C -C ... R A R A : re tin o ic a c id re c e ... L FN G : lu n a tic frin g e h o ... FN D C 1 : fib ro n e c tin ty p e ... M FA P 5 : m ic ro fib rilla r a s ... G R IN 2 D : g lu ta m a te re c e ... S P O N 1 : s p o n d in 1 , e x tr... S C A R A 5 : S c a v e n g e r re c ... C D 6 9 : C D 6 9 m o le c u le g b :N M _ 0 1 4 1 1 4 .1 /D B _ X ... Tra n s c rib e d lo c u s N X N : n u c le o re d o x in C O X 7 A 1 : c y to c h ro m e c ... g b :A K 0 2 2 3 4 3 .1 /D B _ X R ... L O C 2 8 3 0 2 7 : h y p o th e tic ... M R N A ; c D N A D K F Zp 5 6 ... D A R C : D u ffy b lo o d g ro u ... A T A D 1 : A T P a s e fa m ily , ... C lo n e 2 4 9 0 0 m R N A s e q ... FC R L 1 : F c re c e p to r-lik e 1 g b :N M _ 0 1 5 8 7 0 .1 /D B _ X ... U n k n o w n m R N A s e q u e n ... R H O H : R a s h o m o lo g g e ... L TB : ly m p h o to x in b e ta (... g b :N M _ 0 1 8 5 7 5 .1 /D B _ X ... A D R A 2 A : a d re n e rg ic , a l... D P T : d e rm a to p o n tin S C D 5 : s te a ro y l-C o A d e s ... M FA P 5 : m ic ro fib rilla r a s ... C D N A c lo n e IM A G E :5 2 7 ... S P O N 1 : s p o n d in 1 , e x tr... S L F N 5 : s c h la fe n fa m ily ... FA B P 4 : fa tty a c id b in d in ... IG F 2 /// IN S -IG F2 : in s u li... C D N A c lo n e IM A G E :5 2 8 ... FC R L 2 : F c re c e p to r-lik e 2 O G N : o s te o g ly c in (o s te o ... P R K D 2 : p ro te in k in a s e D 2 C O M P : c a rtila g e o lig o m ... C D N A c lo n e IM A G E :4 8 2 ... C A S C 4 : C a n c e r s u s c e p ti... N A R S 2 : A s p a ra g in y l-tR ... A B C A 8 : A T P -b in d in g c a ... D P T : d e rm a to p o n tin g b :B C 0 3 8 6 7 2 .1 /D B _ X R ... S P IN L W 1 : s e rin e p e p tid ... O M D : o s te o m o d u lin g b :A K 0 9 4 9 7 4 .1 /D B _ X R ... S F R P 4 : s e c re te d friz z le ... C D N A F L J 1 2 2 0 4 fis , c lo ... g b :A W 1 1 7 1 8 1 /D B _ X R E ... Tra n s c rib e d lo c u s S F R P 4 : s e c re te d friz z le ... L O C 4 4 1 2 0 4 : h y p o th e tic ... M FA P 5 : m ic ro fib rilla r a s ... A D IP O Q : a d ip o n e c tin , C ... C D C 2 7 : C e ll d iv is io n c y c ... R A B G A P 1 : R A B G T P a s ... P L A 2 G 2 A : p h o s p h o lip a s ... S S X 3 : s y n o v ia l s a rc o m a ... S Q S T M 1 : S e q u e s to s o m ... L O C 6 4 3 6 7 5 : H y p o th e tic ...

Prolonged SD

80 60

Thy1 CD28 CCL19 LTbeta IL-27Ralpha IL-1R

40 20 0 0

10

20

30

40 50 Months

60

70

80 3 .0

2 . 6

2 . 1

1 .7

1 .3

0 .9

0 . 4

0

0 . 4

0 . 9

1 . 3

1 .7

2 . 1

2 .6

T cell markers and chemokines

3 .0

No correlation with immune response in blood Schuler collaboration, ASCO 2009


Impact of gene expression signature on clinical outcome in GSK MAGE3 vaccine trial 11

GSGSGS+ GS+

0.9 0.9 0.8 0.8

%patients patients %

0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 00 00

Number Number at at risk risk GS14 GS14 GS+ 21 GS+ 21

33

66

99

55 18 18

33 15 15

22 12 12

12 15 18 12 15 18 Time (months) Time (months)

21 21

24 24

27 27

11 66

00 22

00 11

00 11

11 55

00 33

30 30

Median GS-: 2.3 months [95% CI: 2.3 - 4.4] GS+: 10.3 months [95% CI: 6.7 - 12.4] HR: 0.31 [95% CI: 0.13 - 0.76] KNN Classifier

Louahed, ASCO 2008


Acknowledgments Melanoma gene array/ chemokines Helena Harlin Ruth Meng Amy Peterson Mark McKee Craig Slingluff Functional genomics core

Type I IFNs Mercedes Fuertes Aalok Kacha Justin Kline

LIGHT adenovirus Ping Yu Yang-Xin Fu Hans Schreiber

Uncoupling negative regulation Justin Kline Yuan-yuan Zha Christian Blank Amy Peterson Ian Brown

Collaborative vaccine/gene array data Gerold Schuler (Erlangen group) Vincent Brichard (GSK-Bio)


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