IL2-Based Fusion Proteins As New Bi-Functional Biovharmaceuticals for the Therapy of Cancer

IL2-based fusion proteins as new bi-functional biovharmaceuticals for the therapy of cancer

Claudia A. Penafuerte-Diaz

Faculty of Medicine, Division of Experimental Medicine

McGill University, Montreal

August 2010

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Doctor of Philosophy

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2 ABSTRACT

The growing number of strategies to specifically eliminate tumors by manipulating the

host's immune system is tangible evidence of the expectation of a cure for cancer may

arise from the field of cancer immunotherapy. The delivery of immune stimulatory genes,

such as pro-inflammatory cytokines, co-stimulatory molecules or suicide genes in the tumor site, has been shown to promote specific anti-tumor responses in mouse models of

cancer. Total tumor eradication was also found to occur despite subtotal tumor

engineering; a phenomenon coined the "bystander effect". The bystander effect in immune competent animals arises mostly from recruitment of a cancer lytic cell-mediated immune response to local and distant tumor cells which escaped gene modification. John

Stagg et al. have previously generated and characterized the murine fusion protein

between Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-

2 (IL2, aka GIFT2), which display novel immunological properties compared to both

cytokines in combination such as greater melanoma site recruitment of macrophages and functional NK cells. Consequently, GIFT2 prevent tumor formation in mice implanted with genetically modified B16 melanoma cells expressing GIFT2. In the Chapter 2 of my thesis, I evaluated the bystander effect of the murine GIFT2 in vivo to induce an effective

antitumor response against non-genetically modified B16 cells present in the tumor site. I

observed that GIFT2 secreted by genetically engineered B16 tumor cells induces a bystander effect on non modified B16 cells and such effect was mediated by recruited

NK cells in the tumor site. However, the immune bystander effect was completely lost as the total B16 cell number was increased from 104 to 106 which correlated with a sharp

reduction in the number of tumor-infiltrating NK cells. I identified that active TGF|3 is the main tumor-derived suppressive factor that downregulated IL-2RJ3 expression and

3 IFNy secretion by NK cells, and therefore attenuated GIFT2-dependent bystander effect.

We demonstrated that in vivo blockade of B16 originating TGF|3 significantly improved the immune bystander effect arising from GIFT2. Based on the potent immunostimulatory properties of the murine GIFT2 on NK cells, in the chapter 3 I

developed, characterized and evaluated the human ortholog of GIFT2, which may serve

as a mean to generate oncolytic NK cells for cell-based therapy of cancer. I observed that

human GIFT2 induces robust NK cell activation ex vivo with significant secretion of pro inflammatory cytokines, chemokines and upregulate the expression of activation markers

and NK cell activating receptors. This phenotype correlates with significantly greater

cytotoxicity against tumor cells. At the molecular level, the human GIFT2 leads to a

potent activation of Jak/STAT signaling pathway downstream of IL-2 receptor. In

conclusion, hGIFT2 fusokine possesses unique biochemical properties and constitutes a

novel and potent tool for ex vivo NK cell activation and maturation. Based on our results,

I propose that cancer gene immunotherapy of pre-established tumors will be enhanced by

blockade of active TGF|3, which acts as a potent pro-oncogenic factor and suppresses

antitumor immunity. To antagonize TGF|3 dependent effects in tandem with a pro inflammatory immune stimulus, I described in chapter 4, the generation of a new

chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-

|3 receptor II (aka FIST). FIST acts as a decoy receptor trapping active TGF-J3 in solution

and directly interacts with IL-2-responsive cells, inducing a distinctive hyperactivation of

STAT1 downstream of IL-2 receptor, which in turn promotes SMAD7 overexpression.

STAT1 hyperactivation further induces significant secretion of CXCL10, upregulates T-

Bet and T-Bet target gene expression in NK cells. The synergism of TGF|3 blockade

4 coupled to IL-2(R)-dependent STAT1 hyperagonism leads to potent immune activation

contemporaneous to a dominant NK cell-dependent antiangiogenic effect in the B16

murine model of melanoma. Consequently, FIST prevent tumor formation not only in immunocompetent mice but also in several immunodeficient mice including CD4 KO,

CDS KO, B-cell deficient (|iMT) and NK-defective beige mice, whereas mice with NK

defective functions such as nonobese diabetic-severe combined immunodeficient (NOD-

SCID) mice and Rag2/yc KO mice developed tumors. In the chapter 5, I took advantage

of this new technology to generate and characterize FIST-stimulated B cells. FIST-

stimulated B cells upregulate co-stimulatory molecules, activation markers and MHC

class II molecule expression, which is also supported by robust hyperactivation of

Jak/STAT signaling pathway. FIST-stimulated B cells maintain B cell identity based on the expression of PAX5 and CD19 and act as effective APC that induce the activation

and cell proliferation of antigen-specific CD4+ and CDS T cells. Interestingly, FIST-

stimulated B cells confer complete protective immunity to EG.7 tumor challenge in vivo.

Therefore, FIST can also be used as stimulator to generate B cells with APC features

useful for the cell-based therapy of cancer.

In conclusion, these bi-functional chimeric proteins are potential biopharmaceuticals for the therapy of cancer. Specifically, FIST constitute an advanced technology that not only target several immune system components to generate an effective antitumor but also

FIST triggers angiostatic mechanisms that suppresses tumor-derived angiogenesis, an

essential process required for tumors to growth and metastasize.

5 RESUME

Le nombre croissant de strategies visant a eliminer une tumeur cancereuse en manipulant

specifiquement le systeme immunitaire de l'hote indique que la possibility de guerison

depend en grande partie du domaine de l'immunotherapie du cancer. Le transfer! tumorale de genes impliques dans l'immunostimulation, tels que des cytokines pro- inflammatoires, des molecules co-stimulatoires, ou des genes de suicide, a ete demontre

chez la souris a promouvoir une reponse anti-tumorale. De plus, l'elimination complete

d'une tumeur peut meme se produire avec un transfer! genique partiel, un phenomene

appele "effet spectateur" (bystander effect). "L'effet spectateur" chez un animal immunocompetent survient lorsqu'une reponse immunitaire mene aussi a la lyse de

cellules cancereuses qui n'ont pas ete modifiees genetiquement. Stagg et collaborateurs

ont genere et caracterise une proteine de fusion comprenant le facteur stimulant les

colonies des granulocytes-macrophages murins (GM-CSF) et l'interleukine-2 (IL2).

Cette proteine de fusion appelee "GIFT2" possede de nouvelles proprietes immunologiques par rapport a l'utilisation des deux cytokines, telles que le recrutement

d'un plus grand nombre de macrophages et de cellules NK dans un melanome. Par

consequent, GIFT2 empeche la formation de tumeurs chez la souris implantee avec des

cellules de melanome B16 genetiquement modifiees pour exprimer GIFT2. Pour le

chapitre 2 de ma these, j'ai evalue l'effet spectateur de GIFT2 et plus precisement sa

capacite a induire une reponse anti-tumorale in vivo contre des cellules B16 non-

modifiees genetiquement. J'ai remarque que GIFT2 secretee par des cellules tumorales

B16 genetiquement modifiees induit un effet spectateur sur les cellules B16 non-

modifiees, et que cet effet est medie par les cellules NK. Toutefois, cet effet spectateur immunitaire se perd lorsque le nombre total de cellules B16 passe de 104 a 106. Avec ce

6 plus grand nombre de cellules B16, j'ai observe une reduction substantielle du nombre de

cellules NK infiltrants. J'ai determine que le facteur principal produit par la tumeur qui

supprimait l'effet spectateur de GIFT2 est le TGF|3 actif. J'ai observe que le TGF|3 actif a

diminue I'expression du recepteur (3 de l'IL-2 (IL-2R|3) ainsi que la secretion de 1'IFNy par les cellules NK. J'ai demontre que lorsque la secretion de TGF|3 par les cellules B16 est inhibee, l'effet spectateur de GIFT2 est considerablement ameliore. Due aux proprietes immunostimulantes de la proteine de fusion GIFT2 murine, j'ai developpe, caracterise et

evalue 1'orthologue humain de GIFT2. Ceci est decris dans le chapitre 3. La GIFT2

humaine peut servir comme un moyen de generer des cellules NK oncolytiques pour la therapie cellulaire du cancer. J'ai remarque que la GIFT2 humaine induit une activation

robuste de cellules NK ex vivo avec une secretion significative de cytokines/chemokines

pro-inflammatoires et une expression augmentee de marqueurs d'activation et de

recepteurs activateurs de cellules NK. Ce phenotype est correle a une plus grande

cytotoxicity contre les cellules tumorales. Au niveau moleculaire, la GIFT2 humaine

mene a une activation puissante de la voie de signalisation Jak/STAT en aval du

recepteur de l'IL-2. La proteine de fusion GIFT2 possede done des proprietes

biochimiques uniques et constitue un outil innovateur et puissant pour l'activation et la

maturation de cellules NK. Suivant ces resultats, j'ai propose que l'inhibition du TGF|3

actif, un facteur oncogenique et suppresseur d'immunite tumorale, pourrait ameliorer l'immunotherapie genique d'un cancer existant. Dans le chapitre 4, je decris la production d'une nouvelle proteine de fusion entre l'IL-2 et le domaine extracellulaire du recepteur II de TGF|3 (TGFJ3RII soluble). Cette proteine chimerique appele FIST a ete generee pour

contrarier les effets du TGF|3 et aussi pour agir comme stimulus immunitaire pro-

7 inflammatoire. J'ai observe que FIST agit comme recepteur du TGF|3 actif qui se trouve

en solution, et aussi interagit directement avec les cellules IL-2-sensibles. Ceci induit

une hyperactivation de STATl en aval du recepteur de l'IL-2, ce qui donne lieu a une

surexpression de SMAD7. De plus, l'hyperactivation de STATl induit une secretion

significative de CXCL10, et augmente I'expression de T-bet et des genes cibles de T-bet

dans les cellules NK. La synergie entre 1'inhibition de TGF|3 et 1'activation IL2(R)-

dependante de STATl conduit a une activation immunitaire puissante en meme temps

qu'un effet anti-angiogenique NK-dependant, dans le model e murin de melanome B16.

Par consequent, FIST empeche la formation de tumeurs non seulement chez la souris immunocompetente, mais aussi chez differentes souris immunodeficientes, y compris les

souris CD4" CDS % cellules B-deficientes (|iMT), et souris beige NK-defectueuses. Par

contre, les souris avec fonction NK defectueuse, telles que les souris diabetiques non

obeses presentant une immunodeficience combinee severe (NOD-SCID) et les souris

Rag2/yc"" ont developpe des tumeurs. Tel que decrit dans le chapitre 5, j'ai profile de

cette nouvelle technologic pour generer et caracteriser les cellules B stimulees par FIST.

J'ai remarque que les cellules B stimulees par FIST ont une expression augmente de la

molecule de co-stimulation, du marqueur d'activation, et de la molecule de MHC classe

II. Ceci est supporte par une hyperactivation robuste de la voie de signalisation

Jak/STAT. Les cellules B stimulees par la proteine FIST maintiennent des

caracteristiques de cellules B. Elles expriment PAX5 et CD 19, et agissent comme des

cellules presentatrices d'antigenes (APC) efficaces qui induisent l'activation et la

proliferation de cellules T CD4+ et CDS antigene-specifiques. Ce qui est aussi tres interessant est que les cellules B stimulees par FIST conferent une immunite protectrice

complete contre le "challenge" de la tumeur EG.7 in vivo. Par consequent, FIST peut

8 egalement etre employe comme stimulus pour generer des cellules B avec fonctions

d'APC qui seraient utiles pour la therapie cellulaire du cancer.

En conclusion, ces nouvelles proteines chimeres bi-fonctionnelles sont des produits biopharmaceutiques potentiels pour le traitement du cancer. Plus precisement, FIST

constitue une technologic de pointe qui cible non seulement des composantes du systeme immunitaire pour generer un effet antitumoral efficace mais aussi qui declenche des

mecanismes angiostatiques qui suppriment le processus d'angiogenese essentiel a la

croissance des tumeurs et de leurs metastases.

9 ACKNOWLEDGMENTS

The achievement of my doctoral studies would not have been possible without the

support and advices of a number of people. First of all, I would like to thank my

supervisor, Dr Jacques Galipeau. Dear Jacques, it's hard for me to find the words to

express my deepest gratitude to you. I thank you for the outstanding supervision, your ideas and advices were crucial in the development of my research projects. Your

enthusiasm for research, kindness, humanity and perseverance as a scientist are inspirational and encourage everyone to be like you. Thank you for giving me the

opportunity to become a scientist and publish in very prestigious journals. I am very fortunate for being part of your research team!

I want to thank my parents Maria C. Diaz-Borrego and Zoilo A. Penafuerte-Perez for

providing me with a great education, support and love always. I thank my love Collin

Horner for being very supportive in my endeavors and encouraging me always in the

most difficult moments. I thank my sister Anay Penafuerte, my aunts Maritza Perez and

Emma Perez for being part of my life and supporting me.

I would like to thank all my talented colleagues, Norma Bautista-Lopez, Elena Birman,

Kathy Forner, Moira Francois, Manaf Bouchentouf, Daniel Coutu, Jessica Cuerquis,

Francois Mercier, Patrick Willians, Jeremy Hsieh, Nicoletta Eliopoulos, Shala Yuan and

Janik Jacmain.

I would like to thank all my friends: Maite Hernandez, Eva Sosa, Manuel Justino,

Esteban Alfonso, Yosvany Martinez, Manuel Flores, Mariaelena Santamaria, Zuzet

Martinez, Nelly O'Farril and their families for many happy moments and for being great friends!

10 PREFACE

Cancer remains one of the leading causes of morbidity and mortality worldwide. The

WHO organization predicts by 2020 that the number of new cases of cancer in the world will increase to more than 15 million, with deaths increasing to 12 million

(http://www.who.int/mediacentre). Much of the burden of cancer incidence, morbidity,

and mortality will occur in the developing world. These dramatic epidemiological

changes in which the burden of chronic, non-contagious disease that once upon a time was limited to industrialized nations is now increasing in less developed countries. In

addition to the pre-existent risks associated with diet, tobacco, alcohol, obesity, and industrial exposures, the developing world is already burdened by cancers some of which

are attributable to infectious diseases.

Current therapies for cancer based on chemotherapy or radiation that kill dividing cells or

block cell division have also shown severe side effects on normal proliferating cells in

patients with cancer. Therefore, the potential for treatment of cancer patients by immunologic approaches that target specifically tumor cells without injuring most normal

cells has great promise.

In an effort to develop new strategies that offer hope for the treatment and eradication of

cancer, I proudly present my Ph. D thesis to the committee members.

In the course of my doctoral studies, I have tested four original hypotheses that have led to the development of novel therapeutic strategies. My research outcomes have been

published in three first-author original peer-reviewed papers, which are presented in this thesis in accordance with McGill guidelines concerning thesis preparation:

11 > Chapter 2: Claudia Penafuerte and Jacques Galipeau. TGFp secreted by B16

melanoma antagonizes cancer gene immunotherapy bystander effect. Cancer

Immunol Immunother. 2008 Aug; 57 l: 1197-206.

> Chapter 3: Claudia Penafuerte, Norma Bautista-Lopez, Boulassel Mohamed-

Rachid, Jean-Pierre Routy and Jacques Galipeau. The human ortholog of

granulocyte-macrophage colony-stimulating factor and interleukin-2 fusion

protein induces potent ex vivo NK cell activation and maturation. Cancer Res.

2009 Dec 1; 69^: 9020-8.

> Chapter 4: Claudia Penafuerte, Norma Bautista-Lopez, Manaf Bouchentouf,

Elena Birman, Kathy Forner, Jacques Galipeau. Novel TGF|3-antagonist inhibits

tumor growth in a mouse model of melanoma by inducing angiostatic-NK cells.

2010. Submitted to Journal of Experimental Medicine.

> Chapter 5: Claudia Penafuerte, Norma Bautista-Lopez, Manaf Bouchentouf,

Elena Birman, Kathy Forner, Jacques Galipeau. FIST-activated B cells act as

potent antigen presenting cells. Manuscript in preparation.

12 TABLE OF CONTENTS

Abstract 3

Resume 6

Acknowledgments 10

Preface 11

Table of contents 13

List of figures 21

Chapter 1: General information 24

1.1 Clasification 25

1.2 Cancer immunology 26

1.2.1 Immunosurveillance 26

1.2.2 Innate immunity 27

1.2.3 Adaptive immunity 28

1.2.4 Innate and adaptive immunity effector molecules 30

i. Receptors 30

ii. Granule-dependent exocytosis pathway 32

iii.Pro-inflammatory cytokines with anti-tumor properties 33

iv. Chemokines with anti-tumor activity 35

1.2.5 Tumor escape mechanisms 36

i. Alterations of MHC molecules and tumor associate antigens (TAA)

expreee&MOM 36

13 ii. Alterations of adhesion and accessory molecule expression on tumor cells or

immune cells 37

Hi. Secretion of suppressive factors by tumor cells or regulatory cells 37

iv. Transforming growth factor /? (TGFfi) family 38

v. Induction of tolerance or clonal deletion of effector cells 40

vi. Induction or recruitment of suppressor cells 40

vii. Contribution of the immune system as promoter of tumor aggressiveness and

progression 41

1.2.6 Immunoediting 42

1.3 Cancer invasion and metastasis 43

1.3.1 The epithelial to mesenchymal transition (EMT) 44

1.3.2 Tumor associated angiogenesis 46

1.4 Immunotherapy 48

1.4.1 Cell based therapy 49

i. Genetically modified tumor cell vaccines 49

ii. Dendritic cell based therapy 50

Hi. B cell based therapy 51

1.4.2 Cytokine and costimulatory molecule based therapy 51

i. IL-2 based therapy 52

ii. Granulocyte-macrophage colony stimulating factor (GM-CSF) based

f/zerqpy 53

1.4.3 Peptide vaccines 54

14 1.4.4 Recombinant virus based vaccines 55

1.4.5 Fusion protein and antibody based therapy 55

1.4.6 DNA vaccines 56

1.5 Specific research aims 58

Chapter 2: TGFfi secreted by B16 melanoma antagonizes cancer gene immunotherapy

bystander effect.

2.1 Abstract 60

2.2 Introduction 61

2.3 Materials and methods 62

2.3.1 Animals, cell lines, and reagents 62

2.3.2 Murine B16F0 tumor implantation in immunocompetent C57B1/6 mice and immune infiltrate analysis 63

2.3.3 Flow cytometry analysis of non modified and genetically modified cytokine expressing B16 cells 64

2.3.4 Cytokine-dependent CTLL-2 proliferation assay 64

2.3.5 Murine NK cell isolation 65

2.3.6 In vivo blockade of TGF|3 65

2.4 Results 66

2.4.1 MHC class I and II expression in B16 melanoma cells 66

15 2.4.2 Immune bystander effect is lost with increased B16 melanoma tumor burden .66

2.4.3 Host derived cellular immune response to B16 melanoma 70

2.4.4 B16 tumor cells secrete biologically active TGF|3 74

2.4.5 B16 derived TGF|3 blocks NK cell recruitment and function 78

2.4.6 In vivo blockade of TGF|3 and effect on immune bystander effect 82

2.4.7 Interferon y pre-treatment of B16 melanoma upregulates expression of MHC I and

MHC II but does not improve immune bystander effect in vivo 86

2.5 Discussion 91

2.6 Acknowledgments 95

Chapter 3: The human ortholog of granulocyte-macrophage colony-stimulating factor and interleukin-2 fusion protein induces potent ex vivo NK cell activation and

maturation.

3.1 Abstract 97

3.2 Introduction 98

3.3 Materials and methods 99

3.3.1 Cell lines, recombinant proteins, antibodies and ELISA kits 99

3.3.2 PBMC and human NK cells 100

3.3.3 Vector construct and transgene expression 101

3.3.4 Analysis of cell surface marker on leukocytes 101

16 3.3.5 Flow cytometric analysis of NK cell expression markers, IFNy expression,

apoptosis and cell proliferation 102

3.3.6 In vitro cytotoxicity assay 102

3.3.7 Intracellular signaling 103

3.3.8 Statistic evaluation 103

3.4 Results 104

3.4.1 Design and expression of hGIFT2 fusokine 104

3.4.2 HGIFT2 induces the proliferation of IL-2 and GM-CSF dependent cell lines.107

3.4.3 Effect of HGIFT2 on primary lymphocytes 107

3.4.4 hGIFT2 induces activation of NK cells 111

3.4.5 hGIFT2 upregulates activating receptor expression, promotes NK cell

maturation and significant greater NK cell cytotoxicity 118

3.4.6 hGIFT2 induces hyperactivation of Jak/Stat signaling pathway in NK

cells 118

3.5 Discussion 128

3.6 Acknowledgments 132

Chapter 4: Novel TGF^-antagonist inhibits tumor growth in a mouse model of

melanoma by inducing angiostatic-NK cells.

4.1 Abstract 134

17 4.2 Introduction 134

4.3 Materials and methods 136

4.3.1 Mice 136

4.3.2 FIST fusokine design, expression and functionality 136

4.3.3 Immune cell isolation and cytokine production 137

4.3.4 Cell signaling and receptor expression 137

4.3.5 Immune cell infiltration in the tumor site 138

4.3.6 Endothelial cell isolation, in vitro angiogenesis, proliferation and migration

assays 138

4.3.7 Statistical analysis 139

4.4 Results 139

4.4.1 Generation and characterization of murine IL-2/sT|3RII fusion protein:

FIST 139

4.4.2 FIST desensitizes immune cells to TGF|3 mediated suppression 140

4.4.3 FIST induces a robust immune bystander effect and inhibits tumor growth in

vivo 141

4.4.4 FIST antitumor effect in immunodeficient mice 144

4.4.5 FIST inhibits tumor-derived angiogenesis 145

4.4.6 FIST mediates upregulation of STATl target genes 159

18 4.5 Discussion 166

4.6 Acknowledgments 169

Chapter 5: FIST-activated B cells act as potent antigen presenting cells.

5.1 Abstract 171

5.2 Introduction 171

5.2 Materials and methods 174

5.2.1 Mice, reagents and antibodies 174

5.2.2 B cell isolation, generation and characterization of FIST-stimulated B cells... 174

5.2.3 Intracellular signaling 175

5.2.4 In vitro antigen presentation assay (APC) 175

5.2.5 In vivo antigen presentation assay (APC) 175

5.2.6 Statistic evaluation 176

5.3 Results 176

5.3.1 FIST induce potent B cell activation 176

5.3.2 FIST promote potent B cell activation via Jak/STAT signaling pathway 177

5.3.3 FIST-stimulated B cells promote potent CD4+ and CDS T cell activation in

antigen specific manner 177

5.3.4 FIST-stimulated and OVA pulsed B cells induce protective immunity against tumor challenge 178

5.4 Discussion 188

19 5.5 Acknowledgments 190

Chapter 6: Conclusions 192

Chapter 7: Contribution to the original knowledge 200

Chapter 8: References 201

20 LIST OF FIGURES

Figure 1: Immunophenotypic analysis of B16 cells and genetically modified B16-GIFT2

cells.

Figure 2: In vivo immune bystander effect of GIFT2-secreting cells

Figure 3: Immune infiltrated analysis of tumor implants

Figure 4: B16 cells secrete active TGF|3

Figure 5: Blockage of immunosuppressive effects of TGF|3 enhances the activation state

of NK cells in response to GIFT2

Figure 6: Blockage of TGF|3 suppressive effects improved the bystander effect of GIFT2.

Figure 7: IFNy upregulates the expression of MHC class I and MHC class II molecules

on B16 cells in vitro, but did not improve the immunogenicity of these cells in vivo

Figure 8: Design and expression of hGIFT2 fusokine

Figure 9: Bioactivity of hGIFT2

Figure 10: hGIFT2 increases NK survival but does not induce NK cell proliferation

Figure 11: hGIFT2 induces NK cell activation

Figure 12: hGIFT2 similarly activate CD56dim and CD56bright cells

Figure 13: hGIFT2 upregulates NK cell activating receptors, promotes NK cell

maturation and greater NK cell cytotoxicity than cytokine controls

21 Figure 14: HGIFT2 does not affect the NK cell inhibitory receptor expression (CD43 and

KIR) and the NK activating receptor NKG2D

Figure 15: hGIFT2 promotes greater NK cell cytotoxicity than cytokine controls over time.

Figure 16: hGIFT2 induces a hyperactivation of JAK/STAT pathway downstream of

GM-CSF and IL-2 receptors in NK cells

Figure 17: Generation and characterization of FIST

Figure 18: FIST induces significant Thl cytokine production by activated immune cells

Figure 19: FIST antagonizes TGF|3 mediated inhibition of c-myc expression

Figure 20: FIST induces a potent anti-tumor response and immune bystander effect

Table 1: FIST treated mice do not display signs of toxicity

Figure 21: NK cells as essential mediator of FIST anti-tumor effects.

Figure 22: Qualitative expression profile of angiogenesis related proteins secreted by

FIST-activated NK

Figure 23: FIST inhibits angiogenesis in vivo and in vitro

Figure 24: Pictures of capillary like structures formed by wild type or CXCR3 knockout

endothelial cells cultured in the presence of the secretome of FIST or control stimulated

NK cells.

Figure 25: FIST mediates upregulation of statl target genes

22 Figure 26: FIST-stimulated B cell phenotype

Figure 27: FIST-stimulated B cells display potent activation of STAT3 and STATS

Figure 28: FIST-stimulated B cells act as effective antigen presenting cells

Figure 29: FIST-stimulated and OVA pulsed B cells promote complete protective immunity against tumor challenge

23 CHAPTER 1

GENERAL INFORMATION

24 CHAPTER 1: GENERAL INFORMATION

Cancer includes a wide range of diseases in which abnormal cells proliferate without

control and invade other tissues and organs forming metastases. Cancer cells can spread to other parts of the body through the blood and lymph systems. Several environmental

and heredity factors are important in the carcinogenic process.

1.1 SSThe nature of cancer depends on the tissue from which it was derived. The vast

majorities of tumors arises from epithelial tissues and are known as carcinomas. The incidence of carcinomas is approximatly 80% of human cancer in the western world, these types of cancers derived from epithelial cells from a variety of tissues such as lung, liver, esophagus, uterus, stomach, as well as epithelial cells lining the glandular tissue in the breast. Based on the biological function of the original epithelial, the carcinomas can be subdivided in two major categories, the squamous cell carcinomas, which are derived from epithelial sheets that normally function to seal cavities or channels protecting the under-lying cell population (i.e epithelial cell lining the skin and the esophagus) and the

adenocarcinomas, which contains specialized cells that produce and secrete substance into the ducts where they are located (i.e. epithelial cells in the lung, uterus and cervix).

The first group of nonepithelial tumors includes the sarcomas, which are derived from various connective tissues but share the common origin from the mesoderm of the

embryo. The sarcomas are originated from a variety of mesenchymal cells such fibloblasts, adypocytes, osteoblasts, myocytes and precursor of endothelial cells, which give rise to one of most uncommon type of tumor, the angiosarcomas. The second group

of nonepithelial cancer arises from the hematopoietic tissues, including leukemias

derived from "white blood" cells such as B and T cells that move freely in circulation,

25 whereas lymphomas include lymphoid lineages tumors that form solid tumor masses found in lymph nodes. The most common hematopoietic malignancies include acute lymphocytic and myelogenous leukemias, chronic lymphocytic and myelogenous leukemias, multiple myeloma, non-Hodgkin's lymphomas and Hodgkin's disease. The third group of nonepithelial tumors arises from cells of the central and peripheral nervus

system. According to the cell type that gives rise to the tumors, they can be clasified as gliomas, glioblastomas, neuroblastomas, schwannomas, and medulloblastomas. Other types of tumors do not fit into the major classifications. For instance, the melanomas are

originated from melanocytes, which arise from neural crest. Despites having similar

origins to neuroectodermal cells, melanocytes develop as non nervous system cells that

settle in the skin and the eyes and whose function is to produce melanine for these

organs.

1.2 CANCER IMMUNOLOGY

1.2.1 Immunosurveillance

In 1967, Burnet coined the term of "immunosurveillance" to define the nature of tumor

recognition by the immune system3. Similarly, Lewis Thomas supported the role of the immune system as mediator of homograph rejection as primary defense against

neoplasia4. These two hypotheses invoke for the first time the concept of tumor immunosurveillance. Initially, this was questionable, due to the absence of tumors in

several compromised mouse strains5 and because the cancer incidence in long-term

suppressed organs transplant recipients was believed to be caused by viral transformation.

In the mid 1990, the concept of immunosurveillance was reconsidered by the observation that IFNy receptor (IFNyR) contributes to immunity against spontaneous and inducible

26 tumors6' 7 This essential role of IFNy in tumor immunity could be associated with the

action of natural killer cells8. Further support of the immunosurveillance theory came from the experiments performed with knockout mice for perforins and effector molecules that mediate NK cell killing activity9, which display an elevated incidence of spontaneous and inducible tumors. The best evidence for immunosurveillance was demonstrated in

knockout mice for RAG-2 proteins, which are responsible for rearranging the genes encoding soluble antibodies molecules and the T-cell receptors expressed on the surfaces

of T cells. RAG-2 or RAG-1 knockout mice lack of T lymphocytes, B lymphocytes and y5 T and MKT cells. These mice spontaneously develop gastrointestinal malignancies by the age of 18 months10.

1.2.2 Innate immunity

Exposure to environmental stresses including pyrogens, inflammatory cytokines and

oxidants may predispose to neoplastic transformation. However, normal cells have

developed complex mechanisms to avoid malignant transformation, which may result in

successful DNA repair and survival or, alternatively apoptosis if these survival

mechanisms fail to repair the damage DNA. If cells fail to repair the DNA and skip

apoptosis, the effective system of innate immunosurveillance acts to defend the organism from tumor initiation. The innate components of the immune response are able to

recognize and attack foreign proteins and malignant or virus- infected cells without

previous exposure to these agents. The immune cells recognize characteristic molecular

pattern that can be expressed by malignant or transformed cells, but not in normal cells.

NK cells are key players in this immune recognition. NK cells were discovered in 1975

and named in honor of their innate 'natural killer' ability to recognize and eliminate

27 malignant cells. It has been well documented in vivo that NK cells spontaneously kill

MHC class I deficient tumor cells and the metastases11. In addition, in response to

cytokine stimulus such IL-2, IL-12, IFNs and IL-15, NK cells increase their cytolytic,

proliferative and anti-tumor properties. In turn, NK cells produce a variety of pro inflammatory cytokines including IFNy, which intensify the anti-tumor response

promoting the recruitment of other innate and adaptive effector cells such as

macrophages, neutrophils, NKT cells, T cells, dendritic cells and B cells. More importantly, through the production of cytokines, NK cells shape the adaptive immune

response by modulating dendritic cell functions12. Macrophages are part of the innate immune system and play important role in tumor immunity. Depending on their

phenotype macrophages kill tumor cells (Ml) or facilitate tumor growth and metastasis

(M2). Macrophages destroy tumor cells through their production of nitric oxide and type

1 cytokines and chemokines. Similarly, NKT cells, characterized by the expression of

both NK and TCR, come in two flavors. Type I NKT cells, which express the invariant

Val4Jal8 TCR VP chain, promote tumor rejection; and type II NKT cells, which express

a non-Val4Jal8 TCR VP chain, promote tumor growth13. NKT cells recognize lipid and glycolipid antigens presented by nonclassical MHC class I CD Id molecules and rapidly

produce large amounts of Thl and Th2 cytokines bridging the innate and adaptive immnunity. NKT cells can regulate both CTL and NK cell anti-tumor activity14.

1.2.3 Adaptive immunity

The recognition of tumor cells by innate components of the immune system cause the generation of danger signals such as extracellular matrix break-downs products, heat

shock proteins (HSPs) derived from cells undergoing necrosis or pro-inflammatory

28 cytokine released as result of immune cell activation15. These danger signals cause the

activation of the antigen presenting dendritic cells 16. This first step in the generation of

adaptive immunity is supported by the danger model, which postulates that the immune

system has the capacity to sense danger by using "professional" antigen presenting cells

(APCs) as sentinels of tissue distress17. The cardinal role of dendritic cells (DC) in

priming adaptive immunity and in orchestrating immune responses against tumors has

been well established. Upon antigen uptake, a process facilitated by specialized receptors with high endocytic capacity, dendritic cells mature and migrate to the regional draining lymph nodes to present the processed antigens to CDS and CD4+ T cells in the context

of MHC class I or II respectively18. The T cell priming capacity of DC, however, depends

not only on antigen presentation but also on other features of DC, such as cytokine

production and expression of costimulatory molecules expressed by mature DC19' 20.

Consequently, activated antigen specific CDS T cells differentiate into cytotoxic T lymphocytes (CTL), which lyse tumor cells in an antigen-specific manner or memory

CDS T cells. On the other hand, activate CD4+ T cells differentiate into helper or

memory CD4+ T cells. Helper CD4+ T cells play a crucial role in orchestrating immune

response 20'21. Helper CD4+ T cells in turn can differentiate into the cell subtypes type 1

(Thl), type 2 (Th 2), Treg or Type 17 (Thl7) cells, which through their secreted

cytokines (Thl, Th2 and Thl7) dictate the fate of the immune response22"25. B cells, also

as components of adaptive immunity, have the capacity to present exogenous proteins to

T cells in association with MHC class II molecules, hence stimulating CD4+ T cell help26.

Harris et al. have identified two populations of "effector" B cells (Bel and Be2) that upon

encountering antigens and T cells produce polarizing cytokines such as IL4 and IFNy that

subsequently regulate the differentiation of naive CD4+ T cells to Thl and Th2 cells27. B

29 cell activation is initiated following engagement of B cell receptor (BCR) by a specific

antigen, which promote B cell maturation, proliferation and differentiation to generate

plasma cells that produce high-affinity antibodies following affinity maturation or

memory B cells, which confer long-lasting protection against antigen challenge28' 29.

Finally, macrophages although behave mainly as innate effector cells, also can function

as antigen presenting cells to activate cytotoxic CD8+ T cells30'31.

1.2.4 Innate and adaptive immunity effector molecules

i. Receptors

The essential functions of NK cells in immunosurveillance are regulated by the integration of signals from inhibitory and activating receptors. The inhibitory receptors

are characterized by the presence of an immunoreceptor tyrosine-based inhibition motif

(ITIM) in their cytoplasmatic domains32, which dominate and control positive signals induced by activating receptors via recruitment of tyrosine phosphatases such as SHP-133.

The inhibitory receptors, such as the human natural killer cell immunoglobulin like

receptors (KIR) or Ly49 molecule family of C-type lectin proteins in rodents recognize

adequate levels of MHC class I molecules expressed on normal and healthy cells and

protect normal tissue from unnecessary NK cell activation34. Accordingly, NKG2A and

B have ITIM motif and therefore act as inhibitors35. NK cells express an array of

activating receptors such as NKG2C, NKG2D, and NKG2E. In general, the activating

receptors are associated with a 12 kDa DNA-activating protein (DAP-12) 36, a factor

containing an immunoreceptor tyrosine-based activating motif (ITAM) that provides

cellular activating signals37. Although NKG2D lacks ITAMs, it forms an activating

receptor complex with a 10 kDa DAP (DAP-10)38. The binding of activating receptors to

30 stress-induced ligands molecules expressed on malignant cells trigger NK cell cytolytic

activity. For instance, the activating receptor NKG2D interacts with H-60 minor

histocompatibility antigen and retinoic acid early inducible (Rae-1) family of cell surface

proteins, as well as with the original identified MHC class I chain-related glycoproteins

(MICA and MICE) promptly induce the activation of PI3K pathway39'40, which in turns

has been reported to be critically involved in NK cell cytotoxicity41. Activating receptors

are also expressed by y5+ T cells and a|3 CDS T cells. However, the expression of these

receptors on T cells is induced only after T cell activation by antigens, while the

expression of the receptor on NK cells is constitutive. This highlights an important

difference between adaptive and innate immunity39' 42' 43. CTL also induces apoptosis of

cancer cells via Fas-FasL receptor interaction system. T cells and NK cells express FasL

(CD 178), whereas tumor cells may express Fas (CD95 or Apo-1), thus tumor cells may be susceptible to apoptosis mediated by this pathway44. Binding of Fas with FasL causes trimerization of Fas-associated death domain (FADD) proteins via homotypic death

domain interactions. This even leads to recruitment of either procaspase 8 or 10, which undergo a process of autoproteolysis to become an activated caspase. Assembly of these

components promotes in the formation of death-inducing signaling complex (DISC).

Caspase 8 in turns activates several procaspases such as 3, 6, or 7 by transproteolysis.

Finally, these effector caspases cleave DNA. In addition, Caspase 8 hydrolyzes Bid by

causing damage in the mitochondrial which trigger cytochrome-C release45"47. At the

molecular level, FasL expression is positively regulated by NFAT, Egr2/Egr3, NFKB,

AP-1, c-myc SP1, and Bl/Cdkl and negatively regulated by c-Fos and CIITA48"50. The

TNF-related apoptosis-inducing ligand (TRAIL) and its specific receptor system TRAIL-

R1 (death receptor 4, DR4) and TRAIL-R2 (DR5) constitute another member of the

31 TNFR family that promote cell death. The receptor-ligand interaction engages FADD

proteins in their cytoplasmic tail, which recruits procaspase 8 that is activated within the

DISC. Activated caspase 8 trigger a similar cascade of reactions as the one previously

reported for the Fas-FasL system51"54. Moreover, TNF-TNFR system also promotes

apoptosis by formation of DISC and the contribution of FADD molecules, which trigger

as previously described the caspase activation cascade and mitochondrial changes. The

receptors of TNF (TNFR) can be grouped into three classes: 1) having cytoplasmic death

domains, 2) linked to adaptor molecules denominated TNF receptor associated factors

(TRAFs), and 3) soluble receptors. TNF-TNFR complex has also been reported to increase NADPH oxidase activity, which promote oxidative stress and therefore necrotic

cell death55"58.

II. Granule-dependent exocytosis pathway

Lysis of tumor cells by cytotoxic lymphocyte (cytotoxic T cells and NK cells) involves

exocytosis or degranulation, a process that require the mobilization of microtubules that

bring the preformed granules or lysosomes of the cytotoxic cell towards the point of

contact with the target cell, releasing stored lytic molecules59' 60. As result of

degranulation, the lysosomal-membrane-associated glycoproteins such as CD107 a,

CD 107b, and CD63, which are found in the granule-membrane inner surfaces, are exposed onto the lymphocyte surface61'62. The lytic molecules contained in granules are

perforin, granzymes (Grzs), and granulysin. Upon encountering of cytotoxic cell-target

cells, perforin is released by exocytosis. Once it is anchored on the target cell surface,

perforin induces the polymerization of the membrane in the presence of Ca2+ to form

cylindrical pores, which allow the flow of granzymes and granulysin through the target

cell membrane provoking ionic exchange and therefore osmotic unbalance and cell

32 death63'64 The development of perforin knockout mice has highlight the important role of this molecule in tumor immunosurveillance9' 65. Granzymes belong to the serine-

proteases family. The Grz-A and Grz-B are the most abundant within lytic granules.

Grazymes induce apoptosis by caspases-independent or -dependent pathways. Based on their functions they are grouped as a) inflammatory, b) initiator (of stress signals), and c)

effector (of apoptosis)66"68. Granulysin is contained in granules from NK cells, cytotoxic

and helper T cells, and NKT cells. It has been reported that granulysin once interacts with

the negative charges from target-cell mitochondrial membrane lipids, induce cell

membrane damage, which provoke the release of cytochrome-C and therefore the

activation of caspase cascade69. hi. Pro-inflammatory cytokines with anti-tumor properties

Cytokines are extracellular messenger molecules secreted by cells involved in immunity, inflammation, differentiation, fibrosis and repair70. A distinctive characteristic of

cytokines is that they are produced in response to stimulation. Typically, they have short

half life, usually in the order of minutes and have a short action radius associated with a very high affinity for the receptors. More important, cytokines can act in networks or

cascades affecting different immune cell types once they are secreted in the inflammation

site. Interleukin-2 (IL-2) is an essential cytokine that promote an innate antitumor

response due to its effectiveness at inducing loco-regional tumor rejection, acting as

autocrine factor for T cells and supporting the development of cytotoxic T cells,

stimulating NK cells proliferation and cytolytic activity71"73. IL12 is among the

proinflammatory cytokines required for the development of Thl cell mediate tumor immunity. Similarly to IL-2, IL-12 induces the production of IFN-y from NK and T cells,

acts as a growth factor for activated T and NK cells, enhances NK cell cytotoxicity, and

33 favors cytotoxic T lymphocyte generation. IL-12 favors Thl cell differentiation by

priming CD4+ T cells for IFN-y production in response to tumor antigens. The early

preference expressed in the immune response depends on the balance between IL-12, which favors Thl responses, and IL-4, which favors Th2 responses. Therefore, IL-12

represents a functional bridge between the nonspecific innate immunity and the

subsequent antigen-specific adaptive immunity74. IL-12 has been reported to possess

potent therapeutic effects at nontoxic doses in several mouse tumor models.

Administration of IL-12 can result in antiangiogenic effects that may also contribute to its

antitumor activity in vivo75, 76. IFNy is a pleiotropic cytokine with essential roles in cell-

mediate immunity against tumor. IFNy upregulates cell-surface class I and II MHC

expression, thus promoting peptide specific activation of CDS and CD4+ T cells

respectively77' 78. IFNy is a major product secreted by activated immune cells such as

CD4+ T helper type 1 lymphocytes, cytotoxic T cells, NK cells, B cells, NKT cells and

professional APC79"81. IFNy promotes characteristic Thl effector mechanisms, such as innate cell-mediated immunity via activation of NK cell effector functions, macrophage tumoricidal activity and specific cytotoxic immunity via T cell:APC interactions. The latter is also enhance by IFNy dependent inhibition of growth of Th2 cell populations82.

IFNy exerts potent inhibitory effects on tumor cell proliferation by inducing cell growth inhibition and apoptosis83. IFNy inhibit proliferation primarily by increasing protein levels of Ink4, p21 and p27Kipl84'85. IFNy orchestrates lymphocyte trafficking to the

sites of inflammation through upregulating expression of adhesion molecules and

chemokines77'86. In addition, IFNy suppresses tumor derived angiogenesis by inhibiting

cell growth and tube formation of endothelial cells directly via STAT1 activation.

34 Moreover, IFNy antagonizes the biological activity of VEGF by inhibiting the expression

of genes required for VEGF response such as angiopoietin-2, urokinase plasminogen

activator, tissue inhibitor of matrix metalloproteinase-1, cyclooxygenase -2, and VEGF

receptor 287. Indirectly, IFNy promotes the production of potent angiostatic chemokines88'

89. Other cytokines with potent anti-tumor activity include IL-15, which is a well known to support NK cells maturation and survival90'91; GM-CSF, besides to chemoattracts APC and macrophages, GM-CSF act as initiator of an adaptive immune response against tumor associated antigens92. iv. Chemokines with anti-tumor activity

Chemokines are small proteins, 70-120 residues long, that contain 1-3 (usually 2)

disulfides. Although, their sequence homology is variable, all share very similar

secondary and tertiary structures with 20-40% sequence identity across the whole

superfamily, but the quaternary structure dramatically differs between subfamilies. Their

classification is based on the number and the spacing of the first two cysteine residues in the aminoterminal part of the protein and includes four structural branches (C, CC, CXC,

and CX3C). In particular, the chemokines CXCL9, CXCL10, and CXCL11, which are interferon-inducible ELR2 CXC chemokines, inhibit tumor growth, not only by acting as

potent angiostatic factors but also they promote cell-mediated immunity favoring lymphocyte trafficking and activation of Thl cells, natural killer cells, macrophages and

dendritic cells89. The appropriate anti-tumor response along with the subsequent generation of angiostatic factors that locally affect tumour associated angiogenesis has given rise to the concept of immunoangiostasis93.

1.2.5 Tumor escape mechanisms

35 Despites of the existence of an effective mechanism of immunosurveillance, tumor cells

evolve with different strategies to escape from immune system recognition and

elimination. These strategies can be grouped in the following major categories: i)

alterations of MHC molecules and tumor associate antigens expreeession, ii) alterations

of adhesion and accessory molecule expression on tumor cells or immune cells, iii)

secretion of suppressive factors by tumor cells or regulatory cells, iv) induction of tolerance or clonal deletion of effector cells, v) induction or recruitment of suppressor

cells, vi) Immune effectors contribution in the sculpt tumors for aggressiveness and

progression.

i. Alterations of MHC molecules and tumor associate antigens (TAA) expression

Tumor cells may down-regulate or lose MHC class I expression and hence avoid

elimination by MHC-restricted cytotoxic T lymphocytes. This tumor phenotype has been

reported associated with poor prognosis in B cell lymphomas94. However, other reports in

colorectal cancer patients indicate that tumor lacking MHC class I expression are

susceptible to NK cell recognition, and therefore predict better survival95. Moreover, tumor cells may upregulate molecules such as HLA-E, which binds inhibitory receptors

(KIR) on NK cells and CTLs. Cytokine secreted by tumor cells and immune such as IL-

15 and TGF|3 also upregulate the expression of these inhibitory receptors96'97 In addition,

Thl cytokines such as IFNy can act as double edged swords promoting tumor progression

by upregulating KIR ligand expression on tumor cells via upregulation of HLA-G

molecule expression98. High serum levels of soluble HLA-G protein in melanoma

patients has been associated with tumor load and disease progression99.

36 II. Alterations of adhesion and accessory molecule expression on tumor cells or immune cells

The appropriated presentation of tumor antigens by dendritic cells is a crucial step for the generation of CTL, therefore adequate adhesion and accessory molecule expression is

essential for this process. It has been reported that tumor infiltrating dendritic cells

promote T cell anergy by downregulating the expression of the costimulatory molecules

CD80 and CD86100. Disregulation of adhesion molecule expression, such as ICAM-1, as well as costimulatory molecule expression such as CD40, has been reported on tumor

cells from different histologies101"103. Actually, CD40 ligation on melanoma cells by

antigen specific CTL increase the susceptibility of tumor cells to lysis104'105. As another

example, the downregulation of CD28 expression in parallel with a reduction of T cell

receptor (TCR) signal transduction may provoke T cell replicative senescence caused by

continuous antigen activation, which favor tumor progression and metastasis106. Effective levels of IFNy in the tumor site are essential in controlling tumor progression. To escape to this immunosurveillance mechanism, tumor cells may downregulate IFNy receptor

(IFNyR) expression or express factor blocking IFNy activity at downstream signaling

pathways107. hi. Secretion of suppressive factors by tumor cells or regulatory cells

The sera of cancer patients contain a variety of suppressive factors and cytokines. For instance, soluble form of adhesion molecules can be associated with disease progression

since they decrease cell mediated cytotoxicity108. Gangliosides secreted from human

melanomas impair dendritic cell differentiation and survival109. Soluble Fas derived from tumor and immune cells promotes antigen specific CD4+ T cells and CTL lysis110' m.

37 Tumor cells also induce non specific suppressive activity by producing adenosine as

result of their hypoxic metabolism. Adenosine in turn can inhibit IL-12 and promote IL-

10 production by monocytes112. IL-10 and TGF|3 are the most suppressive cytokines

secreted by tumors or immune cells. IL-10 affects varies immune functions, inhibits T

cell proliferation, Th-1 type cytokine production, lymphokine-activated killer cell

cytotoxicity and antigen presentation. Elevated levels of IL-10 concentrations in the

serum of patients with solid tumors and haematological malignancies constitute a

prognostic factor of disease progression113' 114 Similarly, TGF|3 exert potent deleterious

effects on several components of the immune system. iv. Transforming growth factor fi (TGFfi) family

The members of the Transforming Growth Factor (3 (TGF|3) family are cytokines involved in essential cellular functions such as proliferation, differentiation, apoptosis, tissue remodeling, angiogenesis, immune response, cell adhesion, and also play a key role in pathophysiology of disease states such chronic inflammatory conditions and cancer115'

116. The members of this family includes the three isoforms of TGF|3s, (31, (32, (33; bone

morphogenetic proteins (BMPs) and activins115. TGFJ3 isoforms are produced as latent

complexes. The large latent complex consists of the small latent complex (TGF-beta and its propeptide) and a high molecular weight protease resistant binding protein known as latent TGFJ3 binding protein (LTBP). LTBPs are required for secretion and proper folding of TGFJ3. The release and activation of TGFJ3 from latent complexes can be

mediated by several mechanisms such as proteases, integrins, and components of

extracellular matrix117. TGFJ3 family members signal through heteromeric complexes of type I ( TJ3RI, also known as activin receptor-like kinase, ALKl, ALK2, ALK5) and type

38 II transmembrane serine/threonine kinase receptor (TJ3RII) 118. TGFJ3 binding induces the

phosphorylation of type II receptor, which phosphorylate type I receptor 119. Once

activated, the type I receptor propagates intracellular signals by inducing the

phosphorylation of receptor-regulated (R-)Smads that include Smadl, Smad2, Smad3,

SmadS and SmadS. TGFJ3 and activins mainly induce Smad2 and Smad3

phosphorylation, whereas BMPs stimulate Smadl, SmadS and SmadS phophorylation120.

Receptor regulated Smads form a complex with the co-mediator Smad4, and translocate to the nucleus where such complexes cooperate with transcriptional co-activators and co

mpressors to regulate the expression of TGFJ3 target genes121' 122. TGFJ3 pathway is

negatively regulated by the inhibitory I-Smads (Smad6 and Smad7), which interfere with

TGFJ3 signaling by competing with receptor regulated Smads (R-)Smads for receptor interaction123. As part of TGFJ3 pathway, co-receptor such as betaglycan and endoglin

modulate TGFJ3 signaling by presenting or sequestering ligands to the type I or type II

receptors. Betaglycan and endoglin have transmembrane domains but lack of intracellular

enzymatic motifs. Betaglycan has affinity for three TGFJ3 isoforms and facilitates their

binding to the signaling receptors (TJ3RI and TJ3RII)124. On the other hand, endoglin

expression has inhibitory effects on TGFJ3/ALK5 signaling whereas enhances

TGFJ3/ALK1 and BMP signaling125' 126. In addition, to the Smad signaling, TGFJ3 and

BMP may activate or inhibit other non-Smads signaling pathways such as MAPK and

PI3K signaling pathways121. TGFJ3 is considered as a tumor suppressor factor because it

promotes cell growth inhibition, apoptosis and differentiation1 . However, an extensive

number of studies attest to the fact that TGFJ3 acts as a potent tumor promoter in

established carcinoma116'127"129. In late stage disease, cancer cells synthesize and secrete

39 high levels of active TGFJ31 protein that can be found in both tumor cells and in plasma

of cancer patients, which is associated with poor prognosis130. As tumor progresses, tumor derived TGFJ3 becomes oncogenic by constitutively inducing epithelial to

mesenchymal transition (EMT), tumor associated angiogenesis and suppressing

antitumor immunity, the sum of whose effect are to promote tumor growth and

metastasis128'13L

v. Induction of tolerance or clonal deletion of effector cells

The generation of nonresponsive tumor infiltrated lymphocytes (TIL) occurs by two main

mechanisms, the first one is the clonal deletion of tumor infiltrated T cells through the upregulation of Fas expression on their cell surface shortly after activation (previously

discussed ), and therefore becoming susceptible to tumor derived FasL mediate cell

death. For instance, apoptosis of TIL due to FasL secretion has been reported for

metastatic gastric carcinoma, breast and cervical cancer and melanoma132"137. The second

mechanism of T-cell unresponsiveness is carried out without destruction of tumor

specific T cells, but instead there is an induction of anergy of antigen specific T cells. In this case, tumor cells express peptide sequences that act as agonist for tumor antigens.

For instance, melanoma cells can secrete self-proteins act as agonist for the melanoma

antigen MART-1 /Mel an-A and anergise anti-tumor T cells by crosspresentation138.

vi. Induction or recruitment of suppressor cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that

consists of myeloid progenitors, immature macrophages, immature granulocytes and immature dendritic cells. In the pathology of cancer, MDSCs not only suppress T cell

responses, but also they exert potent suppressive effects on adaptive and innate immune

responses by modulating cytokine production of macrophages139. Non-immunological

40 functions of MDSC have also been described such as the promotion of tumour derived

angiogenesis, tumour-cell invasion and metastasis140. MDSCs may induce other

suppressor cells. MDSCs promote the de novo development of FOXP3+ regulatory T

(TReg) cells in vivo. This property requires the activation of tumour-specific T cells141.

Regulatory T cells are characterized by different T-cell subpopulations with regulatory

properties including naturally occurring CD4+CD25hlgh Treg cells, induced Treg cells, eg

Trl and TH3 cells, as well as CD4+CD25hlgh Treg cells generated in the periphery by

conversion of CD4CD25" T cells. These different Treg cell populations with regulatory function coexist and contribute to immune suppression142"144. The distinctive feature of these cell types is the expression of the transcription factor FOXP3. They have the ability to actively inhibit CDS T cells, dendritic cells (DCs), natural killer (NK) cells, natural

killer T (NKT) cells, and B cells in a cell-to-cell contact and dose-dependent manner145"

151. The suppressive effect of naturally occurring Treg cells against tumor-specific CDS

T cells was observed in mouse model of B16 melanoma and in a transgenic murine colon

carcinoma model. Treg cells efficiently suppressed CTL mediated immunity against tumor challenge, which indicates that precursor Treg cells in naive hosts gave rise to

effective suppressors during tumor development. Selective accumulation of Treg cells in the tumor site was observed in a murine fibrosarcoma model where the majority of tumor-infiltrating lymphocytes at late stage of tumor progression were Treg cells152'153.

vii. Contribution of the immune system as promoter of tumor aggressiveness and progression

Phagocytic cells such as macrophages are among the first cells recruited to the tumor

site. For example, the amount of macrophages can increase 20-fold in invasive

malignant tissues compared to benign proliferative lesions of breast cancer154. The

41 presence of high numbers of M2 macrophages is associated with poor prognosis. They

modulate the tumor microenvironment, notably by producing survival and growth factors

such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), which promotes an aggressive phenotype. Several chemokines and cytokines can be

released by tumor cells that favor the influx and differentiation of macrophages, including GM-CSF, TGF-pi and CCL3155-157 Accumulating evidence suggests that

neutrophils may also contribute to increase tumor aggressiveness. Neutrophils are

attracted into lesions by tumor-derived cytokines and once in the tumor site, neutrophils

produce suppressive factors that favor invasion and metastasis158"160.

1.2.5 Immunoediting

The cancer immunoediting process comprises of three phases: elimination, equilibrium,

and escape, also known as the "three Es of cancer immunoediting" 161' 162. The

elimination phase is based on the original concept of cancer immunosurveillance, whereby cancer cells are successfully recognized and eliminated by the immune system.

Several studies have reported the involvement of cells of the innate and adaptive immune

system in this phase161"163. Tumor cells that survive the immune system attack, proceed into a phase of equilibrium, characterized by a tumor latency, in which the immune

system anti-tumor response although control tumor development, is not sufficiently

effective to completely destroy cancer. The constant interaction between the immune

system and the tumors over time "edits or sculpts" the phenotype of developing tumors,

resulting in the selection of less immunogenic tumor variant, then the immune attack thus progress into the third phase of the immunoediting process, termed "escape."161"164.

As evidence of the equilibrium phase, there are reported cases of unintentional

42 transplantation of cancer from organ donors, who were in clinical remission or from

donors with no clinical history of malignancy. In those cases, tumor progression was

restrained by anti-tumor immune response, but once latent malignant cells are transplanted to immunosuppressed donors, the immune system pressure disappears giving rise to tumor development165'166.

1.3 CANCER INVASION AND METASTASIS

In the early phases of multi-step tumor progression, cancer cells multiply close to the site where their ancestors began uncontrolled cell proliferation. The intial step of tumor inavasion is characterized by breaching the basement membrane, which allows the tumor

cells to intravasate into either lymphatic or blood microvessels. Once in the blood

circulation, tumor cells can travel to distant anatomical sites, where eventually they get trapped and subsequently extravasate to form micrometastases. Micrometastases can form macrometastases if they colonize the tissues where they have nested. This complex

sequence of steps is known as "the invasion-metastasis cascade"167. Carcinomas begin their development on the epithelial side of the basement membrane, where they growth

and promote angiogenesis even in the stroma side of the basement membrane before

breaching the basement membrane, probably by releasing angiogenic factors through this

barrier to endothelial cells within the stroma. The breakdown of the basement membrane

enhances their aggressiveness due to the direct access to nutrient and oxygen carried by the blood vessels found only in the stromal side of the basement membrane168. The invasion of cancer cells into the blood vessels is termed intravasation. Once lodged in the capillary vessels of tissues, malignant cells must escape from the lumina of the blood vessels and penetrated in the surrounded tissues, process known as extravasation. Tumor

cells use several strategies to extravasate. For instance, the establishment of cancer cells

43 and formation of small tumors within the lumen of the capillary breaks through the

capillary basement membrane and favors the invasion to the surrounding tissues169. Once

cancer cells are settled on the parenchyma of a new tissue, they proliferate to form a tumor mass, such process is termed colonization. The acquisition of local invasiveness implicates phenotypic changes of malignant cells within the primary tumor. In case of

carcinomas, these phenotypic changes toward an invasive and metastatic phenotype are

known as "epithelial to mesenchymal transition (EMT)".

1.3.1 The epithelial to mesenchymal transition (EMT)

Epithelial cells, form organized layers of cells that are adjoined by membranous

structures such as adherens junctions. In addition, normal epithelial cells display apical

and basolateral polarization, which is a cellular architecture that guarantees tissue-

specific vectorial functions. During the development of cancer, EMT begins with loss of

apico-basal polarity as tight junctions dissolve. As part of the process, other cell junctions

such as adherens and gap junctions disassemble and the underlying basement membrane is degraded 170'171. The molecules that mediate epithelial connections to neighboring cells and the basement membrane such as E-cadherin and integrins are replaced by N-cadherin and integrins, which are more transient adhesive molecules. Moreover, epithelial cells

undergo cytoskeleton changes, mainly characterized by the replacement of peripheral

actin by stress fibres, whereas cytokeratin intermediate filaments are replaced by vimentin. Consequently epithelial cells acquire a spindle shape and very motile

capacity172'173. During this process epithelial cells become resistant to anoikis, a form of

apoptosis that is triggered by detachment of cells from extracellular matrix174. Once

reached their destination, mesenchymal cancer cells undergo the reverse process of

mesenchymal-epithelial transition174. In conclusion, EMT provides the mechanism for

44 cancer cells to invade the local tissue and blood vessel setting the basis for the metastatic

spread. In vivo, EMT induction is regulated by complex interplay of extracellular signals, which include growth factors, cytokines and extracellular matrix molecules. Indeed,

clinical evidence indicates that regulators of EMT in cancer cells correlate with cancer

aggressiveness and poor patient outcomes175' 176. Growth factors commonly associated with EMT include transforming growth factor (3 (TGF-(3)177, fibroblast growth factor

(FGF), HGF, EGF and Wnt family members178. In particular, TGFJ3 is considered the

classical inducer of EMT, which induces all the phenotypic changes that characterize the

process, such as loss of apico-basal polarity, downregulation of cell-cell adhesion,

expression of mesenchymal cytoskeletal proteins and extracellular proteases that favor

cell invasion and migration172'179. Previous studies about the mechanism of disassembly

of tight junctions stimulated by TGFJ3, indicate that TGFJ3 is an essential direct

deregulator of Par6, a key component of epithelial polarity complex regulating the

assembly of tight junctions180. Upon TGFJ3 binding to TGFJ3 receptor, promotes the

activation of type II TGFJ3 associated receptor kinase, which is in turns phosphorylates

Par6, which is associated with occluding tight junctions. This protein-protein interaction is independent of Smad proteins. Phosphorylated Par6 can recruit Smurfl, which

mediates the ubiquitination and degradation of RhoA, small GTPase family member

responsible for stress fiber formation and the maintenance of apico-basal polarity and junctional stability181,182 Rho activation also promotes the formation of focal adhesions that mediate communication of fibroblastoid cells with extracellular matrix. Therefore,

Rho family GTPases and their regulatory molecules play essential roles in epithelial

plasticity and important effectors of EMT induced by TGF (3(401). Remodeling of cell

45 contact with basal lamina requires activation of proteolytic enzymes, such as MMP2 and

MMP9, which can be activated in response to TGFJ3 to allow the degradation of collagen type IV component of basement membrane183. A number of in vivo studies demonstrate that the blockade of TGFJ3 or TGF|3 receptor reduces the metastatic potential of a variety

of experimental cancers presumably by interfering with the activation of EMT pathway

duringi • cancer progression• 184-186

1.3.2 Tumor associated angiogenesis

The vasculogenesis is the de novo development of blood vessels from endothelial cell 1 C7 1 CO precursors called angioblasts, which differentiate into a primitive capillary network '

The primitive capillary network is remodeled by a process known as angiogenesis, which is characterized by the formation of new blood vessel sprout from the pre-existing

capillaries189. The process of angiogenesis comprises two phases. The first phase is the

activation or initiation, characterized by destabilization of the blood vessels and increased

permeability and degradation of the extracellular matrix favoring endothelial cell

proliferation and migration. The second phase, the resolution phase is when endothelial

cells stop proliferating and pericytes and vascular smooth muscle cells are recruited to guarantee stabilization, remodeling and maturation of the new blood vessels190. To grow

beyond 1-2 cubic millimeters, tumors induce angiogenesis in order to get a supply of

nutrients and oxygen191. This process is known as an angiogenic switch and is regulated

by a variety of pro and anti-angiogenic factors192. In particular, members of TGF|3 family

are essential regulators of tumor angiogenesis. TGF|3 family members and their receptors

are expressed by a variety of cell types including endothelial cells, mural cells (pericytes

and vascular smooth muscle cells), tumor cells and immune cells upon activation117. In

46 most of the cell types, ALK5 is the predominant type I receptor mediating TGF|3

response. However, in endothelial cells, TGF|3 signal through two distinct type I

receptors, the ALK5 and ALKl dependent pathways, which results in the activation of

Smad2/3 and Smadl/5/8 respectively193. Previous studies indicate that ALKl regulates the expression of genes implicated in endothelial cell proliferation and migration, whereas ALK5 regulates the expression of genes involved in cell adhesion, cell-cell interaction and extracellular matrix remodeling193"195. Interestingly, ALKl/Smadl

signaling has been shown to act as an antagonist of ALK5/Smad2/3 and the latter is

required for optimal ALKl activation196. In addition to its essential role on endothelial

cells proliferation and migration, TGF|3 affects angiogenesis by acting on other cell

components of blood vessels such as smooth muscle cells, in which TGF|3 via ALK5

pathway promotes vascular smooth muscle differentiation by inducing expression of

smooth muscle alpha-actin and myosin197' 198. Several genetic studies emphasize the important role of TGF|3 family members in angiogenesis and vasculogenesis. In

particular, TGFJ31 deficient mice display fragile vessels and die due to defective yolk sac vasculogenesis199' 200. Similarly, knockout mice for TJ3RII, T|3RI and ALKl die at

midgestation due to severe defects in angiogenesis and vasculogenesis201"203. Vascular

disorders described in patients such as hereditary hemorrhagic telangiectasia have been linked to mutations in ALKl and endoglin genes204' 205. TGF|3 expression is upregulated in many types of carcinomas and high levels of this cytokine in the serum of cancer

patients correlate with poor prognosis206"208. The role of TGF|3 as inducer of tumor

angiogenesis has been attested in several tumor models. For instances, cancer cells

overexpressing TGFJ31 display increased tumor angiogenesis in tumor in vivo 209.

47 Consequently, treatment with TGF|3 antagonists such as neutralizing antibodies to

TGFJ31 or ectopic expression of soluble TGF|3 receptor III (TJ3RIII) reduced angiogenesis

and growth of tumors formed by human colon carcinoma and breast carcinoma cell lines

(HCT116 and MDA-MB-435 respectively)210' 211. The mechanism by which TGF|3 induces tumor angiogenesis is a product of both direct and indirect effects. Directly,

TGF|3 activates endothelial cell proliferation and migration, promotes in vitro capillary formation of endothelial cells when they are cultured in collagen matrix as well as

angiogenesis in vivo in the chicken chorioallantoic membrane assay212. Indirectly, TGF|3

can induce the expression of vascular endothelial growth factor (VEGF) in a variety of

cell types in the tumor site such as tumor cells, stromal fibroblasts and recruited

macrophages213. Moreover, TGF|3 induces the expression of extracellular matrix

components that play crucial roles in both the initiation and resolution phase of

angioegenesis, such as MMPs, collagen, plasminogen and integrins214'215. Based on the

dynamic roles of TGF|3 in tumor progression and angiogenesis, the use of

TGF|3 antagonists as part of a treatment for carcinomas may significantly contribute to tumor regression and clearance.

1.4 IMMUNOTHERAPY

Immunotherapy is a therapeutic modality that utilizes the patient's own immune system to recognize and eliminate malignant cells. The main goal of immunotherapy is to induce

an effective immune response to tumors in patients 216. Several strategies to overcome the immune escape mechanisms of tumor cells have been considered. In particular, the identification of tumor-associated antigens (TAA) has prompted the development of

different strategies for antitumor vaccination. The specific recognition of TAA may elicit

48 a persistent immune memory that eliminate residual tumor cells and protect recipients from relapses.

1.4.1 Cell based therapy

i. Genetically modified tumor cell vaccines

A cancer vaccine made of tumor cells represents the entire antigenic repertoire therefore

constitute a native source of TAA. The anti-tumor potential of tumor cell vaccines has

been tested in the clinic with very promising outcomes. One of the classic examples is the

allogeneic melanoma whole cell vaccine composed of three melanoma cell lines

established in vitro, which significant improved the five years survival rate in patients

suffering of stage IV melanoma217. Antigens belonging to allogeneic cells are processed

and presented by host antigen-presenting cells in the context of MHC class I and are

recognized by host T-lymphocytes218 (a phenomenon called cross-priming). In the

autologous setting, irradiated melanoma and modified with dinitrophenyl were used to treat phase III patients who remain tumor-free after resection of lymph node metastases

and as results, 50-60% of them remained 4 years relapse free and prolonged survival219.

Allovaccines have several advantages over autologous vaccines, for instance, cell lines that are well characterized and can be easily expended to treat higher number of patients included in a clinical study. In addition, cell lines can be genetically modified to increase their immunogenicity and there is no need to isolate cells from every tumor. On the other hand, the rate of success in isolating and culturing tumor cells from a primary tumor varies according to the type of tumor and experience of the operator. In contrast, cell lines can be transduced and monoclonal population can be selected for the production of

a certain amount of protein, which assures homogeneity of the vaccine preparations220.

For instances, genetically modified tumor cells to express both GM-CSF and CD40L

49 have been shown to induce substantial infiltration of dendritic cells and enhance the

r\r\ -I r)r)r) immune anti-tumor response initiated by these cells ' . The way cellular antigens are

released and captured by DC for T cell priming depends on how tumor cells die after injection. Irradiation of the cell vaccine induces apoptotic bodies that can be captured by

DC. However, the generation of tumor antigens by a non-apoptotic pathaway was

associated with higher immunogenicity and induction of heat shock protein (hsp) expression223. Antigen uptake is followed by dendritic cell maturation and migration to the lymph node, where ensure correct antigen presentation 224

II. Dendritic cell based therapy

The professional antigen presenting cells, dendritic cells (DCs) present antigens to CDS

T cells either through endogenous processing of intracellular protein or by uptaking and

processing exogenous antigens, a process known as crosspresentation. In the context of

cancer, the process of crosspresentation is the most important natural mode of

presentation of soluble proteins or particulate matter from apoptotic or necrotic cancer

cells225' 226. DC can internalize complete tumor lysates or apoptotic cells and present

derived antigen in an HLA I-restricted manner227. Both Langerhans cells and interstitial

DCs crosspresent melanoma-associated antigens to CTL and activate them in vitro228.

Vaccination of DCs loaded with TAA has shown positive outcomes in the clinic. For instance, complete and partial responses as indicative of tumor-specific immunity has been observed in patients with metastatic melanoma vaccinated with antigen-specific

pulsed dendritic cells 229. The engineering of DC with expression vectors encoding for

TAA genes allow constitutive expression of TAA peptides, which induce a long-term

antigen presentation in vivo and generation of specific CTLs against TAA epitopes230.

The generation of monocyte-derived DC from acute myelogenous leukemia cells (AML)

50 or chronic myelogenous leukemia (CML) have shown to stimulate autologous anti- leukemia T cell responses, which constitute a promising strategy for the treatment of these malignancies 231"233. hi. B cell based therapy

B cells have the capacity to present antigens to either CD4+ or CDS T cells, such

antigens can be internalized by binding to their cell surface immunoglobulins or by endocytosis in an immunoglobulin independent manner234. In particular, CD40-activated

B cells are comparable to DC in their antigen presentation capacity235. In addition, B cells

display several advantages over DC. For instance, B cells are more abundant in the

peripheral blood than DC (5-10% versus 0.5-1% of peripheral white blood cells)236 and expandable in vitro from small blood volumes, which has great value for pediatric immunotherapy ' . In addition, activated B cells upregulate and maintain high co-

stimulatory molecule expression and cytokine production such IL-2, IFNy and TNFa

required for an appropriated T cell activation and differentiation into Th type I

phenotype239. Activated B cells have been shown to prime naive and expand memory

CDS T cells in cancer patients240. Lapointe et al. demonstrate that activated B cells can

effectively generate tumor-antigen specific CD4+ T cells in cancer patients when B cells

are pulsed with tumor cell lysates241. As other example, the induction of myeloid leukemia-specific CD4+ and CD8+ T cells has also been reported for CD40-activated B

cells, which have been genetically modified to express tumor antigens242.

1.4.2 Cytokine and costimulatory molecule based therapy

The role of cytokines implicated in the activation of anti-tumor responses has been

previously shown in experimental models. For instance, the number of APC in the site of tumor infiltration can be increased by the chemoattractant activity of cytokines such as

51 GM-CSF (granulocyte-macrophage colony-stimulating factor) and IL-4 (interleukin-4), which promote the differentiation of DC precursors. IL-12 and IL-2 in turn increase B-

and T-cell responses and IL-2, IL-12, IFN-y (interferon y) and TNF-a (tumor necrosis factor a) increase T-cell cytotoxicity, while NK cytotoxicity is enhanced by IL-12 or

FLT 3-L 243 244 Based on clinical results obtained in patients with advanced melanoma or

renal cell carcinoma indicate that GM-CSF and IL-12 secreted by tumor cell based vaccines promote anti-tumor effects by inducing marked infiltration of dendritic cells,

CD4+ and CDS T-lymphocytes into tumor lesions. Costimulatory molecules such as

CD40:CD40L are essential for the activation of T, B and NK cell responses. Previous

studies have shown that activation of CD40 promotes activation of host APC and

converts tolerogenic T cells into cytotoxic T cells, which may induce the regression of

established tumors245' 246. On the other hand, the blockade of inhibitory signals such as

CTLA-4, which physiologically blocks T cell activation, has been shown to retard tumor growth in experimental models247.

i. IL-2 based therapy

IL-2 was the first cytokine formally approved by the U.S. Food and Drug Administration for the therapy of cancer in May 1992248. The antitumor effect of IL-2 results from its

ability to expand lymphocyte population in vivo and enhances the effector function of these cells249. IL-2 has shown promising outcomes in the treatment of renal carcinoma

and melanoma250. Prevention of growth of established tumor has been observed with local infusions of IL-2251. Intratumorally infusion of IL-2 is more effective strategy than

systemic application. The latter has been characterized by the occurrence of serious

adverse reactions including damage of the blood vessels of the body (capillary leak

syndrome)252'253.

52 II. Granulocyte macrophage colony stimulating factor based therapy

The secretion of GM-CSF by irradiate tumor cells as part of a tumor cell based vaccine

approach has shown to prevent the growth of tumor challenge with non-irradiated wild type cells. GM-CSF secreted by irradiated tumor cells promote a substantial recruitment

of dendritic cells, macrophages and granulocytes to the tumor site254. Specifically, injection sites of metastatic melanoma patients vaccinated with irradiated, autologous tumor cells genetically modified to secrete GM-CSF induce dense infiltrates of dendritic

cells expressing high levels of co-stimulatory molecules. These results demonstrate the important ability of GM-CSF to enhance dendritic cell functions in vivo, which has

relevant implications for an effective anti-tumor response255. Autologous and allogeneic

GM-CSF producing tumor cell vaccines have been used in the clinic with very promising

outcomes. For instances, renal carcinoma patients treated with autologous GM-CSF

producing cells display a clinical response characterized by regression of pulmonary

metastases. However, the percent of complete responses was very low mainly due to limitations to obtain the desired number of viable cells for gene transfer256. Similarly, tumor regression and prolonged survival were observed in phase I/II clinical trials with

patients suffering of Non-Small-Cell Lung (NSCL) carcinoma and metastatic melanoma.

In all these cases the duration of survival and tumor regresions were GM-CSF dose-

dependent257' 258. Despite its potent chemoattractant capacity for antigen presenting cells,

GM-CSF has shown to suppress certain immune functions such as downregulate the

activity of NK cells259.

1.4.3 Peptide vaccines

The aim of clinical protocols based on peptide antigens for active vaccination is to induce

strong CTL response against the tumor expressing such peptide antigens. CTLs mainly

53 recognize peptides of 8 to 10 amino acids derived from intracellular or endogenous

proteins that form complexes with MHC class I molecules260' 261, whereas CD4+ T lymphocytes recognize peptides of 12-24 amino acids presented by APC in the context of

MHC class II molecules262.

Peptide vaccines have several advantages over other vaccine approaches: 1) specificity of the immune response against few unique antigens thus limiting the potential risk of

autoimmune cross-reactivity or immunosuppressive activity often observed with more

complex immunogens, 2) existence of new technology that allow to sequence and prepare larger quantities of tumor antigen peptides for both laboratory and clinic use, 3) use of

synthetic peptides reduces the risk of bacterial or viral contamination that might be

present in autologous or allogeneic tissues. However, the disadvantages of peptide immunization are: 1) lack of universal applicability since each peptide is restricted to a

single HLA molecule; 2) poor immunogenicity of most native peptides and therefore the

need of adjuvants; and consequently 3) the risk of inducing antigenic tolerance. As exemples of tumor specific peptides that have been used in clinical trials P210-derived

peptides from BCR-ABL oncogene were able to induce peptide-specific T cell immunity in both normal donor and CML patients263' 264 Other report show tumor regressions in

patients with metastatic melanoma treated with an antigenic MAGE-3-derived peptide265.

Although strong peptide-specific CTL and CD4+ T cell responses have been seen in vitro for many tumor peptides, few promising reports on T-cell induced immunity generated

after peptide vaccination in patients have been observed266'267.

1.4.4 Recombinant virus based vaccines

Viruses constitute interesting vehicles for the delivery of therapeutic agents because they

can induce antibody, T helper and CTL responses without co-stimulation268. However,

54 the utilization of viral vectors is restricted due to possible recombination with wild type virus, oncogenic potential, or virus induced immunosuppression. Vaccinia virus (VV) is

one of the viral vector more commonly used due to the large amounts of DNA that can be inserted its genome269. The administration of VV encoding human melanoma antigen gplOO in transgenic mice for the human HLA-A* 0201 allele was effective in inducing

specific CDS T cell for the epitope 270. Adenoviruses have been extensively used as vector, in which critical genes that required for viral replication have been deleted and

replaced by genes encoding tumor associated antigens271. The use of recombinant virus in

the clinic has encountered some intrinsic limitations such as the presence of high

neutralizing antibody titers in the sera of patients previously vaccinated for smallpox

prevention, and against Adenoviruses, which may neutralize the recombinant virus and impair the ability of these viruses to immunize against the tumor antigens272.

1.4.5 Fusion protein and antibody based therapy

Potent proinflammatory cytokines and chemokines have been combined with antibodies

specific for tumor-antigens or tumor-derived proteins to generate fusion proteins, which localize the cytokine activity in the tumor site and guarantee effective anti-tumor immune

response. As examples, the fusion of cytokines, chemokines or co-stimulatory molecules with monoclonal antibody targeting necrotic regions of the tumor (TNT therapy). These

antibodies were generated using stable intracellular antigens, which are present in all cell types but are released as result of necrosis. Since 30-80% of the tumor mass is necrotic or degenerative region, this strategy guarantees the delivery of therapeutic agents to the core of tumor, a site abundant in tumor antigens273. Fusion proteins not only may recapitulate

synergistic effects as result of the combination of two bioactive proteins, but also may

possess unheralded biopharmaceutical properties not seen in the combination of both

55 components of the fusion. For instance the fusion of GM-CSF and IL-2 for the therapy of

melanoma not only display the characteristic anti-cancer effects of both GM-CSF and IL-

2, but also is endowed of immunological properties distinct of GM-CSF and IL-2 used

alone or in combination2' 274. Other strategies include the use of antibodies to deplete

suppressive immune cells such as regulatory T or suppressive cytokines. As examples, the IL-2 receptor therapy include monoclonal antibodies specific for IL-2 receptor alpha

chain such as Basiliximab, a chimeric antibody approved by the US Food and Drug

Administration (FDA); and Daclizumab, the first humanized antibody also approved by the FDA275' 276. In particular, Daclizumab has shown to be a promising therapy for the treatment of patients with adult T cell leukemia (ATL), who are profoundly immunosuppressed due to their leukemia cells behave as regulatory T cells that express 077 07C CTLA4 and therefore suppress the immune functions of normal lymphocytes '

1.4.6 DNA vaccines

The potency of DNA immunization is significantly inferior to that one of recombinant viruses, probably because DNA does not undergo autoreplication in the transfected cells, which limits the amount of tumor associated antigen produced and therefore inflammatory reactions are scarce. Consequently, multiples DNA inoculation and the use

of adjuvants are required to induce optimal response. DNA vaccines has been used for the treatment of established pulmonary metastases, but recombinant cytokines were

required to enhance the therapeutic effects279. Although induction of tumor antigen

specific immunity has been observed in vivo by using DNA immunization280"282. The

main limitation of this strategy is the induction of central and peripheral tolerance to self

antigens, which may restrain the use of DNA vaccines in the therapy of cancer283.

56 1.5 SPECIFIC RESEARCH AIMS

The main objective of the research projects presented in this thesis was to develop new biopharmaceuticals for the immunotherapy of cancer. The specific research aims were:

1. Evaluated the ability of the murine GIFT2 fusion protein in vivo to induce an

effective anti-tumor response against null or non-modified B16 cells present in

the tumor site (bystander effect), as well as determine the factors implicated in

GIFT2-mediated anti-cancer activity.

2. Generate and characterize the human ortholog of GIFT2 and evaluate its use as a

means to generate oncolytic NK cells that may serve as an effective cellular

platform for cancer cell therapy.

3. To test the hypothesis that the blockade of TGF|3 signalling combined with

cytokine-driven immune activation will couple antiangiogenesis to an effective

immune antitumor response, resulting in potent anticancer properties. To address

this hypothesis, we generated and characterized a new TGF|3 antagonist and

immunostimulator (aka FIST) for the therapy of cancer.

4. Evaluate the APC properties of FIST-stimulated B cells, as well as their ability to

induce protective antitumor immunity.

57 CHAPTER 2

TGFp secreted by B16 melanoma antagonizes cancer gene immunotherapy bystander effect.

Reference: Claudia Penafuerte and Jacques Galipeau. TGFp secreted by B16 melanoma antagonizes cancer gene immunotherapy bystander effect. Cancer Immunol Immunother.

2008 Aug; 57 1197-206.

58 CHAPTER 2: TGFp secreted by B16 melanoma antagonizes cancer gene

immunotherapy bystander effect.

2.1 ABSTRACT

Tumor-targeted delivery of immune stimulatory genes, such as pro-inflammatory

cytokines and suicide genes, has shown to cure mouse models of cancer. Total tumor

eradication was also found to occur despite subtotal tumor engineering; a phenomenon

coined the "bystander effect". The bystander effect in immune competent animals arises

mostly from recruitment of a cancer lytic cell-mediated immune response to local and

distant tumor cells which escaped gene modification. We have previously described a

Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin 2 (IL2) fusokine (aka GIFT2) which serves as a potent anticancer cytokine and it here served as a means to understand the mechanistic underpinnings to the immune bystander effect in an immune competent model of B16 melanoma. As expected, we observed that GIFT2

secreted by genetically engineered B16 tumor cells induces a bystander effect on non

modified B16 cells, when admixed in a 1:1 ratio. However, despite keeping the 1:1 ratio

constant, the immune bystander effect was completely lost as the total B16 cell number was increased from 104 to 106 which correlated with a sharp reduction in the number of tumor-infiltrating NK cells. We found that B16 secrete biologically active TGF|3 which in turn inhibited GIFT2 dependent immune cell proliferation in vitro and downregulated

IL-2RJ3 expression and IFNy secretion by NK cells. In vivo blockade of B16 originating

TGF|3 significantly improved the immune bystander effect arising from GIFT2. We

propose that cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of tumor-derived TGF|3.

59 2.2 INTRODUCTION

Many immunogene therapy strategies have been developed for the treatment of malignant

melanoma. These approaches include the introduction of "suicide genes", the expression

of tumor suppressor genes by tumor cells or the inactivation of oncogene expression, as well as the introduction of genes encoding pro-inflammatory proteins such as co-

stimulatory molecules and cytokines. In particular, the genetic modification of melanoma

cells to secrete pro-inflammatory cytokines enhances the immunogenicity of these cells by providing signals required to trigger an effective cell mediate immune response284.

Since it is not possible to modify all pre-existing tumor cells with suicide or

proinflammatory genes in situ by any contemporary gene transfer technology, an important feature to consider for cancer gene immunotherapy is the bystander effect285.

Whereas a small fraction of tumor is gene modified to initiate an immune response in vivo against a vastly bulkier pre-established native cancer. Indeed, a cytokine-secreting live cell cancer vaccine approach is also wholly dependent upon a robust immune

"bystander" effect for clinical effectiveness286. A vast array of pro-inflammatory

cytokines and derivatives has been studied as a means to initiate an anticancer immune

response in animal models of melanoma and in clinical trials. However, despite tightly

controlled conditions, we - as others 287- have observed treatment failures in mouse

models of melanoma where the immune "bystander" effect was lost as the experimental tumor burden was increased at onset of treatment. As a possible explanation for this

observation, we may invoke the balance between inhibitory and stimulatory signals

essential in the maintenance of homeostasis and in the regulation of the immune response

as possibly antagonistic to the immune bystander effect. During cancer progression this

60 balance is disrupted and inhibitory signals may prevail, leading to an immunosuppression in the tumor-microenvironment and resulting in tumor growth. Various mechanisms have been proposed for tumor cell evasion from physiological immunosurveillance and these include the dysregulation of MHC class I and tumor antigen expression as well as

adhesion/accessory molecules expression, induction of anergy or clonal deletion of

effector cells, and the secretion of suppressive soluble factors.

Previous studies from our laboratory have shown that the fusion protein between GM-

CSF and IL-2 - aka GIFT2 - has novel immunological properties compared to both

cytokines in combination, such as greater melanoma site recruitment of macrophages and functional NK cells, and circumvents the limitations of each individual cytokine2. With the use of GIFT2 fusokine as means to initiate an anticancer immune response, we here

analyzed the immune bystander effect of GIFT2-secreting melanoma cells on non-

modified B16 present in the tumor site in vivo. We observed that the bystander effect is lost as tumor burden increases and that B16-derived TGF|3 was responsible in good part

of this acquired refractoriness by its direct effect on innate effector cells despite local

production of a potent pro-inflammatory fusokine. These data strongly support the need to target tumor-derived suppressor cytokines - such as TGF|3 - for an optimal immune bystander response to a cancer immunogene platform.

2.3 MATERIALS AND METHODS

2.3.1 Animals, cell lines, and reagents

All experimental mice were females 6 to 8 weeks old (Jackson Laboratory, Bar Harbor,

ME). The C57Bl/6-derived B16F0 (B16) mouse melanoma cells (American Type Culture

61 Coleccion [ATCC], Manassas, VA) as well as a polyclonal population of B16 derivative

(B16GIFT2 (23)) were maintained in Dulbecco's modified Eagle's medium (Wisent

Technologies, Rocklin, CA), supplemented with 10% fetal bovine serum (Wisent

Technologies) and 50 U/ml Pen/Strep (Wisent Technologies). The cell lines CTLL-2 and

mouse embryonic fibroblasts (MEF) (American Type Culture Collection [ATCC],

Manassas, VA) were grown according to ATCC's recommendations. Recombinant

mouse TGF|3 and IL-2, as well as TGF|3 neutralizing antibody (anti-TGF|31, 2, 3 isoforms) and soluble TGF|3 receptor II (TJ3RII) were obtained from R&D Systems,

Minneapolis, MN; antiphosphorylated SMAD2 and SMAD3 antibodies were obtained from Cell Signalling Technology, Danvers, MA; a-tubulin antibody was obtained from

Santa Cruz Biotechnology, Santa Cruz, CA. Anti-mouse FcR III/II, CD3, CDS, CD4,

CD25, NK1.1, CD80, CD86, CD 105, MHC class I, MHC class II, CD 122 (IL-2R(3 chain)

and the isotype control antibodies for flow cytometry were obtained from BD

Biosciences, San Diego, CA. The enzyme-linked immunosorbent assay (ELISA) kit for

mouse IFN-y was obtained from BD Biosciences.

2.3.2 Murine B16F0 tumor implantation in immunocompetent C57B1/6 mice and

immune infiltrate analysis.

Non-modified and genetically modified GIFT2 fusokine-secreting B16 cells (0.7 ± 0.2

pmol per 106 cells per 24 hours) were injected subcutaneously in C57B1/6 mice, and tumor growth was monitored over time. For immune infiltrate analysis, 104 or 106 genetically modified cytokine secreting and/or non modified B16 cells were mixed with

500 |iL Matrigel (BD Biosciences) at 4°C and injected subcutaneously in C57B1/6 mice.

Implants were surgically removed 6 days after transplantation and enzymatically

62 dissociated as reported previously2. After incubation with anti-FcR III/II mAb for 1 hour, infiltrated cells were incubated for 1 hour at 4°C with appropriate antibodies and

analyzed by flow cytometry using a FACS Calibur cytometer.

2.3.3 Flow cytometry analysis of non modified and genetically modified cytokine expressing B16 cells

Flow cytometry analysis was performed in phosphate-buffered saline (PBS) with 2%

FBS with the following antibodies: R-phycoerythrin PE-conjugated anti-mouse H-2 Kb

(MHC class I, clone AF6-88.5), I-Ab (MHC class II, clone AF6-120.1), CD80 (clone 16-

10A1), CD86 (clone GL1) and CD105. Isotype control analysis was performed in

parallel. Non modified and genetically modified B16 cells expressing mouse GIFT2 were incubated with appropriated antibodies for one hour at 4°C and the expression of these

cell surface markers was determined by using FACS Calibur cytometry (BD) and

analyzed using Cellquest software (BD biosciences).

2.3.4 Cytokine-dependent CTLL-2 proliferation assay

CTLL-2 cells were pre-incubated in medium conditioned by non-modified B16 cells and

pretreated with TGF|3 neutralizing antibody or isotype control, as well as in medium

conditioned by MEF. The cells were plated at 104 cells/well of a 96-well plate with increasing concentration of mouse recombinant IL-2. The cells were incubated for 48

hours and 20 pL of 5 mg/ml 3 -(4,5 -dimethylthiazol -2-yl)-2,5 -diphenyltetrazolium

bromide (MTT) solution were incorporated for the last 4 hours of incubation. The

reaction was stopped by adding 200 pL of dimethyl sulfoxide and absorbance read at 570

nm. The expression of CDS and CD 122 (IL-2R|3 chain) on CTLL-2 cells pre-incubated as

63 previously described were determined by flow cytometry analysis using FACS Calibur

Cytometer (BD).

2.3.5 Murine NK cell isolation

Mouse NK cell population was obtained by resuspending 107 splenocytes/ml in PBS

containing 0.5% bovine serum albumin and treated with biotin-antibody cocktail

containing anti-CD5 (Ly-1), CD8a (Ly-2), CD4 (L3T4), Gr-1 (Ly-6G/C), CD 19 and Ter-

119 MicroBeads (Miltenyi Biotec, Gladbach, Germany). After incubation for 15 min at

4°C, cells were washed and the cell populations were depleted by magnetic cell sorting

(MACS) system with an autoMACS™ column (Miltenyi Biotec) according to the

manufacturer's instructions. NK cell population purity assessed by flow cytometry was

90% (data not shown). NK cells were incubated in the conditioned media from non-

modified or genetically modified B16 cells expressing GIFT2 in the presence or absence

of TGF|3 neutralizing antibody for 72 hours and IL-2RJ3 (CD 122) expression was

determined by flow cytometry. After 72 hours, the supernatant was collected and IFNy

production was determined by ELISA. NK cell extracts were immunoblotted using anti-

phosphorylated SMAD2 and SMAD3 antibodies and anti a-tubulin as loading control.

2.3.6 In vivo blockade of TGFP

104 genetically modified cytokine secreting and/or non modified B16 cells were mixed with 500 |iL Matrigel (BD Biosciences) at 4°C plus 40 p,g TGFP neutralizing antibody or isotype control, and injected subcutaneously in C57B1/6 mice. Tumor volume was

monitored over time by performing external measurements in two dimensions and

calculating using the equation volume=lengthxwidth2x0.5 and statistic analysis was

64 performed. Similar experiments were carried out using 10 p,g of soluble TGF|3 receptor II

as a TGF|3 blocking agent.

2.4 RESULTS

2.4.1 MHC class I and II expression in B16 melanoma cells.

B16 melanoma derived from C57B1/6 mice 288'289 is known as "poorly immunogenic" 290 yet under certain circumstances, it is possible to upregulate MHC I and MHC II expression in these cells in vitro 291. To verify the immune phenotype of B16 and

B16GIFT2 cells here utilized as model systems, we performed flow cytometric analysis for cell surface expression of MHC I, MHC II and the co-stimulatory molecules CD80

and CD86. As shown in the Figure 1, B16 cells and their B16GIFT2 derivatives do not express detectable levels of these surface proteins, yet robustly express CD 105 (endoglin)

a co-receptor for TGF|3. This phenotype is consistent with low immunogenicity and is unaffected by expressing GIFT2 fusokine.

2.4.2 Immune bystander effect is lost with increased B16 melanoma tumor burden

To test the effect of tumor burden on immune bystander effect, we admixed B16

melanoma cells with a polyclonal population of GIFT2-secreting B16 cells (hereafter

B16GIFT2) at a constant 1:1 ratio. We measured tumor growth in mice having received

an initial tumor cell inoculum of either: lxlO4, lxlO5 or lxlO6 of each cell type. As

previously reported, Figure 2A shows that mice implanted with B16GIFT2 melanoma

cells remain tumor free long term and mice implanted with B16 cells promptly develop

palpable tumors within 20 days. The cohort of immunocompetent mice implanted with

lxlO4 B16GIFT2 cells mixed with lxlO4 wild type B16 cells displayed the highest

65 percentage of survival and cure (40% of mice), indicating that the paracrine secretion of

GIFT2 from B16GIFT2 cells induced a local bystander antitumor response against wild type B16 cells present at the tumor site. However, this bystander effect is lost in a cell

dose dependent manner as the number of total B16 cells increases (despite a constant 1:1

66 Figure 1: Immunophenotypic analysis of B16 cells and genetically modified B16-

GIFT2 cells

The expression of CD 105, CD80, CD86, MHC class I and MHC class II was evaluated on B16 cells and B16GIFT2 cells by flow cytometry.

67 CD 105 CD80 CD86 MHC II MHC I

Counts Counts Counts Counts Counts 0 40 SO 120 160200 0 40 SO 120 160 200 (i 40 SO 120 160 200 0 40 60 nl120 160 200 0 40 80 120 160 £00

Counts Counts Counts Counts Counts 3 40 80 120 160 200 0 40 60 120 160 200 )AT" 40 SO 120| 160200 0 40 80 120 160 200 0 40 80 120 160 200 -o V o

—* : 2 z sJ i > J f 8~ § v m h O at ; CD O - o 5 i r o 55 u xg % % 5> -b. O J

01 00 mix of B16 and B16GIFT2 cells), with a complete loss of the immune bystander effect with a lxlO6 cell dose. Figure 2BC details a replicate in vivo experiment where

B16:B16GIFT2 at 1:1 ratio either lxlO4 (B) or lxlO6 (C) per cell type were implanted at

day 0 and tumor growth monitored over time. Whereas mice receiving solely B16GIFT2

remained tumor-free independently of the size of the day 0 inoculum, virtually all mice

receiving lxlO6 of each B16B16GIFT2 at 1:1 grew tumors as quickly as B16 controls.

Only in the "low tumor burden" group receiving lxlO4 B16B16GIFT2 at 1:1 of each cell type had a long term 50% cure rate (p<0.05 log rank).

2.4.3 Host derived cellular immune response to B16 melanoma

We observed that the bystander effect was operative in implants of lxlO4 of each

B16B16GIFT2 at 1:1 yet lost in similar implants inoculated at a dose of lxlO6 cells as

shown in Figure 1C. We speculate that the host-derived cell mediated immune response

must be involved in this discrepancy. To analyze the differential response to low (lxlO4)

and high (lxlO6) dose implants, we performed an in vivo matrigel cell infiltrate analysis

as previously described2. In brief, melanoma cells were embedded in matrigel, injected in mice subcutaneously, surgically retrieved 6 days later and enzymatically dissociated to

produce a cellular suspension amenable to flow cytometry analysis. We analyzed the

recruitment of host-derived CD4+, CDS , NK, NKT cells, and CD4 CD25 cells to the tumor site. Immune infiltrate analysis of the matrigel plugs of lxlO4 B16 B16GIFT2 at

1:1 admixed cells per implant, exhibited a pattern of cell migration similar to that seen in implants where all B16 cells express GIFT2 (Figure. 3A). There was no significant

difference in the proportion of CD4, CDS or CD4/CD25 cells between B16GIFT2 and

B16 B16GIFT2 admixed cells at low and high tumor cell dose. The cohort of mice

69 injected with wild type B16 shows overall a poor recruitment of immune effector cells to the tumor site.

70 Figure 2: In vivo immune bystander effect of GIFT2-secreting cells

Kaplan-Meier survival curve of: (A) Cohort of 10 C57B1/6 mice per each experimental group were injected subcutaneously with (filled circle) lxlO5 B16GIFT2 cells (positive

control), (filled triangle) lxlO5 B16 cells (negative control), or a mixture of both at 1:1

ratio varying the cell number: (filled squared) lxlO4, (open circle) lxlO5 and (open cross)

lxlO6 of each cell type. (B) Cohorts of 10 mice per each experimental group were injected subcutaneously with (filled circle) lx 104 B16GIFT2 cells, (filled triangle) lxlO4

B16 cells and (filled squared) lxlO4 admixed cells at 1:1 ratio (2xl04 total cell number).

(C) A similar experiment was performed using a cohort of 10 mice per group injected with (lilies squared) lxlO6 admixed cells at 1:1 ratio (2xl06 total cell number), (filled

circle) B16GIFT2 cells and (filled triangle) B16 cells. These experiments were repeated three times with similar results and statistic analysis indicated significant differences between the test groups (p<0.05 log rank).

71 Figure 2

A 100 • 90 80 70 60 50 40 30 20 ^6< 10 C>O<>CHXHOCK>OO 0 kA MX MX IfM *~X 0 10 20 30 40 50 60 > B 100 90 3 80 (Z2 70 4- O 60 50 | 40

Days post tumor implantation

72 However, the cohort of mice implanted with lxlO6 B16:B16GIFT2 at 1:1 admixed cells

revealed a significantly reduced NK and MKT cell recruitment to the tumor site (Figure

3B), suggesting a selective suppression of innate cellular effectors as B16 tumor burden increases and immune bystander effect is lost. We also performed a cell infiltrated

analysis in implants containing lxlO5 of each B16:B16GIFT2 and we observed a pattern

of cell migration similar to that seen in lxlO6 cell dose, although no statistically

significant difference was observed (P=0.08 log rank, Figure 3).

2.4.4 B16 tumor cells secrete biologically active TGFJ3

The loss of the immune bystander effect with tumor cell dose is associated with a

decrease of host-derived NK and MKT cells infiltration at tumor site. This observation

suggests that a secreted inhibitory factor is released by B16 cells (and their GIFT2

derivatives) which acts as a dominant negative modulator of the immune bystander effect

driven by the GIFT2 fusokine. B16 have been previously shown to release latent TGF|3 which would be a likely candidate suppressor of immunogene-driven bystander effect 292.

We here determined whether B16 cells produced functionally active TGF|3. An IL-2

dependent cell line (CTLL-2) was used to analyze this immunosuppressive property and was used as a bioassay for active TGF|3. CTLL-2 cells cultured in medium conditioned

by B16 cells and increasing doses of recombinant IL-2 showed a significant reduced

proliferation in MTT assay compared to the control (medium conditioned by mouse

embryonic fibroblasts). The addition of TGF|3 neutralizing antibody rescued the ability of

these cells to proliferate in response to IL-2 (Figure 4A). Active TGF|3 induces de novo

expression of CDS on CTLL-2 cells and on normal immature thymocytes 293. Based on

73 this property, we observed the expression of CDS on CTLL-2 cultured in medium

conditioned by B16 cells. Similarly, recombinant TGF|3 (1 ng/ml) induced de novo

74 Figure 3: Immune infiltrated analysis of tumor implants

Immunocompetent C57B1/6 mice were injected subcutaneously with (filled bars) B16

cells, (open bars) B16GIFT2 cells, (dotted bar) admixed cells and (chequered bar) mouse

embryonic fibroblasts (MEF) embedded in matrigel. Implants were retrieved 6 days post implantation and digested with collagenase to collect immune infiltrated cells, which were analyzed by flow cytometry. (A) Cohorts of 6 mice per each experimental group were implanted with lxlO4 cells. (B) Cohorts of 6 mice per each experimental group were implanted with lxlO6 cells. Significant differences were observed of NK cells tumor recruitment between implants contained lxlO6 admixed cells and lxlO6 B16GIFT2

cells (p<0.05). These experiments were performed in triplicate with similar results.

75 Figure 3

B 0.8 0.8 , Low burden High burden 0.7 0.7 - to ° 0.6 O 0.6 - X I J/> 8 05 0O 0 5 - o O o 0.4 O 0.4 - z z 0.3 0.3 -

0.2 0.2 - E* 0.1 0.1 - E i 0 I 0 . L A.J1I > / # o

by B16 cells (Figure 4C). We analyzed surface expression of components of the interleukin-2 (IL2) receptor complex by CTLL-2 in response to B16 conditioned media.

We found that CTLL-2 cells cultured in medium conditioned by B16 cells downregulated the expression of IL-2R|3 after 72 hours of incubation and such expression increased

significantly after treatment with TGF|3 neutralizing antibody (Figure 4D). The expression of the IL-2Ra on CTLL-2 was not altered by B16 conditioned media (data not shown).

2.4.5 B16 derived TGF|3 blocks NK cell recruitment and function

IL-2/IL-2R interaction on NK cells has relevant implications in the expansion and

development of NK cells, as well as in the cytotoxicity and cytokine production by these

cells. In particular, the common IL-2/IL-15R|3 signaling activates transcription factors involved in the control of perforin expression and secretion of proinflammatory cytokines

such as GM-CSF and IFNy 73'294 Since we observed a reduced recruitment of NK cells to the tumor site as B16 tumor burden increases, we evaluated the responsiveness of

mouse NK cells to IL-2 by culturing NK cells in medium conditioned by B16 and

B16GIFT2 cells. As shown in figure 5A, the expression of IL-2RJ3 chain was

significantly reduced on NK cells cultured in medium conditioned by B16 cells

supplemented with recombinant IL-2 (23.5 pmol/ml). Pre-treating medium conditioned

by B16 cells with anti-TGF|3 neutralizing antibody restored the expression of IL-2RJ3 on

NK cells to similar levels as the positive control. On the other hand, NK cells cultured in

77 medium conditioned by B16GIFT2 cells containing equimolar concentration of GIFT2, display a significantly greater expression of IL-2RJ3 despite the presence of a similar

78 Figure 4: B16 cells secrete active TGF0

(A) CTLL-2 cells were cultured in the absence of serum with medium conditioned by:

(filled circle) B16 cells plus isotype control, (filled diamond) B16 cells plus TGF|3

neutralizing antibody and (open square) mouse embryonic fibroblast (MEF). MTT assay was performed to assess the proliferation ability of CTLL-2 cells in the presence of increasing concentration of recombinant IL2. The results plotted represent the average of two independent experiments performed in triplicate. (B) The ability of active TGF|3 of inducing the novo CDS expression on CTLL-2 was determined by culturing these cells with recombinant TGF|3 as control in the presence of recombinant IL2. TGF|3

neutralizing antibody was used to assess the specificity of this property. CDS expression

on CTLL-2 was measured by flow cytometry. (C) CTLL-2 cultured in medium

conditioned by B16 cells plus recombinant IL2 with or without TGF|3 neutralizing

antibody. (D) IL-2RJ3 expression on CTLL-2 cultured with IL2 in medium conditioned

by B16 cells with and without TGF|3 neutralizing antibody also was assessed. Significant

differences (p<0.05) are indicated.

79 Figure 4

p<0.05

or* 0 20 40 60 80 100 II 2 units

B IL2 lL2+rlGFPi !L2+rTGFp 1+ocTGFp Ab

<1% 67% RPMI ,s -i 2% f M2 s§- isj A »• 2 3 tfp 10' CDSFtTC10 10 1*4 CDSFiK f5« T»' ' II' CD8FITC^ ^ • • To"

aIGHp Ab

17.6% <1% r4V BI6CM Isi T 3 V. J 3 1 2 3 ti" "IF CWFITCTo* IO 10 io° id CD6FITCIO io io< s 1 <1% .8 i MEF CM ' A, 1 3 4 10° 10 CDSFITC10* 10 ID

* = p<0.05

70 * fit) + ac

ttTGFp Ab

80 concentration of active TGF|3 secreted also by B16GIFT2 cells. This expression was not

altered by TGF|3 neutralizing antibody pre-treatment. This increase in the expression of

IL-2RJ3 demonstrated that GIFT2 fusokine released by B16GIFT2 cells overrides the

suppressive effects of TGF|3 on NK cells. We also evaluated the expression of IL-2Ra

and IL-2Ry on NK cells, and in both cases we detected low expression that was not

altered with anti-TGF|3 neutralizing antibody (data not shown). Although these cells

secreted IFNy in response to the stimulatory effect of the GIFT2 fusokine, a substantially greater amount of IFNy was obtained with TGF|3 neutralizing antibody pre-treatment

(figure 5B). We investigated the direct effect of TGF|3 on NK cells by analyzing the

phosphorylation levels of Smad3 and Smad2 signalling molecules activated by TGF|3.

Despite the fact that B16 and B16GIFT2 cells secrete equal amounts of active TGF|3 (200

pg/ml), NK cells pre-cultured in medium conditioned by B16 cells show significant

higher levels of Smad3 phosphorylation than those observed in the cell extracts of NK

cells cultured in medium conditioned by B16GIFT2 cells (figure 5C). These results

suggest that GIFT2 antagonizes the suppressive effect of TGF|3 on NK cells. Regarding

Smad2, we observed similar phosphorylation levels in cell extracts of NK cells cultured in medium conditioned by B16 and B16GIFT2 cells (figure 5C). Smad3 and Smad2

phosphorylation was completely abolished by anti-TGF|3 neutralizing antibody.

2.4.6 In vivo blockade of TGF0 and effect on immune bystander effect

We assessed the contribution of TGF|3's immune suppressive features, as well as its

property to reduce GIFT2's bystander effect in vivo, by embedding lxlO4 of each

B16B16GIFT2 at 1:1 ratio admixed in matrigel along with soluble TGF|3 receptor II

81 (TJ3RII) or anti-TGF|3 neutralizing antibody. We observed a significant delay of tumor growth in mice injected with admixed cells

82 Figure 5: Blockage of immunosuppressive effects of TGF0 enhances the activation state of NK cells in response to GIFT2

(A) Percentage of IL-2R|3 expressing NK cells cultured in medium conditioned by: B16

cells supplemented with 23.5 pmol/ml of recombinant IL-2 in the presence or absence of

TGF|3 neutralizing antibody, RPMI supplemented with 23.5 pmol/ml of recombinant IL-2 in the presence or absence of 1 ng/ml of recombinant active TGF|3, and B16GIFT2 cells

containing equimolar concentration of GIFT2. Significant differences are indicated

(p<0.05 log rank). (B) IFNy production by NK cells pretreated as described in panel A.

pretreated with medium conditioned as described in the figure. Whole NK cell extract were subjected to gel electrophoresis and immunostained with an anti-phospho-Smad3-

specific or an anti-phospho-Smad2-specific antibody. Nitrocellulose membrane were

stripped and reprobed with anti-atubulin antibody.

83 Figure 5

6 p<0.05 100 (,000 90 5000 80 1 p<0.05 u 70 — 4000 p<0.05 yj 60 I 3 50 tt 3000 c 40 %H a 2000 1 30 : 20 : 1000 10 0 • 0 I rTGFp rTOF|i aTGFp aTGFp Ab

IsotypeAb — "H — — — Isorype Ab +-+---

616 B16-GIFT2 RPMI B16 B16-GIFT2 RPMI

P-Smad2

ti-Tubulin

84 and sT|3RII (figure 6A) or TGF|3 neutralizing antibody (figure 6B), indicating that active

TGF|3 secreted by B16 cells is an important variable which contributes to the reduction of the immune bystander effect of a potent pro-inflammatory fusokine such as GIFT2.

2.4.7 Interferon y pre-treatment of B16 melanoma upregulates expression of MHC I and

MHC II but does not improve immune bystander effect in vivo.

B16 melanoma cells can adopt an antigen presenting cell (APC)-like phenotype after treatment with interferon y (IFNy) in vitro 29 \ To test whether IFNy pre-treatment of

melanoma cell would enhance their immunogenicity and secondarily the immune bystander effect in vivo, we performed an experiment similar in design to that shown in figure 7 except B16 and B16GIFT2 and their admixed implants were pre-treated with

IFNy. As shown in Figure 7A, B16 and B16GIFT2 cells readily and robustly upregulate both MHC I and MHC II expression. These cells were then utilized for in vivo testing.

However, despite an enhanced immune phenotype following IFNy treatment, the bystander effect using lxlO6 of each B16B16GIFT2 at 1:1 admixed cells was not improved compared with control cells untreated with IFNy (Figure 7B). The TGFJ31 and

TGFJ33 co-receptor, (CD105/endoglin), is highly expressed by B16 cells (supplementary

data 1), which suggests that these cells may be sensitive to TGFJ31 or TGFJ33 dependent

effects. However, IFNy induced MHC class II expression was not downregulated by

adding recombinant active TGFJ31 (Figure 7A), which indicated that B16 cells are

resistant to TGF|3 dependent inhibition of class MHC II expression.

85 Figure 6: Blockage of TGF(3 suppressive effects improved the bystander effect of

GIFT2. (A) Cohorts of 10 C57B1/6 mice per group were implanted subcutaneously with

matrigel plugs containing (filled circle) lxlO4 B16GIFT2 cells, (filled triangle) lxlO4

B16 cells, and (open diamond) lxlO4 admixed cells at 1:1 ratio embedded in matrigel

alone or (filled square) with soluble TGF|3 receptor (TJ3RII) protein. (B) Similar experiments were performed using (filled square) TGF|3 neutralizing antibody as TGF|3 blocking agent. In both cases tumor volume was measure over time and statistic analysis was performed. These experiments were repeated three times and significant differences

are indicated (p<0.05 log rank).

86 Figure 6

2000 sTpRII 1800 1600 1400 1200 u E 1000 > P<0 3 I 800 600 H 400 200 0 7 11 14 20 22 25

Days post tumor implantation B 2000 1800 aTGFpAb 1600 1400 1 1200 1000 p<0.05 0 800 1 600 400 200 0 7 12 14 17 21 Days post tumor implantation Figure 7: IFNy upregulates the expression of MHC class I and MHC class II

molecules on B16 cells in vitro, but did not improve the immunogenicity of these

cells in vivo. (A) MHC class I and class II expression was analyzed by flow cytometry on

B16 cells and B16GIFT2 cells pretreated with IFNy (lng/ml) for 48 hours in the presence or absence of recombinant TGF|3. (B) B16 cells were pretreated with IFNy for 48 hour

before subcutaneous implantation into immunocompetent C57B1/6 mice. Cohorts of 10

mice per group were injected with IFNy (filled circle) pretreated or (open circle) not

pretreated B16GIFT2 and B16 cells mixed at 1:1 ratio.

88 Figure 7

MHC I MHC II IFNy + rTGFp

CD mi , 94% 8% 5

io° !»' io2 to3 io4 10° 101 102 1G3 104 11^ ID1 IO2 103 104 10° ll)1 10* IO3 104 10° ifl' ir nr 104 MKIPE M4CMK

0% 98% 98% I£ 1 2 3 4 10° 101 IO2 ID3 IO4 to9 101 102 TO3 104 io1 to2 io3 io4 ioO To' 1*2 IO3 10" 10° 101 102 103 104 10° I0 ID IO 10 MHC IPE MKIIPE mciiPE

B 100 «—# # #

70 Igo gs. 50 40

30 20 10 0 0 5 10 15 20 25 30 Days post tumor implantation

89 2.5 DISCUSSION

B16 melanoma cells implanted in C57B1/6 mice serves as a robust and popular animal

model system to test therapeutic platforms which recruit an immune anti-melanoma

response. In previous published work by our group, we utilized this in vivo system to test the utility of a GMCSF & IL2 fusokine (aka GIFT2) and showed that gene transfer of

GIFT2 encoding cDNA to B16 melanoma cells could serve as part of a cancer immunogene therapy strategy2. However, we had made the observation that as B16 tumor burden was increased the immune stimulation effects of GIFT2 were attenuated. Close

reading of reports by other groups exploiting the B16-C57B1/6 animal model in immunotherapy strategies reveals that an increased B16 tumor burden is apparently

antagonistic to the use of immune rejection treatment strategies 295, including graft versus

tumor arising from allogeneic marrow transplantation. Cures are readily achieved with

small tumor implants, treatment failures abound when even modest increases in tumor

cell dose are tested. We have observed that tumor-associated immunosuppresion is more

prominent as B16 burden increases. Specially, the significant reduction in the number of

NK and MKT cells recruited at the tumor site observed in the implants of lxlO6 of each

admixed cells (B16 B16GIFT2) compared to the implants comprising equal cell number

of B16GIFT2, indicate that tumor derived suppressive factors are antagonistic to the

GIFT2 dependent immune bystander effect. Several factors secreted by tumor cells have been described to have deleterious effects on the anti-tumor immune response. One of the most potent immunosuppressive cytokine secreted by tumor cells is transforming growth factor |3 (TGF|3), which stimulates tumor growth while abolishing effector functions of

macrophages, NK cells, CTL and dendritic cells as well as cytokine secretion. Melanoma tumor cells release high levels of TGFJ31, 2 and 3 to the tumor microenvironment.

90 However, these cells are resistant to TGF|3-induced growth inhibition by overexpressing

SMAD inhibitors such as Ski and Sno 1. In concordance with previous studies, we

observed that B16 tumor-derived active TGF|3 antagonizes the immune stimulatory

effects of IL-2 on a murine IL-2 dependent CDS T cell line (CTLL-2). TGF|3

preferentially antagonize IL-2 induced CTLL-2 cell proliferation and not IL-2 induced

cell survival in a Smad3 dependent manner. Through the inhibitory activity of Smad3,

TGF|3 inhibits IL-2 induced expression of promitogenic genes such as c-myc, cyclin D,

and cyclin E without affecting the activation status of upstream IL-2 receptor associated

kinase (JAK1) and signaling molecules She and STAT5296. TGFJ31 also inhibits IL-2 induced proliferation in splenocytes and thymocytes through SMAD3 independent

mechanism297, which may involve suppression Cdc25 expression298 and inactivation of

phosphatase 2A (PP2A)299. We also observed a downregulation in the expression of IL-

2R|3 on CTLL-2 cultured in medium conditioned by B16 cells, which correlated with a

dramatically decreased ability of these cells to proliferate in the presence of IL-2. These

results confirm that B16 cells secrete active TGF|3, which antagonizes IL-2- induced

proliferation and gene expression in lymphoid cell lines. B16GIFT2 cells are always totally immune rejected independently of tumor cell dose, and this rejection is driven by

GIFT2 mediated recruitment and activation of NK/NKT cells as well as CDS T cells2.

Since we observed suppression of NK cell recruitment at tumor site in vivo, we tested whether B16-derived TGF|3 would be a direct cause of this phenomenon. In order to

evaluate the responsiveness of NK cells to IL-2 and GIFT2 in the presence of tumor

derived TGF|3, we analyzed the expression levels of IL-2 receptor complex subunits. IL-

2R|3 plays an important role in NK cell function since IL-2RJ3 deficient mice display a

91 reduction of NK1.1+CD3" cells in the circulation and a complete absence of NK

cytotoxicity activity in vitro300 We found that B16-derived TGF|3 will markedly suppress

IL-2RJ3 expression on NK cells despite the presence of high concentrations of

recombinant IL-2. In contrast, NK cells cultured in medium conditioned by B16GIFT2

cells, which also secrete similar concentrations of tumor derived TGF|3, display a striking expression of IL-2R|3. These results suggest that GIFT2 exerts potent immunostimulatory effect on NK cells. Despite the greater immunostimulatory effect of GIFT2 on NK cells, tumor derived TGF|3 down modulates IFNy production by NK cells cultured in medium

conditioned by B16GIFT2 cells. Notwithstanding TGF|3 antagonism, we observed a

substantial production of IFNy (2 ng/ml) by NK cells cultured in medium conditioned by

B16GIFT2 cells, which increased more than two fold by blocking TGF|3 with specific

antibody. In addition, the phosphorylation status of SMAD3 in this cell extract was

significantly reduced despite B16GIFT2 cells secreting equal amounts of active TGF|3 as

non-modified B16 cells. These results suggest that GIFT2 antagonizes the suppressive effect of TGF|3 on NK cells. GIFT2, in addition to directly affecting SMAD3

phophorylation, and therefore the responsive of NK cells to tumor derived TGF|3, also

acts as potent chemoattracting factor for NK cell in vivo and promotes the secretion of

substantial amount of IFNy by NK cells. IFNy inhibits TGF|3-induced phosphorylation of

SMAD3 through JAK/STAT pathway, which induces the expression of SMAD7, an

antagonist of receptor regulated SMADs. SMAD7 acts by preventing the interaction of

SMAD3 with the TGF|3 receptor301. The suppressive effects of tumor derived TGF|3 on

NK cells have been reported in several tumor models other than melanoma. For example, breast tumor cell line (MDA-231) derived TGF|3 exerts deleterious effects on NK

92 mediated cytotoxicity. Monoclonal antibody inducing blockade of all three mammalian

TGF|3 isoforms inhibited MDA-231 primary tumor or metastases formation in immunocompetent mice, but this effect was completely abolished in beige NK cell

QQAO deficient nude mice ' . In addition, TGF|3 antagonizes the immunoregulatory effects

of several cytokines including IL-2, because of the crosstalk between the signaling

pathways. This pleiotropic cytokine is secreted by a variety of cell types as a latent

complex unable to bind to TGF|3 signaling receptor and initiate signaling transduction.

Different mechanisms have been proposed to induce the activation of latent TGF|3 such

as proteolytic cleavage, enzymatic deglycosylation and/or conformational changes of latent TGF|3 complex, which expose the TGF|3 receptor binding site 304 Indeed,

TGF|3 may serve as a powerful means for B16 to escape immune surveillance but to also

actively antagonize pro-inflammatory cancer gene therapy as we show here, a

phenomenon which parallels human clinical experience in melanoma therapy. Indeed,

recent observations indicate the presence of increased production of suppressive

cytokines such as TGFb and IL-10 in patients bearing more than one invaded lymph

node305.

These observations conciliate an apparent paradox: GIFT2 drives the activation of NK

cells - as demonstrated by the upregulation of IL-2R|3 on effector NK cells - whilst

contemporaneous production of TGF|3 specifically blocks this effect. Reciprocally,

GIFT2 can inhibit TGF|3-mediated signaling in NK cells. However, this reciprocal

antagonism is skewed in favor of the TGF|3 effect since as tumor burden increases

suppression of NK cells wins out. The effect of GIFT2 can be rescued in part by specific

blockade of TGF|3 in vivo. We propose the following model: GIFT2 secreted by

93 genetically modified B16 tumor cells, induces a potent chemotactic stimulus on NK cells, which respond to GIFT2 immunostimulatory effect by upregulating the expression of IL-

2R|3 on their cell surfaces and producing substantial amounts of IFNy. This creates a

cytokine circuit that recruits other IFNy-secreting immune cells, as well as GIFT2

responder cells such as macrophages and cytotoxic T cells. All these cytokines cooperate to antagonize the immunosuppressive effects of tumor-derived TGF|3. In contrast, at

elevated tumor burden, the amount of active TGF|3 in the tumor microenvironment

overcomes the immunostimulatory effects of GIFT2 and prevents the recruitment of tumor infiltrating NK cells by blunting the expression of IL-2R|3 on their cell surface and therefore impairing their responsiveness to GIFT2 chemotactic effects, resulting in insufficient IFNy secretion and recruitment of other immune effector cells.

In summary, eliciting an immune response to melanoma - and other types of cancers as well - will be antagonized by tumor-secreted TGF|3. The resistance of tumor to immune

clearance will increase proportionally to cancer burden306. Blockade of TGF|3 inhibitory effect on cell-mediated immune response will likely be an important component in cancer gene immunotherapy of bulky malignant disease.

2.6 ACKNOWLEDGMENTS

This work was supported by a Canadian Institute for Health Research operating grant

MOP-15017. CP is recipient of Montreal Centre for Experimental Therapeutics in

Cancer Scholarship and US Army Graduate study Scholarship and JG is a Fonds de recherche en sante du Quebec chercheur-boursier senior. We thank Nicoletta Eliopoulos,

Moira Francois and John Stagg for technical assistance.

94 CHAPTER 3

The human ortholog of granulocyte-macrophage colony-stimulating factor and

interleukin-2 fusion protein induces potent ex vivo NK cell activation and

maturation.

Reference: Claudia Penafuerte, Norma Bautista-Lopez, Boulassel Mohamed-Rachid,

Jean-Pierre Routy and Jacques Galipeau. Cancer Res. 2009 Dec 1; 69 1. 9020-8

Preface to Chapter 3: We have shown in Chapter 2 that the expression of the murine fusion protein GIFT2 secreted by genetically modified B16 cells induces a robust anti tumor bystander effect in vivo against non-modified malignant cells present in the tumor

site. However, GIFT2- mediated bystander effect is attenuated by tumor-derived active

TGF|3 in a dose-dependent manner as tumor burden increase. The blockade of tumor-

derived TGF|3 suppressive effect on NK cells will definitively improve the potency of

GIFT2 mediated immune bystander effect in the condition of high tumor burden. In the

next chapter, we aim to generate and characterize the human ortholog of GIFT2 and its use as a means to generate oncolytic NK cells that may serve as an effective cellular

platform for cancer cell therapy.

95 CHAPTER 3: The human ortholog of granulocyte-macrophage colony-stimulating factor and interleukin-2 fusion protein induces potent ex vivo NK cell activation and maturation.

3.1 ABSTRACT

NK cells are an appealing cellular pharmaceutical for cancer therapy because of their innate ability to recognize and kill tumor cells. Therefore, the development of methods which can enhance the potency in their anticancer effect would be desirable. We have

previously shown that a murine GM-CSF/IL-2 fusion protein displays novel antitumor

properties in vivo compared to both cytokines in combination due to recruitment of NK

cells. In the present work, we have found that human ortholog of the GM-CSF/IL-2 fusion protein (aka hGIFT2) induces robust NK cell activation ex vivo with significant

secretion of RANTES and a 37 fold increase in IFNy production when compared to either

IL-2 or GM-CSF single cytokine treatment or their combination. Moreover, hGIFT2

upregulates the expression of NK cell activating receptors NKp44, NKp46 and DNAM-1

(CD226), as well as CD69, CD 107a and IL-2RJ3 expression. In addition, hGIFT2

promotes NK cell maturation, based on the downregulation of CD117 expression and

upregulation of CDllb. This phenotype correlates with significantly greater cytotoxicity

against tumor cells. At the molecular level, hGIFT2 leads to a potent activation of JAK

kinases downstream of both IL-2 and GM-CSF receptors (JAK1 and JAK2 respectively)

and consequently leads to a hyperphosphorylation of STAT1, STAT3 and STATS. In

conclusion, hGIFT2 fusokine possesses unique biochemical properties distinct from IL-2

and GM-CSF and constitutes a novel and potent tool for ex vivo NK cell activation and

maturation and may be of use for cancer cell immunotherapy.

96 3.2 INTRODUCTION

NK cells are innate effector cells that react against virus infected or transformed cells before the onset of adaptive immunity. These cells express an array of inhibitory and

activating receptors that sense the quality and quantity of self major histocompatibility

complex (MHC) class I molecules expressed at the cell surface, which mediate the

recognition of target cells and regulate the activation state of NK cells. Upon cytokine

stimulation, NK cells become lymphokine-activated killer (LAK) cells that proliferate,

produce cytokines (IFNy and TNFa) and upregulate effector molecules such as adhesion

molecules, perforin, granzymes, Fas ligand (FasL), and TRAIL. These features endow

NK cells with the ability to kill tumor cells and act as essential promoters of an adaptive immunity against tumor challenge 307 Indeed, several in vitro as well as in vivo studies indicate that tumor cells are recognized as NK cell targets. NK cells not only directly eliminate tumor cells, but also induce the subsequent development of tumor-specific T

cell responses to parental tumor cells. For example, the potential of NK cell-based therapy has been evoked in patients suffering of acute myeloid leukemia. In this case,

adoptive transfer of allogeneic NK cells with mismatched NK inhibitory receptors and

HLA class I ligands produce graft-vs-leukemia in the absence of graft-vs-host disease due to the ability of donor NK cells to attack recipient dendritic cells, which are known to be

responsible for priming donor T cells and induce graft-vs-host disease 116. Therefore,

clinical interest in NK cells as a cancer cell pharmaceutical is rapidly growing, with more than 200 clinical trials examining either autologous or allogeneic NK cells as well as universal donor NK cell lines. However, there are some theoretical limitations to the use

of NK cells for cancer therapy. Tumor cells expressing low levels of MHC class I may

97 fail to trigger NK cell activation 308. NK cells from patients affected by advanced bulky

malignancies display impaired cytolytic activity, which can be attributed to tumor-

derived suppressive factors such as TGF|3 that exert deleterious effects of NK cell effector functions 121. Therefore, the infusion of primed and activated NK cells would be

an approach which may enhance the effectiveness of these cells in the treatment of

cancer. The use of cytokines and combinations thereof would be an obvious method to

activate NK cells ex vivo. Better still, pharmacological activation of NK cells in a

manner unachievable with native cytokines may lead to desirable acquired anticancer

properties. In this line of thought, we have previously shown that a murine GM-CSF/IL-

2 fusion protein (aka GIFT2) displays novel antitumor properties in vivo compared to both cytokines in combination regarding to tumor site recruitment of significant functional NK cell infiltration2. In the present study, we demonstrate that the human

ortholog of the GM-CSF/IL-2 fusion protein (hGIFT2) induces potent human NK cell

activation and cytotoxicity distinct from that achieved by IL-2 or GM-CSF. The use of

hGIFT2 as a means to generate oncolytic NK cells may serve an enhanced cellular

platform for cancer cell therapy.

3.3 MATERIALS AND METHODS

3.3.1 Cell lines, recombinant proteins, antibodies and ELISA kits

293T cells were cultured in DMEM (Wisent Technologies, Rocklin, CA) supplemented with 10% FBS (Wisent Technologies). The cell lines TF-1, CTLL-2, Daudi, K562 and

U266 (American Type Culture Collection [ATCC], Manassas, VA) were grown

according to ATCC s recommendations. Recombinant proteins (hIL-2 and hGM-CSF,

R&D Systems, Minneapolis, MN); anti a-tubulin (Santa Cruz Biotechnology, Santa

98 Cruz, CA); polyclonal anti-phosphorylated Jakl, Jak2, Statl, Stat3, StatS, ERK and

IkBa, as well as their respective antibodies against full length proteins (Cell Signalling

Technology, Danvers, MA); anti-human FcR III/II, CD3, CD4, CDS, CD56, NKG2D,

NKp44, NKp46, NKp30, CD 107a, CD117, CD 122, CD69, CD226, CDllb, CD43, KIR,

IFNy, or their isotype control antibodies mlgGl and m!gG2a for flow cytometry (BD

Biosciences, San Diego, CA); enzyme-linked immunosorbent assay (ELISA) kits for

human IL-2 and GM-CSF (Invitrogen and eBioscience), for RANTES, IFNy and TNFa

(R&D systems). Cytotoxicity detection kit LDH (Roche applied science, Mannheim,

Germany).

3.3.2 PBMC and human NK cells

Blood was drawn from healthy donors, after informed consent had been obtained, into

heparin- or citrate-coated CPT tubes (BD Biosciences, San Jose, CA) and peripheral

blood mononuclear cells (PBMC) were isolated by density centrifugation according to the manufacturer's recommendation. NK cells were obtained by negative purification

using the untouched NK cells isolation kit (Miltenyi Biotec, Bergisch Gladbach,

Germany). Cells were treated with NK cell biotin-antibody cocktail and magnetic

microbeads reagents and were depleted by magnetic cell sorting (MACS) system with an

autoMACS™ column (Miltenyi Biotec) according to the manufacturer's instructions. NK

cell population (CD56 CD3") purity assessed by flow cytometry was 96%. Three

different donors were used for each experiment and an average of two different

experiments was performed with the same donor.

99 3.3.3 Vector construct and transgene expression

The human GM-CSF cDNA (Invivogen, San Diego, CA) was modified to remove the 3'

nucleotides encoding the stop codon and subsequently cloned in frame with the cDNA encoding the human IL-2 devoid of start codon to generate the cDNA for hGIFT2 fusokine. The hGIFT2 was incorporated into a bicistronic vector (pCMV) allowing the

expression of both hGIFT2 and green fluorescent protein (GFP) 309. The nucleotide

sequence of the hGIFT2 cDNA was confirmed by DNA sequencing at the Guelph

Molecular Supercenter (University of Guelph, Ontario, Canada). Concentrated media

conditioned by 293 T cells stably transfected with pCMV encoding for hGIFT2 was used to test the bioactivity of hGIFT2. The IL-2-responsive CTLL-2 or GM-CSF-responsive

TF-1 cell lines were plated at a density of 105 cells/well in a 96-well plate and treated with increasing concentration of cytokines for 48 hours. Cell proliferation was assessed with a 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

3.3.4 Analysis of cell surface marker on leukocytes

PBMC were cultured with hGIFT2 and equimolar concentrations of cytokine controls

(IL-2, GM-CSF, IL-2 combined with GM-CSF, 2 pmol). After seven days in cultured, the

cells were resuspended in phosphate-buffered saline (PBS) with 2% FBS and incubated with anti-human FcR III/II for 30 mins. Subsequently, cells were labelled with a mixture

of conjugated antibodies specific for cell surface markers (CD3, CD4, CDS, CD56 and their respective isotype controls) for 1 hour at 4°C and analyzed by flow cytometry using

a Becton Dickinson FAC Scan (San Jose, CA).

100 3.3.5 Flow cytometric analysis of NK cell expression markers, IFNy expression, apoptosis and cell proliferation.

For cell surface marker staining, NK cells stimulated with hGIFT2 and equimolar

concentrations of cytokine for seven days were resuspended in phosphate-buffered saline

(PBS) with 2% FBS, incubated with anti-human FcR III/II for 30 mins and labelled with

conjugated antibodies specific for NK cells activating receptors (NKp30, NKp44,

NKp46, NKG2D, CD226), inhibitory receptors (CD43, KIR), maturation markers

(CDlib, CD 117), NK cell activation markers (CD69, CD107a) and IL-2 receptor

components (a subunit CD25, (3 subunit CD 122 and y subunit CD 132). The expression of these cell surface markers was determined by FACS Calibur cytometer (BD) and

analyzed using Cellquest software (BD). For IFNy intracellular staining, NK cells

previously stimulated with cytokines for two hours were treated with Brefaldin A for four

hours, fixed, permeabilized and stained with IFNy conjugated antibody. For Annexin V

and PI staining, NK cells cultured with cytokines (2 pmol) for seven days were incubated with Annexin V/propidium iodide (PI) for 15 minutes at room temperature and analyzed

by flow cytometry. For CFSE staining, NK cells pre-stained with Carboxyfluorescein

succinimidyl ester (CFSE) were cultured with cytokines for four days and cell

proliferation was assessed by flow cytometry. The data were analyzed using Cellquest

software (Becton & Dickinson).

3.3.6 In vitro cytotoxicity assay

lxlO5 NK cells (effector cells) previously stimulated with hGIFT2 or cytokine controls for 3 days were washed three times and co-cultured with lxlO4 target cells (Daudi, K562,

101 U266) at effectontarget cell ratio of 10:1 for 4 hours. Supernatants were collected and

used to measure the lactate dehydrogenase (LDH) released from the cytosol of damaged

or lysed cells using a cytotoxicity detection LDH kit. As controls, we measured the

spontaneous LDH released from target and effector cells. The percentage of cytotoxicity was calculated according to the formula:

cytotoxicity (%) = (Experimental - Effector Spontaneous)/(Target Maximum -Target

Spontaneous) x 100.

Target Maximum: maximum LDH released from Triton X-100 treated target cells.

3.3.7 Intracellular signaling

For signaling analysis, media conditioned by stably transfected 293 T cells expressing

hGIFT2, as well as media conditioned by non transfected cells containing equimolar

concentration of cytokine controls (IL-2, GM-CSF or IL-2 combined with GM-CSF, 5

pmol each) were added to 106 NK cells for 20 minutes before being lysed and probed by

Western blot (WB) with rabbit anti-phosphorylated Jakl, Jak2, Statl, Stat3 and StatS,

ERK and Akt antibodies. Antibodies against total proteins were used as loading controls

as well as anti a-tubulin antibody. Three independent experiments were performed with

NK cells from different donors.

3.3.8 Statistic evaluation

P values were calculated by paired student T test. P<0.05 was considered statistically

significant.

102 3.4 RESULTS

3.4.1 Design and expression of hGIFT2 fusokine

The human GMCSF/IL-2 (hGIFT2) construct was created by cloning a modified human

GM-CSF cDNA missing the nucleotides encoding for the stop codon in frame with the 5'

end of the human IL-2 cDNA devoid of its start codon. The final fusokine hGIFT2 cDNA

encodes for a single polypeptide chain of 302 aminoacids (Figure 8A). Denaturing immunoblotting performed on the supernatant of 293T cells stably transfected to express

hGIFT2 (5 ng per million cells in 24h) showed that the chimeric protein was secreted and

has an apparent molecular weight (MW) of 45kDa by SDS-PAGE under reducing

conditions (Figure 8B). We detected and quantified hGIFT2 by IL-2 and GM-CSF

ELISA and we found that 1 pmol310 of hGIFT2 is equivalent to 97 units, whereas 1 pmol

311 of recombinant IL2 is equivalent to 221 units (Figure 8C). Both techniques confirm that only the full-length hGIFT2 fusokine of predicted MW is secreted by transfected

cells.

103 Figure 8: Design and expression of hGIFT2 fusokine

(A) Schematic representation of the hGIFT2 aminoacid sequence. (B) Denaturing immunoblot using conditioned media (CM) from stably transfected 293T cells expressing hGIFT2 probed with both anti-GM-CSF and anti-IL2 antibodies. CM from 293T cells transfected expressing GM-CSF or IL2 was used as positive control. (C) Detection and

quantification of hGIFT2 by GM-CSF and IL-2 ELISAs. In the panel C, standard curve for each recombinant cytokine are represented by white circles and serial dilutions of

hGIFT2 are represented by black diamonds.

104 Figure 8

[IuiLiaii GM-CSF MWLQSI LLLGTVAC SFSAP AR SP SP S TOPWFH VN AI GM-CSF QEARRLLNLSRDTAAEMNETVEVISEMFD LQEP TC LQTRLELYKQGLRGSLTKLKGPLTMM ASHYK.QHC 0.4 PPTPETSC AIQlllTCSr KEN1KDFLLV IHl'DCWEPV QE- G I inker I S I LMYRMQI .I.SCIAI.ST.AI VTNSAPTSSSTKKT Human IL-2 QLQLEHLLLDLQMlLh G IN N YK N PK L'i R.M L TFKFYMPKKATELKHLQCLEEELKPLEEVL 0 200 400 600 NI.AQSKNFHLKPRDLISMNTO'T-ELKGSET TFMCEYADETAITVEFLKR.W ITFC Q SII STLT p$'ml

II.-2 B I

.0 0.8 0.6 50 - A SO _ 0.4 0.2

0.0 0 200 400 600 20 - I pg/ml Anti-human GM-CSF Anti-hum.™ TL2

105 3.4.2 hGIFT2 induces the proliferation of IL-2 and GM-CSF dependent cell lines

The bioactivity of GM-CSF and IL-2 domains as part of hGIFT2 was confirmed by

proliferation of GM-CSF-dependent cell line, TF-1 (Figure 9A) and IL-2 dependent cell line, CTLL-2 (Figure 9B). The proliferation level of each cell line was determined by

MTT incorporation for the last 4 hours of incubation with cytokines. As result, hGIFT2 induces similar proliferation levels of these cell lines compared to equimolar

concentration of each cytokine controls, which indicate that both GMCSF and IL2 as part

of the fusion preserve the ability to induce an appropriate proliferative response.

3.4.3 Effect of hGIFT2 on primary lymphocytes

The effect of hGIFT2 on lymphocytes was determined by culturing peripheral

mononuclear blood cells (PBMC) with equimolar concentrations of cytokines (2 pmol).

After seven days in culture, we observed that hGIFT2 promotes a significant increase in the number of NK cells compared to equimolar concentration of cytokine controls, whereas the number of CDS, CD4, and NKT cells did not show significant differences with each cytokine treatment (Figure 10A). Similarly, we observed a significant increase

of NK cell percentage in the hGIFT2 treated PBMC fraction from three different donors

(Figure 10B). Based on these results, we investigated whether hGIFT2 induces NK cell

proliferation or enhances NK cell survival. To test these hypotheses, purified NK cells were pre-stained with CFSE, cultured with equimolar concentrations of cytokines and

analyzed by flow cytometry. After four days in culture, IL-2 as well as IL-2 combined with GM-CSF induces NK cell proliferation (Figure IOC). However, after seven days in

culture with cytokine controls the NK cell number decreased, even though the media was

106 changed at day 4 to avoid cell starvation. In contrast, hGIFT2 does not induce NK cell proliferation but rather protect NK cells from apoptosis (Figure 10CD). Based on these

107 Figure 9: Bioactivity of hGIFT2

To test the bioactivity of hGIFT2, proliferation assays were performed by MTT incorporation after 48 hours of incubation with increasing equimolar concentration of

cytokines. (A) CTLL-2, IL-2 dependent cell line (P 0,05 between hGIFT2 and IL-2). (B)

TF-1, GM-CSF dependent cell line (f >0.05 between hGIFT2 and GM-CSF). Media

conditioned by transfected 293T cells containing equimolar concentrations of IL-2 or

GM-CSF was used as controls. Results are shown as mean of triplicates + SEM of one

representative experiment out of three performed. No significant differences between

hGIFT2 and controls (P>0.05) were observed.

108 Figure 9

TF1 -O— GM-CSF ~m~ hGIFT2 —X— IL-2

0.05 0 1 0 15 pmols/ml -o- GM-CSF —hGIFT2 IL-2

1.5 pmols/ml

109 results and in agreement with previous reports 312'312, we suggest that IL-2 activated NK

cells undergo apoptosis in long term culture.

3.4.4 hGIFT2 induces activation of NK cells

Purified human NK cells pretreated with hGIFT2 displayed an enhanced activation

profile compared to NK cells stimulated with equimolar concentration of cytokine

controls based on the expression levels of the early activation marker (CD69) and a

marker of degranulation (CD107a; Figure 4A). In order to evaluate the responsiveness of

NK cells to hGIFT2, we analyzed the expression levels of the IL-2 trimeric receptor

subunits (a,|3,y) . IL-2Ra and IL-2Ry expression levels were not modified by any

cytokine treatment (data not shown), whereas IL-2R|3 expression was upregulated by

hGIFT2 and downregulated by GM-CSF (Figure 11 A). Intracellular staining for IFNy indicates that hGIFT2 induces a robust expression of IFNy in the first 6 hours of cytokine treatment (Figure 1 IB, left panel). By ELISA, we quantified that hGIFT2 promotes more than thirty seven fold greater IFNy production than equimolar concentration of IL-2 after

3 days in culture (Figure 1 IB, right panel), as well as significant secretion of RANTES

Figure 11C, lower panel). However, we did not detect significant differences regarding to

TNFa production (Figure 11C, upper panel). We also observed that GM-CSF inhibits

NK cell derived IFNy, TNFa and RANTES production. These results indicate that

hGIFT2 displays superior properties as a tool for NK cell activation and overcomes GM-

CSF mediated immunosuppression. In addition, hGIFT2 similarly affect both CD56bnght

and CD56dim subsets of NK cells, which upon hGIFT2 stimulation become highly

activated and granulated (Figure 12).

110 Figure 10: hGIFT2 increases NK survival but does not induce NK cell proliferation

PBMC or NK cells were cultured with of 2 pmol of hGIFT2 or equimolar concentrations

of cytokine controls (IL-2, GM-CSF or both IL-2 and GM-CSF combined, 2 pmol each).

(A) PBMC in cultured with cytokines for seven days were labeled with conjugated

antibodies specific for cell surface markers CD3, CD4, CDS, CD56 and cell type number was quantified by flow cytometry. (B) The percentage of NK cells from three different

donors was determined in the PBMC fraction cultured with equimolar concentrations of

cytokines for seven days. Cells were labeled with conjugated antibodies specific for cell

surface markers CD3 and CD56 and the percentage of NK cells was quantified by flow

cytometry. (C) NK cells pre-staining with CFSE were incubated with cytokines for four

days. The decrease of CFSE intensity as indicative of cell proliferation was assessed by flow cytometry. The percentage of the parent peak is indicated for each figure. NK cells

cultured with cytokines for seven days were staining with Annexin V and PI and the

percentage of apoptotic cells in each condition was determined by flow cytometry. These

results are representative of three independent experiments performed with blood from three healthy volunteers. Significant differences between hGIFT2 and controls in all figures are denoted by asterisks: *,p<0.05.

Ill Figure 10

A 4 DM • CD4 • donor I 3500 •CD8 •donor 2 5000 • NKT y 30 •donor 3 2500 * • Jvk 2000 1500 • 1000 500 0 h* In llkli

IL-2 GM-CSF IL-2 + GM-CSF HGIFT2 96% 63% 96% 64% 94% £ : H a ; a 1 ! 5 - _.^v • F TT.T- M plffiWiTVYYy-/ ...>T 200 400 600 800 1000 0 200 400 GOO 800 1000 0 200 400 &D0 mI 1000 A C 200 4 DO &0Q 800L 1003 0 200 400 600 800I 10QC CFSE CFSE CFSE CFSE CFSE D

Control RPMI IL-2 GM-CSF TL-2 + GM-CSF HGIFT2 3- •1 ] -I

12% 10% 21% 8% 2% ?on 4nn eoo 800 1000 0 20C 400 6C0 800 1000 0 200 430 <00 SOD 1000 o ?oo 400 firm eon mni 2on 4iin ismv 8(io ifinc 0 200 400 &DC m 1000 Annexin VF(TC -ftVFITt: Anr^Kn V FITC Anna^n YFITC A'ins>ln FITC Anrif>:-

112 Figure 11: hGIFT2 induces NK cell activation

NK cells cultured with 2 pmol of cytokines (hGIFT2 or cytokine controls) were analyzed for the expression of NK cell activation markers and the production cytokines and

chemokines. (A), CD69, the early activation marker; CD 107a, a marker of degranulation

and CD 122, the (3 subunit of IL-2 receptor. (B) Intracellular staining for IFNy was

performed on NK cells cultured for 6 hours with cytokines and treated with Brefaldin A,

IFNy expression was measured by flow cytometry using a conjugated anti-IFNy antibody

and IFNy production from NK cells stimulated with cytokines for three days was

quantified by ELISA. (C) NK cell derived TNFa production was quantified by ELISA,

and no significant differences was observed, whereas RANTES production was

significantly increased by hGIFT2. Results are shown as mean + SEM (n=3). Significant

differences between hGIFT2 and controls in all figures are denoted by asterisks: *, p<0.05. These results are representative of three independent experiments performed with blood from three healthy volunteers.

113 Figure 11

TNFa RP\1[ IL-2 GM-CSF 1L-2 + GM-CSF hGIFT2 c T h Si 600 113 167 153 162 231 500 J 1J 1 i •A 1 • 1 400 A ' .V. O - o o - fli A 2" 300 1 tu 0 KOS 5 MO (I 1JKW 1 mm ? 218 ccse-PE CCM-F-= CM646 Qii^E CI69-PE Z 200 C06!) Kill »i i W; 0 I 137 14« 1+2 15D 21)2 i II s . . 1; f j j 1: SCO - /y A RANTES L A £k— 700 4 0 1 DO! 0 1D0D 0 1000 0 1C0C 0 1000 CDWaFITS CDimFITC C&IWftFITt CD107sFJTC 0)1G?$FITC 600 j m CD 107a I j 3 430 - 253 "i 154 261 225 268 293 | m. - 149 s i : 1 • i 200 - • T V V A L.'"A H i v i \ _#/. V 100 - o- T o- 1D0D 0 1000 0 100] 0 1DOO 1000 0 i CM22-PE 0>IZZ-P= 012£-FE OIZZ-FE CCMH-PE 1L-Z L.-3HM-W MihTZ CDL22

B 30000 - * nm 25003 - MFI liGIFH |1500C IL-2+GM-CSI' I GM-CSF g 10000 - soon - Kr VI 0 • IFNv EM GM-CSF miCM-C$r tlGTITZ

114 Figure 12: hGIFT2 similarly activate CD56dlm and CD56bright cells

NK cells were cultured with 2 pmol of IL2 or hGIFT2 for three days and labelled with

CD56 antibody, both population CD56dim and CD56bnght were identified in a linear

scatter. hGIFT2 similarly stimulated both CD56bnght and CD56dim subsets of NK cells, which display bigger size and more granularity.

115 Figure 12

CD56Jil

CD56brlshl

0 200 400 600 800 1000 hGIFT2 CD56-PE*Cy7

FSC-H

116 3.4.5 hGIFT2 upregulates activating receptor expression, promotes NK cell

maturation and significant greater NK cell cytotoxicity

Spontaneous cytotoxic activity is mainly triggered by the activating receptors natural

cytotoxicity receptors (NKp30, NKp44, NKp46), NKG2D and leukocyte adhesion

molecule DNAX accessory molecule 1 (DNAM-1 (CD226) 307'308. hGIFT2-stimulated

NK cells upregulate NKp44, NKp46 and DNAM-1 (CD226) expression (Figure 13A)

but not NKG2D (Figure 14A) and NKp30 (data not shown) expression. In addition,

hGIFT2 downregulates the expression of the receptor for stem cell factor c-kit (CD117)

and upregulates the expression of integrin CD lib (macrophage antigen-1 [Mac-1];

Figure 13B). hGIFT2 downregulates the expression of the inhibitory receptor CD43 and

does not alter the expression level of the inhibitory receptors KIR (Figure 14BC). This

phenotype indicates that hGIFT2 stimulated NK cells undergo a maturation process, which correlated with significant greater cytotoxicity than cytokine controls against both

NK-resistant Daudi cells and NK-sensitive K562 and U266 tumor cells. As previously

reported 312, we also observed that GM-CSF significantly suppresses NK cell cytotoxicity

(Figure 13C). After seven days in culture with cytokines, a 30% decline of cytotoxicity was observed in hGIFT2 stimulated NK cells, and a 60% reduction of killing ability in

IL-2 stimulated NK cells (Figure 15).

3.4.6 hGIFT2 induces hyperactivation of Jak/Stat signaling pathway in NK cells

NK cells express components of both IL-2 and GM-CSF receptors 313. The intracellular

signaling of IL-2R occurs through the (3 chain and the y chain (JAK3/STAT5), whilst the

GM-CSF receptor signals through its (3 chain (JAK2/STAT5). In order to decipher the

molecular mechanism underpinning hGIFT2 s effect on NK cells, we analyzed the

117 activation status of JAK kinases and STAT transcription factors downstream of IL-2 and

GM-CSF receptors, respectively. We observed that hGIFT2 induces robust hyperphosphorylation of JAKl and JAK2 and this is associated with greater activation of

STAT1, STAT3 and STATS when compared with controls. In contrast, GM-CSF completely abrogates STATS phosphorylation and inhibits IL-2 mediated STATS activation (Figure 16AB). We also measured the activation status of other signaling pathways downstream of IL-2 and GM-CSF receptors, such as MKK/ERK and AKT pathways 72,314. AKT pathway was more activated by hGIFT2 than cytokine controls

(Figure 16C), which supports the observation that hGIFT2 enhances NK survival. In contrast, the activation status of NF-kB and MKK/ERK did not show significant differences in hGIFT2 stimulated NK cells compared to equimolar concentrations of cytokine controls (data not shown).

118 Figure 13: hGIFT2 upregulates NK cell activating receptors, promotes NK cell maturation and greater NK cell cytotoxicity than cytokine controls

NK cells cultured with 2 pmol of cytokines for three days were labelled with conjugated

antibodies specific for (A) NK cell activating receptors (NKp44, NKp46 and DNAM-1

[CD226]). (B) NK cell maturation markers (integrin, CD lib and receptor for stem cell factor c-kit, CD 117). (C) NK cells previously activated with 2 pmol of cytokines for three days were cocultured with target cells (Daudi, K562 and U266) for four hours and the cytotoxicity activity of NK cells was measured by quantifying the levels of lactate

dehydrogenase (LDH) released from the cytosol of damaged or lysed cells. Maximum

LDH level was measured from Triton X-100 treated target cells (Pos Control).

Significant differences between hGIFT2 and controls in all figures are indicated. The

results in all the figures are representative of three independent experiments performed with purified NK cells from three healthy donors.

119 Figure 13

Da ud i P<0.(X)5 I 1

RPMI IL-2 GM-CSF IL-2+GM-CSF hGIFT

232 246 232 240 273 ll l| 1 ll -A A .A % A ,M o (1>226.F,E C022t> PE 10C 00226

117 149 129 142 241 A., ax !'<(). ft? NKp44 PI FE 1L'"'"' " NK g44 Ft NKp44 PE i> h " ^ F1 10(1 NKp44 90 K562 e 80 477 •3 70 267 362 288 357 | 60 % 50 f s NKpJi> ^ 20 10 0 RPMI IL-2 GM-CSF

244 249 240 256 27(1 I: :^v •y\ -A p=— 1000 0 1000 0 100 0 tooo 0 1000 aCDI17PE CD117 re C6117P6 CD 157fE tt>H7P6 cm 17 *v///

120 Figure 14: hGIFT2 does not affect the NK cell inhibitory receptor expression (CD43 and KIR) and the NK activating receptor NKG2D

NK cells cultured with 2 pmol of cytokines for three days were labelled with conjugated

antibodies specific for (A) NK activating receptor, NKG2D. (B) NK inhibitory receptor,

CD43 (C) NK inhibitory receptor, KIR.

121 Figure 14

°^7Tii i| 11 m| i ' o 111,11 i ipTnpwipDWf 0 1000 0 1000 0 1000 0 1# 0 1000 NKG2D PE MKG2D PE NKG20 PE NK520 PE NK62C* PE NKG2B

B RPMI IL-2 GM-CSF IL-2 + GM-CSF hGIFT: 51

«r W 0 1000 0 1000 0 1000 CM3APC CM3APC CM3APC CMS APC CM3APC CD43

261

'i'ffi in 0 1000 0 1000 KIRFITC KTRFITC KIRFITC KIRFITC KIRFITC KIR

122 Figure 15: hGIFT2 promotes greater NK cell cytotoxicity than cytokine controls

over time

NK cells previously activated with 2 pmol of cytokines for three or seven days were

cocultured with target cells (U266) for four hours and the cytotoxicity activity of NK

cells was measured by quantifying the levels of lactate dehydrogenase (LDH) released from the cytosol of damaged or lysed cells. Significant differences are denoted by

asterisks: *,p<0.05.

123 Figure 15

U266 Figure 16: hGIFT2 induces a hyperactivation of JAK/STAT pathway downstream

of GM-CSF and IL-2 receptors in NK cells

5xl06 NK cells were stimulated for 20 mins with 5 pmols of cytokines and cell lysates were probed for (A) phosphorylated JAK1, JAK2 and their respective anti full length

protein antibodies. (B) Phosphorylated STAT1, STAT3 and STATS, as well as total

STAT1, STAT3 and STATS antibodies. (C) Phosphorylated AKT and total AKT

antibody. Antibodies anti full length proteins and a-tubulin were used as loading

controls. Optical density quantifications of immunoblots are shown in each figure. These

results are representative of three independent experiments performed with NK cells from three healthy volunteers.

125 Figure 16

P-Jak2 P-STAT5 — •

Tolal AKT J;ik2 r-STAT5 a 0.8 1.8 "3 0.6 0.08 1.5 2 0.4 1.2 4 0.06 0.9 a 0.2 h 0.04 0.6 Till a o.oz 0.3 i 0 5 o I m P-STAT:* P-Jakl T-STAT3 Juki

3 0.4 7 0.3 r 0.2 i 0.1 Z o I P-STATI

TOIAL-STAT

et-mbulin

126 3.5 DISCUSSION

The clinical effectiveness of GM-CSF and IL-2 combination for the treatment of cancer is controversial. A phase II clinical study of moderate dose IL-2 and GM-CSF in patients with metastatic or unresectable renal carcinoma (RCC) fails to manifest the synergistic therapeutic effect of these two cytokines 315. Indeed patients receiving GM-CSF and IL-2 treatment display an impaired LAK activity compared to IL-2 alone and specifically GM-

CSF has been shown to downregulate certain aspects of innate immune response such as

NK cell cytotoxicity 312. In the present study we have shown that hGIFT2 displays

unheralded novel properties that overcomes GM-CSF mediated NK cell suppression and

promotes potent NK cell activation. hGIFT2 upregulates the expression of the early

activation marker CD69 and promotes greater NK cell degranulation based on acquired

surface expression of LAMP-1 (CD 107a). The potent immune stimulatory effects of

hGIFT2 on NK cells are evidenced by a substantial upregulation of NK activating

receptors NKp44 and NKp46. The human triggering receptors responsible for NK cell

cytotoxicity include the natural cytotoxicity receptors (NCRs) and NKG2D. NCRs

belong to the Ig superfamily and include NKp46 and NKp30, which are expressed by

both resting and activated NK cells, and NKp44, which is expressed only by activated

NK cells 285'316'317 Although, the cellular ligands on tumor cells for NCRs are currently

unknown, NCR have been identified as crucial receptors for target cell recognition and induction of NK cell cytotoxicity toward a wide range of cancer cells. Substantial

evidence indicates that there is a direct correlation between NCR surface densities and the ability of NK cells to kill different tumor target cells 318. hGIFT2 slightly upregulates the expression of DNAM-1 (CD226), which is a transmembrane glycoprotein that

mediates lymphocyte adhesion and signaling. This activating receptor recognizes PVR

127 (CD155) and Nectin-2 (CD 112), two members of the lectin family highly expressed on

carcinomas, melanomas and some hematopoietic cell lines 129,319. Consequently, hGIFT2

stimulated NK promote significant cytotoxic activity against all target tumor cells

analyzed. Interesting, hGIFT2 treated NK cells were able to lyse "NK-resistant" Daudi

cells (Burkitt's lymphoma). These tumor cells are negative for MHC class I expression,

but expresses MHC class II, which correlates with the increase resistant to NK-mediated lysis 320. As expected, hGIFT2-stimulated NK cells also promote greater cell lysis of NK- sensitive K562 and U266 cell lines. K562 leukaemia cells (CML) are negative for both

MHC class I and II expression, whereas U266 cells (myeloma), although are sensitive to

NK-mediated cell cytotoxicity express high levels of MHC class I 321, which supports

"the missing self' hypothesis that NK cells kill certain targets because they fail to express

adequate levels of MHC gene products.

NK cells stimulated with IL-2 for seven days display a remarkable reduction of

cytotoxicity activity against myeloma cells. According to previous reports that IL2 rir)r) QOQ induces NK cells apoptosis in long term culture ' . We suggest that the reduction of

NK cell cytotoxicity over time is due to a decrease of IL-2 stimulated NK viability. On the other hand, hGIFT2 does not induce NK cell proliferation compared to equimolar

concentration of IL-2, but rather protects NK cells from apoptosis, which result in a net increase of NK cell number over time. In addition, hGIFT2 prompts NK cells to secrete

substantial amounts of IFNy (thirty seven fold greater than equimolar concentrations of

IL-2) as well as RANTES. The extremely high production of IFNy may restrain the

proliferation of NK cells and at the same time prevent NK cell apoptosis, since IFNy has

been reported to induce cell cycle arrest in several cell types 312. In lymphocyte activated

128 killer (LAK) cells, IFNy in synergy with IL-2 acts as inhibitor for LAK cell proliferation but not differentiation 324 We also found that distinct from IL-2, hGIFT2 induces a NK

cell maturation process characterized by the downregulation of the expression of the

receptor for stem cell factor c-kit (CD 117), which is expressed by NK cell precursors but is largely diminished on matured NK cells 325'326. CD lib integrin upregulation constitute

another marker of NK cell maturation 327 As molecular mechanisms underpinning

hGIFT2 effect on effector cells, hGIFT2 not only upregulates IL-2R|3 (CD122)

expression but also induces a hyperactivation of JAK1 and JAK2 kinases associated to (3

chains of both IL-2 and GM-CSF receptors, which lead to a robust phosphorylation of

STAT1, STAT3 and STATS transcription factors. By comparing the levels of STATS

phosphorylation in hGIFT2 stimulated NK cells with equimolar concentration of GM-

CSF, we observed that hGIFT2 overcomes GM-CSF mediated suppressive signals on NK

cells. These results suggest that hGIFT2 has higher avidity for NK cells than single

cytokine control maybe due to a bivalent binding of hGIFT2 to both receptors expressed

on NK cells. Interestingly, hGIFT2 fusokine promotes greater activation of (PI3K)/Akt in

NK cells. Activated Akt in turn phosphorylates a number of downstream target genes that

prevent apoptosis and promote cell survival, which could be the mechanism used by

hGIFT2 to protect NK cell from apoptosis and to enhance survival.

Several clinical trials using ex vivo IL-2 expanded and activated autologous NK cells

have shown apparent limited effectiveness in the therapy of cancer, including melanoma,

renal carcinoma, lung carcinoma, ovarian and brain cancer 328. The infusion of high doses

of IL-2 not only is associated with severe toxic side effects, but also sensitizes NK cells to apoptosis when they are in contact with the vascular endothelium, which may cause a

129 decrease of NK cell migration to the tumor 329' 330. Interestingly, allogeneic NK cells

characterized by a KIR/KIR-ligand incompatibility display more cytotoxicity against

solid tumors and leukemia 331' 332. Remarkably, patients with AML treated with

alloreactive NK cells benefit from higher rates of engraftment, survival and reduced incidence of GvHD 333. The low rate of GvHD is a consequence of the inefficient priming

of alloreactive donor T cells consequent to NK cell mediated killing of recipient antigen-

presenting cells (APCs) 334 However, alloreactive NK cells arising from hematopoietic

stem cell transplantation (HSC) require several weeks to mature post-transplantation, which may contribute to leukemia relapse 335. Therefore, we may speculate that the infusion of mature NK cells would more effectively prevent leukemic relapse, promote engraftment by eliminating patient lympho-hematopoietic cells and reduce GvHD by lysing patient APCs. Therefore, biochemical NK priming which enhances their survival,

effector function and maturation would be desirable in the setting of cancer cell immunotherapy - features afforded by NK cell treatment with hGIFT2.

In summary, NK cells are the only lymphoid cells that express components of both IL-2

and GM-CSF receptors 313. Therefore, the selective effect of hGIFT2 on NK cells suggest

that hGIFT2 binds to these cytokine receptors in an atypical fashion, which triggers

aberrant signals downstream of both IL-2 and GM-CSF receptors leading to the

hyperphosphorylation of receptor-associated JAK/STAT proteins. Consequently,

hyperactivated NK cells produce large amount of IFNy and RANTES and overexpress

NCRs involved in NK cell cytotoxicity whilst contemporaneously acquiring features of

mature and apoptosis-resistant cells. The use of GIFT2-primed autologous NK cells may therefore be of interest in cell therapy of cancer.

130 3.6 ACKNOWLEDGMENTS

This work was supported by a Canadian Institute for Health Research operating grant

MOP-15017. CP is recipient of Montreal Centre for Experimental Therapeutics in

Cancer Scholarship and US Army Graduate study Scholarship and JG is a Fonds de recherche en sante du Quebec chercheur-boursier senior. We thank Dr. Manaf

Bouchentouf, Drs N. Eliopoulos and Patrick Williams for technical advice and materials.

131 CHAPTER 4

Title: Novel Transforming Growth Factor |3-antagonist inhibits tumor growth and

angiogenesis by inducing Interleukin-2 receptor-driven STAT1 activation.

Reference: Claudia Penafuerte, Norma Bautista-Lopez, Manaf Bouchentouf, Elena

Birman, Kathy Forner, Jacques Galipeau. Novel Transforming Growth Factor (3-

antagonist inhibits tumor growth and angiogenesis by inducing Interleukin-2 receptor-

driven STAT1 activation. Manuscript submitted to Journal of Immunology (10-03816-

FLR).

Preface to Chapter 4: We have previously demonstrated that the human ortholog of

GIFT2 also displays unheralded novel biological properties that overcomes GM-CSF

mediated NK cell suppression and promotes potent NK cell activation in vitro. However, we do not discard the possibility that potent tumor-derived suppressive factors such as

TGF|3 may antagonize or reduce human GIFT2-mediated immunostimulatory properties in vivo. To antagonize TGF|3 dependent effects accoupled with a pro-inflammatory immune stimulus, we have generated a new chimeric protein borne of the Fusion of IL-2

and the Soluble extracellular domain of TGF-J3 receptor II (aka FIST).

132 CHAPTER 4: Novel TGFP-antagonist inhibits tumor growth in a mouse model of

melanoma by inducing angiostatic-NK cells.

4.1 ABSTRACT

Carcinoma derived transforming growth factor (3 (TGF|3) acts as a potent pro-oncogenic factor and suppresses antitumor immunity. To antagonize TGF|3-mediated effects in tandem with a pro-inflammatory immune stimulus, we generated a chimeric protein borne of the Fusion of IL-2 and the Soluble extracellular domain of TGF-J3 receptor II

(FIST). FIST acts as a decoy receptor trapping active TGF|3 in solution and interacts with

IL-2 responsive lymphoid cells, inducing a distinctive hyperactivation of STAT1

downstream of IL-2 Receptor(R) which in turn promotes SMAD7 overexpression.

Consequently, FIST-stimulated lymphoid cells are resistant to TGF|3-mediated

suppression and produce significant amounts of pro-inflammatory cytokines. STAT1

hyperactivation further induces significant secretion of angiostatic CXCL10. Moreover,

FIST upregulates T-bet expression in NK cells promoting a potent Thl-mediated anti tumor response. As a result, FIST stimulation completely inhibits pancreatic cancer

(PANC02) and melanoma (B16) tumor growth in immunocompetent C57BL/6 mice. In

addition, melanoma cells expressing FIST fail to form tumors in CDS % CD4" ", B

(|xMT) deficient and beige mice, but not in NOD-SCID and Rag2/yc knockout mice,

consistent with the pivotal role of FIST-responsive, cancer killing NK cells in vivo. In

summary, FIST constitutes a novel strategy of treating cancer that targets both the host's

angiogenic and innate immune response to malignant cells.

133 4.2 INTRODUCTION

TGF|3 has been considered as a tumor suppressor factor because it promotes cell growth inhibition, apoptosis and differentiation. However, an extensive number of studies attest to the fact that TGF|3 acts as a potent tumor promoter in established carcinoma 129'336. As tumor progress, cancer derived TGF|3 drives malignant progression by constitutively inducing epithelial to mesenchymal transition (EMT), tumor associated angiogenesis and

suppressing tumor immunity, the sum of whose effects are to promote tumor growth and

metastasis 128' 129'312'337'338. There are three different TGF|3 isoforms (TGF|3 1, 2, 3), which are encoded by different genes, but all function through the same receptor

signalling systems 115. Among them, TGFJ31 is the most frequently overexpressed in

carcinomas 339 In mammalian cells TGF|3 signal through heteromeric complexes of type

I (activin receptor-like kinases, ALK, also known as T|3RI) and type II (TJ3RII) transmembrane serine/threonine kinase receptors 340. Upon ligand binding, the

constitutively active TJ3RII phosphorylates the type I receptor (ALKs), which propagates

signals into the cells by phophorylating receptor regulated (R-)SMADs (i. e. SMAD2 and

SMAD3). Phophorylated R-SMADs form an heteromeric complex with the co-mediator

SMAD4 122, and translocate to the nucleus where this complex cooperate with transcription activators and repressors to regulate the expression of TGF|3 target genes

312. Inhibitory SMADs (i-SMADs, SMAD6 and SMAD7) act as negative regulator of

TGF|3 signalling pathway by preventing the interaction of TGF|3 receptor complex with

R-SMADs 199' 202'341. Moreover, inhibitory SMADs recruit phosphatases and ubiquitin ligases to the activated receptors promoting their dephosphorylation and degradation 342.

As pro-angiogenic factors, TGF|3 family members play an essential role in

134 vasculogenesis and tumor angiogenesis 199' 202' 343. TGF|3 regulate the expression of various extracellular matrix components that play a key role in both the initiation and

resolution phase of angiogenesis 193. Consistent with this notion, we hypothesize that the blockade of TGF|3 signalling combined with cytokine-driven immune activation will

couple anti angiogenesis to an effective immune antitumor response, resulting in potent

anticancer properties. Interleukin-2 (IL-2) has a well documented 344 clinical track record for driving an anticancer innate immune response and was FDA-approved in 1998 for immunotherapy of melanoma 345. We have found in a pre-clinical model of B16

melanoma that IL-2 driven anticancer activity is severely curtailed by tumor-derived

TGF|3 and that blockade of TGF|3 activity markedly enhances immunotherapy 346. We therefore speculated that IL-2 could serve as a rational cytokine partner to blockade of

TGF|3 signalling as part of a "sword and shield" pharmaceutical strategy. To test this

concept, we created a novel pharmaceutical comprised of IL-2 fused to the soluble TGF|3

receptor II (TJ3RII).

4.3 MATERIALS AND METHODS

4.3.1 Mice

B-cell deficient (|iMT), NK, CD4, CDS T cell, IFNy KO mice, beige mice (C57BL/6J-

bg), C57B1/6, Balb/c immunocompetent, CXCR3 KO mice (Jackson Laboratory, Bar

Harbor, ME) and Rag2/yc KO (Taconic New York's Hudson Valley) were female 6 to 8 weeks old. STAT1 KO mice were generously donated by Joan Durbin 347

4.3.2 FIST fusokine design, expression and functionality The mouse IL-2 cDNA (Invivogen San Diego, CA) was modified by removing the 3'

nucleotide encoding the STOP codon and subsequently cloned in frame with TGF|3

receptor II ectodomain cDNA (Invivogen San Diego, CA) to generate the cDNA for IL-

2/sT|3RII fusion protein (aka FIST). The stop codon and poly A was added at the C- terminal region. For the proliferation assay, 104 CTLL-2 cells per well were plated in a

96-well plate and treated with 5 pmol of FIST or controls for 72 hours. Cell proliferation was assessed by MTT assay. FIST and TGF|3 complexes were precipitated using TGFJ31

specific antibodies bound to protein G beads and the precipitated FIST protein was

detected by western blotting with IL-2 specific antibodies. Infectious retroparticles

encoding FIST were generated with 293-GP2 packaging cells (Clontech, Mountain View,

CA) and used to genetically modify C57Bl/6-derived B16F0 melanoma cells. CTLL-2

and B16F0 cell lines were maintained in RPMI supplemented with T-STIM with Con A (rat

IL-2 culture supplement from Becton Dickinson) and 10% fetal bovine serum and DMEM

medium (Wisent Technologies, Rocklin, CA) supplemented with 10% fetal bovine serum

and 50 U/ml Pen/Strep (Wisent Technologies).

4.3.3 Immune cell isolation and cytokine production

Enriched T, B and NK cell populations were obtained from splenocytes of immunocompetent or STAT1 knockout mice by magnetic separation according to the

manufacturer's recommendation (Stem Cell Technology, Vancouver). The purity of each

population was assessed by flow cytometry (for B cells 94%, NK cells 96% and T cells

96%) using specific conjugated antibodies for cell markers (BD Biosciences, San Diego,

CA). Cytokine concentrations were quantified by enzyme-linked immunosorbent assay

(ELISA) kits (eBiosciences, San Diego, CA and R&D, MN).

136 4.3.4 Cell signaling and receptor expression

5xl06 CTLL-2 were cultured with 5 pmol of FIST or controls before being lysed and

probed by western blot with rabbit phospho-specific and total SMAD2, SMAD3, STAT1,

STAT3, STATS (Cell Signalling Technology, Danvers, MA); a-tubulin and SMAD7

antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). For T-bet expression, cytokine

stimulated NK cells were treated with Brefaldin A for four hours, fixed, permeabilized

and stained with T-bet conjugated antibody or isotype control for 1 hour. For CXCR3 and

IL-2RJ3 expression, stimulated NK cells were incubated with FcR III/II blocking antibody for 30 mins and labelled with conjugated antibodies specific for CXCR3 and IL-2RJ3 for

1 hour. The mean fluorescence intensity was measured by flow cytometry and the data were analyzed using Cellquest software (Becton & Dickinson).

4.3.5 Immune cell infiltration in the tumor site

To analyze the immune cell types infiltrated in the tumor site, 106 genetically modified

B16 cells expressing FIST or controls were mixed with 500 |iL Matrigel (BD

Biosciences) at 4°C and injected subcutaneously in immunocompetent C57B1/6 mice.

Implants were surgically removed 6 days after implantation and enzymatically

dissociated as reported previously 348. Infiltrated cells were collected and incubated with

anti-FcR III/II antibody for 30 minutes and with cell type specific or isotypic control

antibodies for 1 hour at 4°C. The expression of surface markers was determined as

previously described.

4.3.6 Endothelial cell isolation, in vitro angiogenesis, proliferation and migration assays

137 Livers of immunocompetent mice were digested with collagenase for 40 minutes at 37°C to prepare a cell suspension. Cells were labelled with PE-conjugated CD31 specific

antibodies to isolate endothelial cells (EC) by positive selection of PE-labelled cells

3 (Stemcell technologies). For the in vitro capillary-like structure formation assay, 5x10

EC were seeded per well in a ninety-six-well plate previously coated with a type IV

collagen matrix (Chemicon, Billerica, MA). EC were incubated at 37°C for 24 hours in the presence of conditioned media (CM) by NK cells previously stimulated with 5 pmol

of FIST or control for 72 hours (CM NK cells). A numerical score was assigned to each

condition according to the degree of angiogenesis progression based on the number and

size of polygons formed, capillary thickness, cell alignment and fusion, as previously

reported (349). For the proliferation assay, 2xl03 cells were cultured in a 0.1% gelatin

coated 96 well plate for 6 days in a low serum EC media supplemented with endothelial

cell growth supplement (ECGS) and epidermal growth factor (EGF) (BD Biosciences) in the presence of CM by stimulated NK cells. Cell number was determined using the

ViaLight Plus kit (Lonza, Rockland, ME). For migration assay, 24-well cell invasion

assay (Millipore, Billerica, MA) was used to examine the migration of endothelial cells

(EC) according to the manufacturer's protocol. Briefly, this modified Boy den chamber is

composed of a well (lower compartment) containing an insert (upper compartment).

8xl03 EC were suspended in 0.5 ml of serum-free medium and loaded into upper

chambers of the transwell. Lower chambers were loaded with 1% RPMI medium in

presence or absence of FIST, IL-2, sT|3RII or IL-2 plus sT(3RII-stimulated NK cell

conditioned medium (NK cells were stimulated with 5 pmol of FIST or control).

Following a 24 hour incubation period at 37°C, cells remaining on the upper surface of the insert (non-migrated cells) were removed gently and placed into a sterile 24-well plate containing a cell detachment solution for 30 min at 37°C. Dislodged cells were treated with a lysis buffer and incubated with the CyQuant GR dye for 15 min at room temperature. The mixture was transferred to a 96-well plate and fluorescence was read at

480 nm. Invaded cell number was determined by running a fluorescent versus cell dose

curve using 8000, 4000, 2000 EC (n=3). To determine FIST angiostatic properties in

vivo, 106 genetically modified B16 cells expressing FIST or controls were mixed with

500 |iL Matrigel (BD Biosciences) at 4°C and injected subcutaneously in immunocompetent C57B1/6 mice. Implants were surgically removed 20 days later and

histological sections of the tumors were labeled with red fluorescence CD31 antibodies to

detect the formation of tumor-derived blood vessels.

4.3.7 Statistical analysis

We used the two-tailed unpaired Student's t test for two experimental group's

comparison, Dunnett's multiple comparisons to compare three or more test groups, and log rank test to compare two survival distributions.

4.4 RESULTS

4.4.1 Generation and characterization of murine IL-2/sT(3RII fusion protein: FIST

We generated a plasmid construct encoding for the fusion of IL-2 and the ectodomain of

TJ3RII splicing variant B (sTBRIIB, from aa Leu 9 to Asp 184). The fusion transgene

cDNA encodes for a single polypeptide chain of 328 aa's (Figure 17A) that migrates as

an approximately 55KDa protein in SDS-PAGE under reducing conditions (Figure 17B,

C). We observed in co-immunoprecipitation assays that FIST precipitated active TGFJ31

(Figure 17D), indicating that FIST acts as decoy receptor trapping active TGF|3. As

139 predicted, IL-2 as part of the fusion protein preserves the ability to recognize and bind its

specific receptor and induce the proliferation of CTLL-2 cells (Figure 17E). We analyzed

the activation status of transcription factors downstream of TJ3RII (SMAD3 and SMAD2)

and observed that FIST exerts a dominant negative effect on TGF|3 canonical pathway

not only by impairing the phosphorylation of SMAD2 and SMAD3 but also by inducing

SMAD7 expression in CTLL-2 (Figure 17F). We also examined the activation of transcription factors downstream of IL-2 receptor. FIST induces similar STATS and

STAT3 phosphorylation levels compared to equimolar concentrations of controls.

Surprisingly, we observed a distinctive STAT1 hyperactivation (Figure 17G). We also

compared the activation levels of other signalling pathways downstream of IL-2 receptor

such as MAPK and PI3K/AKT pathways and no differences were observed between

FIST and equimolar concentrations of controls (data not shown). We investigated whether FIST induces SMAD7 expression via STAT1 activation by using lymphocytes from STAT1 knockout mice, and we significantly abrogated SMAD7 expression

compared to lymphocytes from immunocompetent mice, yet observed a preserved inhibition of SMAD2 phosphorylation compared to the controls (Figure 17H).

4.4.2 FIST desensitizes immune cells to TGF0 mediated suppression

We tested whether FIST can override TGF(3-mediated suppression of lymphocyte

activation. We found that FIST on its own primes 5xl06 unfractionated splenocytes to

produce a significant increase (>35%) of IFNy compared to equimolar concentration of

IL-2 in 96 hours. In contrast to IL-2 alone, this effect was not suppressed in the presence

of 0.5 ng/ml of active TGFJ31 (Figure ISA). In addition, we observed that FIST induces

c-Myc upregulation, another gene suppressed by active TGF|3 (Figure 19). In an attempt

140 to define the immune cell type responsive to FIST; lxlO6 NK cells, T cells or B cells were cultured with 5 pmols of FIST or equimolar concentration of controls for 72 hours

and determined by ELISA that FIST-stimulated T, B and NK produce significant higher

amounts (> 90% increase) of IFNy, TNFa and GM-CSF than controls (Figure 18B-F).

4.4.3 FIST induces a robust immune bystander effect and inhibits tumor growth in vivo

We have previously demonstrated that B16 melanoma secrete large amounts of TGF|3 that results in significant resistance to melanoma-targeted immunotherapy 346. We utilized this TGF|3 biased tumor model system to test the activity of FIST in a

suppressive cancer setting. We found that FIST and IL-2 secreted by a polyclonal

population of genetically modified B16 cells (B16 FIST and B16 IL-2 secrete 10 pmols

per million cells in 24 hours) are equally effective in inducing a potent anti-tumor

response. Mice injected subcutaneously with 5xl05 B16 FIST or IL-2 cells do not

develop tumors. However, a cohort of mice injected with 5xl05 B16 IL-2 cells mixed with 5xl05 null B16 cells developed tumors at a rate comparable to the control group implanted with 5xl05 null B16 cells. In contrast, FIST mediated bystander effect protects

60% of the injected mice with mixed cell (Figure 20A). After 80 days, mice previously implanted with B16 FIST or B16 IL-2 cells were challenged with lxlO6 null B16 cells in the contralateral flank and the percentage of survival was monitored over time. Fifty

percent of mice previously treated with FIST rejected null B16 cells in contrast to the failure of IL-2 in conferring protective immunity (Figure 20B).

141 Figure 17: Generation and characterization of FIST

(A) Amino acid sequence of FIST. (B) Western blotting of conditioned media (CM) of

293T cells transfected with vectors encoding FIST or controls and protein detection with

sTBRII especific antibody. (C) FIST protein detection by western blotting with IL-2

specific antibody. (D) FIST and active TGFJ31 complexes precipitated with TGFJ31

specific antibodies bound protein G beads and detected by western blotting with IL-2

specific antibodies. (E) The bioactivity of IL-2 as part of FIST determined by in vitro

proliferation assay based on 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium (MTT)

uptake and cell number was quantified by an optical density versus cell dose curve (n=3

per group; data are shown as means + s.d). (F) Western blotting for phospho-specific

and total SMAD2, SMAD3, and SMAD7 proteins from cell lysates of 5xl06 CTLL-2

cells previously stimulated with 5 pmols of FIST or controls for 20 minutes for SMAD2

and SMAD3 or 2 hours for SMAD7. (G) Western blotting for phospho-specific and total

STAT1, STAT3, and STAT5 proteins from cell lysates of 5xl06 CTLL-2 cells previously

stimulated with 5 pmols of FIST or controls for 20 minutes. (H) Similar experiments as

Figure IF were performed with unfractionated splenocytes from STAT1 knockout or the

companion strain Balb/c mice as control.

142 Figure 17

RAQASHSAGMYSMQLASCVTLTl.VlXV NSAPTSS6T5537AEAQQQQOQOQOQ QQHLEGLLMDLQELLSRMENYHNLKLP RMLTF KFYLPKQATELKDLQC LECELG IL-2 P LRHVLDLTOSKSFQ LEO AENFISNIRV TWKLKGSDNTFECQFDDESATVVOFL P-5MAD3 RRWIAFC05IISTAS P.SMAD2 1 t 9 F-SMAD7 J RIASTtPPHVPKSDVE J.6MA02J3 MEAQKDAS IML.SC NRTIHPLKHFNSDV MASDNGGAVKLPQLCKFCDVRL5TC D NQ KSCMSNCSl TAIC EKPHEVCVAWVR iTGFpfill KW DKNITLETVCHDPKLTYHGFTLE DAA SPKCVMKEKKRAGE TFF JlilCACH M E EC NDYIIFSE EYTTSSPDL1.LVHQVTDPL P-STAT1

TtSMI

366 P-STAT3 60KD- Tolal-STAT3 4 7KD- P.&TATJ T-SW5

6QKD— rTQfp sT|ien FIST

E" tL-2

hE B i 4 $ I Lll $sr FIST treated mice show normal body weight and behaviour and had normal blood counts

(Table 1). We analyzed FIST mediated recruitment of host-derived lymphocytes as well

as specific immune cells types to matrigel plugs seven days post tumor implantation. We found that FIST induces greater recruitment (>50% increase) of lymphocytes than

equimolar concentration of controls. Specifically, we observed a significant increase of

NK (>56%), MKT (>70%), B (>80%) and CD8+ T (>85%) cells recruited in the tumor

site (Figure 20 C, D).

4.4.4 FIST antitumor effect in immunodeficient mice

To determine the main immune cell type implicated in FIST dependent antitumor

response in vivo, 5xl05 null or B16 FIST cells were injected subcutaneously into an array

of immune defective mice, including: CD4 KO, CDS KO, B-cell deficient (|iMT),

nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, NK-defective

beige mice, and Rag2/yc KO mice and survival was monitored over time. Surprisingly,

B16 FIST cells do not form tumors in CD4 KO, CDS KO, B-cell deficient (|iMT) and

NK-defective beige mice (Figure 21A-D). However, 80% of NOD-SCID mice implanted with B16 FIST cells developed tumors and were sacrificed with a significant delay of two

months compared to the control, whereas Rag2/yc KO mice developed tumors at a rate indistinguishable from control group (Figure 21EF). These results suggest that amongst yc-bearing lymphoid cells, NK cells play an essential role as mediator of FIST anti-tumor

effect. Based on the fact that NK cells from beige mice display severe defect of natural

killing capability, but preserve an intact NK cytokine production 35°, whereas NOD-SCID

mice display defects on both NK cell number and function 312, suggest that FIST anti tumor effect can be mediated by a NK cell-derived soluble factor. Notably, tumors

144 expressing FIST implanted in NOD-SCID display very necrotic appearance suggesting a

defect in angiogenesis as well (data not shown). We investigated the contribution of IFNy in vivo since FIST stimulated NK cells secrete substantial amounts of IFNy. However,

B16 FIST cells also fail to form tumors in IFNy knockout mice (Figure 21G) as well as in

IFNy receptor knockout mice (data not shown). The analysis of the expression profile of

angiogenesis related proteins secreted by FIST stimulated NK cells indicate the presence

of high levels of CXCL10 (IFNy inducible protein, aka IP-10, (Figure 22A). We

measured by ELISA the concentration of CXCL10 in the secretome of NK cells

stimulated by FIST or controls from immunocompetent, Beige, IFNy KO and NOD-SCID

mice and we found a correlation between the levels of IP-10 secreted and the percent of

survival overtime (Figure 21H). In addition, FIST stimulated NK cells from immunocompetent mice secrete significant higher amounts of CXCL9 than control

(Figure 22B).

4.4.5 FIST inhibits tumor-derived angiogenesis

To assess the effect FIST on tumor angiogenesis, lxlO6 B16 FIST cells or controls mixed with Matrigel as described in methods were injected subcutaneously in immunocompetent C57B1/6 mice. Implants were surgically removed 20 days later and

histological sections were labeled with red fluorescence conjugated CD31 antibodies to

detect the formation of tumor-derived blood vessels. In contrast to controls, matrigel

plugs containing B16 FIST cells were devoid of any detectable B16-associated blood vessel formation (Figure 23A). Although we determined that the secretome of FIST-

stimulated NK cells did not decrease the proliferation rate of endothelial cells in vitro

(Figure 23B), it significantly suppresses their migration (Figure 23C) and the formation

145 of capillary like structures in a 3D collagen matrix as indicative of progressive

angiogenesis (Figure 23D).

Figure 18: FIST induces significant Thl cytokine production by activated immune cells

(A) Quantification by ELISA of IFNy produced by 5xl06 unfractionated splenocytes from immunocompetent C57B1/6 mice cultured with 5 pmols of FIST or controls for 96

hours (n=4; data are shown as means + s.d). T, B and NK cells were cultured with 5

pmols of FIST or controls and the supernatant were collected and used to measure the

concentration of IFNy, CXCLIO, TNFa and GM-CSF by enzyme-linked immunosorbent

assay (ELISA). (B) IFNy produced by lxlO6 purified T cells (n=3; data are shown as

means + s.d). (C) and (D) TNFa and IFNy produced by lxlO6 B cells. (n=3; data are

shown as means + s.d). (E) and (F) GM-CSF and IFNy produced by 1X106NK cells (n=3;

data are shown as means + s.d). For each figure, asterisks represent the indicated P values.

146 Figure 18

LYMPHOCYTES

T cells

n d 1 3 n d

rTGFh

sTUFtll RPMI 11-2 iirpil IL2*ST|!RII FIST FIST

IL-2

8 cells 6 Milt 5 ™ T i 32 400 o 3C0

| SCO 7 5 100 r a n d n.d n.d

FLPMI H-2 STI1RII IL2*tT|«ll FIST RPML IL-2 sTURII IL2-'5Timil FIST

NK C*:/S 12 NK cells 180 10 160 s lib 3 Ss • » 1 L, 60 L-7 I V 30 n.d n.d n.d n d n d 0 I RPMI IL-2 sTiifiii IL2»STIMN FIST RPtil IL-Z aTWll IL2«-aTtifi!l FIST

* P<0.« ** P«0 005

147 Figure 19: FIST antagonizes TGF(3 mediated inhibition of c-myc expression

5xl06 CTLL-2 were cultured with 5 pmol of FIST or controls for two hours before being lysed and probed by western blot with rabbit phospho-specific for c-myc (left panel) and

c-myc expression was quantified based on the pixel density of the immunoblot bands

(right panel).

148 Figure 19

149 Figure 20: FIST induces a potent anti-tumor response and immune bystander effect

(A) 5xl05 null or genetically modified B16 cells expressing FIST (B16 FIST cells) or

controls were injected subcutaneously in C57B1/6 immunocompetent mice and the

percent of survival was monitored over time. These results are representative of two independent experiments (n=10 per group; data are shown as means + s.d). (B) Mice

previously implanted with B16 FIST or B16 IL-2 cells were challenged with lxlO6 null

B16 cells and the percentage of survival was monitored over time. (C) Analysis of immune cell recruited in the matrigel plugs. The total lymphocyte number was

determined based on forward and side scattergram (FSC/SSC) pattern for lymphocytes.

(D) The total number of each immune cell type was determined by labeling the infiltrated cells with cell surface specific antibodies (n=3 per group; data are shown as the mean + s.d). For each figure, asterisks represent the indicated P value.

150 eres re K» O

o Survival (%) Survival i%)

f\J O $ S Total number ol lymphocytes {*1D1)

O -1 l>J W U1 o> I I I I 1 I

-rt 8

s E3 % < Si

5 it o •• if- -• if 11 % —L- E E.

! • 1 • Ei w + k yt s

I f t t (• !f H cd m 00 m CD CD 00 CD CD ~n gt 9) o5 a Ch CD CD CD cn •n 3 u> 3 F F C Tl F 0> M (7) K> H 5 U> Ki + $ -t ? ±00 W L CP m c£ 3 66 = C= S Figure 20 continuation

• Null •iL-2

•sTflRii

nlL2+sT0FM • FIST

* P<0.0S ** P*,0Q0&

J. -CHJ

CM CDS NK NKT Treg CDi& vfiTctils

152 Table 1: FIST treated mice do not display signs of toxicity

Total blood cell count of FIST treated and same age non-treated mice used as control.

153 Table 1

Normal range Parameters Non-treated FIST-treated 3.0-15.0 WBC 7.2 x 103/mm3 6.9 x 103/mm3 5.00-12.00 RBC 12.92 x 103/mm3 13.91 x 103/mm3 11.1-18.0 HGB 17.6 g/dl 19.6 g/dl 36.0-52.0 HCT 54.10% 59.40% 140-600 PLT 1060 x 103/mm3 773 x 103/mm3

17.0-48.0 % LYM 31.40% 36.80% 4.0-10.0 % MON 11.50% 1180% 43.0-76.0 %GRA 57.10% 51.40% 1.2-3.2 # LYM 2.2 x 103/mm3 2.5 x 103/mm3 0.3-0.8 # MON 0.8 x 103/mm3 0.8 x 103/mm3 1.2-6.8 # GRA 4.2 x 103/mm3 3.6 x 103/mm3

154 Figure 21: NK cells as essential mediator of FIST anti-tumor effects

(A) 5xl05 null or FIST secreting B16 cells were injected subcutaneously into CD8+ T,

(B) CD4+ T, (C) JJ.MT, (D) Beige, (E) nonobese diabetic-severe combined immunodeficient (NOD-SCID), (F) common y-chain (yc)/RAG-2 double and (G) IFNy

knockout mice and percent of survival was monitored over time (n=5 per group; data are

shown as the mean + s.d). For each figure, asterisks represent the indicated P value. (H)

CXCL10 production by FIST or controls stimulated NK cells from C57B1/6 immunocompetent, Beige, IFNy knockout and NOD-SCID mice. The results are

representative of two independent experiments performed in triplicates (data are shown

as the mean + s.d).

155 Figure 21

CDS KO mice CDd KOmioe ioo

>' 60 a 40 20

0 200

HMT mice Beige mice 100 iOO

80 80

SO §. 40 I 40

20 a

NOD SCI D mice Rag2/TeKO mice

.£ E

G JFN-y KO mice

100 50O • C57Bl«6 flSO 400 • Boyo SO BL 3a) • IFNy KO 1 € 60 MOD SOD ISO 11 100 1,I 40 0000000000 FiST 0 50 IOO 150 ZOO IL-2 Csyi * P

156 Figure 22: Qualitative expression profile of angiogenesis related proteins secreted by

FIST-activated NK

(A) NK cells were cultured with 5 pmols of FIST or IL-2 combinated with sT|3RII as control for 72 hours and supematants were collected to determine the presence of angiogenesis related proteins by mouse angiogenesis array kit. (B) CXCL9 (MIG) produced by FIST stimulated NK cells isolated from immunocompetent mice.

157 Figure 22

• IL2+sTbRII

E 100

RPM sTbR L-2+sTbR

158 To assess the contribution in vitro of CXCL10 as an angiostatic factor, we determined the

ability CXCR3 (CXCL10 receptor) knockout endothelial cells to form capillary like

structures in the presence of the secretome of FIST stimulated NK cells from immunocompetent or STAT1 knockout mice. CXCR3 knockout endothelial cells form

perfect capillary like structures in all the conditions tested. In contrast, wild type

endothelial cells display the lowest score of progressive angiogenesis in the presence of the secretome of FIST stimulated NK cells derived from immunocompetent mice, but not from STAT1 KO mice, which show significant increase of angiogenesis score (Figure

23E and Figure 24). To assess the contribution of CXCL10 angiostatic effect in vivo, we injected subcutaneously CXCR3 KO mice with 5xl05 null or B16 FIST cells and the

percent of survival was determined over time. B16 FIST cells also fail to form tumors in the CXCR3 KO mice (Figure 23F).

4.4.6 FIST mediates upregulation of STAT1 target genes

FIST angiostatic properties arises in part from CXCL10 production by FIST stimulated

NK cells (Figure 25A). Similarly, we observed significant upregulation of T-bet, the

master regulator of Thl cell differentiation, which may serve as an alternative mechanism for FIST mediated potent anti-tumor effect in vivo (Figure 25C). We also observed

significant upregulation of T-bet target genes such as IFNy, CXCR3 and IL-2RJ3 (Figure

25EGI). The expression of CXCL10, T-bet, IFNy and CXCR3 was significant inhibited in FIST-stimulated NK cells from STAT1 KO mice (Figure 25BDFH), whereas IL-2RJ3

expression was not affected by STAT1 deletion and T-bet downregulation (Figure 25J), which suggest that other transcription factors may complement their functions 321.

159 Figure 23: FIST inhibits angiogenesis in vivo and in vitro

(A) Matrigel plugs sections stained with DAPI and red fluorescence conjugated CD31

specific antibody to detect blood vessel formation in matrigel plugs removed from

C57B1/6 immunocompetent mice previously injected with 5xl05 B16 FIST or control

cells embedded in Matrigel. (B) Endothelial cells (EC) proliferation in the presence of

media conditioned (CM) by FIST or controls stimulated NK cells from immunocompetent mice. Cell number was quantified by running a luminescent versus

cell dose curve (n=3, data are shown as the mean + s.d). (C) In vitro EC migration in

response to the CM of FIST or controls stimulated NK cells from immunocompetent

mice. Migrated cell number was quantified by running a fluorescent versus cell dose

curve (n=3, data are shown as the mean + s.d). (D) In vitro angiogenesis assay of 5x1

EC cultured in the presence of the CM of FIST or controls stimulated NK cells from immunocompetent mice (n=3, data are shown as the mean + s.d). (E) Comparison of

angiogenesis score between of 5x10^ wild type and CXCR3 knockout EC cultured in the

presence of the CM of FIST or control stimulated NK cells from immunocompetent and

STAT1 knockout mice (n=3, data are shown as the mean + s.d). (F) Percent of survival

over time of CXCR3 knockout mice subcutaneously injected with 5xl05 B16 FIST or

null cells (n=5 per group; data are shown as the mean + s.d). For each figure, asterisks

represent the indicated P values.

160 Figure 23

DAPI

CD31

Merge

S 20

imre CKCH3KOEC

•P B

NK NK NKVFIST) NKC^EST) WT STATtKO vn STAT 1 ICO I 100

- 60

|*0 B16 null " BIG FIST. 40

I 20 40 60 S£J 100 ll DFIYI IL-2NKCM

sTftRII NK CM

FislNK CM * !><().05 ** P<0 005

161 Figure 24: Pictures of capillary like structures formed by wild type or CXCR3 knockout endothelial cells cultured in the presence of the secretome of FIST or control stimulated NK cells.

162 Figure 24

WT EC and CM FIST NK

CXCR3 KO EC and CM FIST NK CXCR3 KO EC and CM FIST NK STAT1 KO

.. • V „T>* v joJcT

WTECandCMNK

6r" •) •/1

163 Figure 25: FIST mediates upregulation of statl target genes

(A) and (B) CXCL10 production over time by lxl06 of FIST or control stimulated NK

cells from immunocompetent and STAT1 knockout mice respectively. (C) and (D) T-bet expression over time was determined by intracellular staining of lxlO6 of FIST or

controls stimulated NK cells from immunocompetent or STAT1 knockout mice

respectively. T-bet expression levels were quantified based on the mean fluorescence intensity (MFI) values obtained by flow cytometry. (E) and (F) IFNy production over time by lxlO6 of FIST or controls stimulated NK cells from immunocompetent and

STAT1 knockout mice respectively. (G) and (H) Cell surface CXCR3 expression over time by lxlO6 of FIST or controls stimulated NK cells from immunocompetent and

STAT1 knockout respectively. (I) and (J) Cell surface IL-2RJ3 expression over time by

lxlO6 of FIST or controls stimulated NK cells from immunocompetent and STAT1

knockout mice respectively. The receptor expression levels were quantified based on the

MFI values obtained by flow cytometry. For all the experiments, n=3 per group; data are

shown as the mean + s.d. For each figure, asterisks represent the indicated P values.

164 Figure 25

500 Balb/c

Hours Hours

C 160 Balb/c

_ 120 - 5 Z 80

48 72 24 48 72 96 Hours Hours

100 Balb/c Stati KO 80 I

0.02 0[ =e==8 24 48 72 96 Hours

100 j Balb/c • Stati KO 80 -

Balb/c Stati KO

40 cn 30 CM 30

48 72 Hours Hours

-RPMI - IL-2 • sTpRII • IL-2 + sTpRH —-*-FIST

165 4.5 DISCUSSION

Upon IL-2 binding, the IL-2 receptor (IL-2R) activates two tyrosine kinases, Janus kinase

1 (JAK1) and JAK3, which interact with the cytoplasmic domains of the IL-2R|3 and yc

subunits351. Activated IL-2R becomes phosphorylated on specific tyrosine residues which

serve as docking sites for proteins containing Src-homology 2 (SH2) or phosphotyrosine

binding (PTE) domains including She adaptor and STAT proteins (STATI, STAT3 and

STATS). IL-2R recruits STAT proteins to different subdomains of the IL-2RJ3 chain.

STAT5 associates with phosphorylated Tyr-510 of the IL-2RJ3 C-terminal region. In

contrast, STATI and STAT3 interaction with the IL-2RJ3 chain occurs through its acidic

subdomain and may not require either phosphorylation of the receptor or even the

presence of tyrosine residues of IL-2RJ3352,351. In addition, IL-2-mediated STATs

activation is concentration sensitive since low IL-2 concentration is sufficient to induce

STAT5 activation, whereas STATI activation requires high IL-2 concentrations 352,353.

Our new bifunctional pharmaceutical (FIST) is endowed with the ability to signal through the IL-2 receptor inducing similar STAT5 activation and distinctive STAT3 and STATI

activation compared to equimolar concentrations of IL-2 or IL-2 combined with sT|3RII.

Specifically, FIST induces a partial hyperagonist response in IL-2R-expressing cells

characterized by a potent activation of STATI combined with contemporaneous inhibition of TGF|3 signaling pathway through transcriptional induction of the inhibitory

SMAD7. Based on previous studies, JAK1/STAT1 activation acts as positive regulator of

SMAD7 expression 354 STATI deletion completely abrogated FIST-dependent-SMAD7

overexpression, but not the inhibition of SMAD2 phosphorylation, which indicates that the T|3RII ectodomain moiety of FIST effectively functions as a decoy receptor trapping

166 active TGF|3 in solution. Smad7 directly inhibits TGF-J3 signaling in the nucleus by interacting with transcriptional repressors, or disrupting the formation of the TGF-J3- induced functional Smad-DNA complexes355. Smad7 also recruits HECT type of E3

ubiquitin ligases, Smurfl and SmurfZ to the activated TGF|3 receptor I (T|3RI) leading to the degradation of the receptor through the proteasomal pathway 123.

In a STATI dependent mechanism, FIST acts intracellularly to inhibit TGF|3 signaling

pathway via Smad7 expression. This property characterizes FIST mechanism of action

and is responsible for the superiority of FIST as pro-inflammatory compound versus IL-2

or IL-2 combined with sT|3RII. In contrast, total lymphocyte or purified immune cells

stimulated by IL-2 plus sT|3RII display similar activation as IL-2 stimulated cells indicating that sT|3RII although trap active TGF|3 secreted by immune cells, does not

operate intracellularly and does not enhance the activation status of STAT proteins

downstream of IL-2 receptor. Consequently, FIST-stimulated lymphocytes in vitro

become resistant to TGF|3 mediated suppression, and produce significant greater amounts

of pro-inflammatory cytokines. However, IL-2 dependent cells (CTLL-2) cultured with

FIST display a similar proliferation rate as CTLL-2 stimulated with equimolar

concentrations of IL-2 or IL-2 combined with sT|3RII. This confirms the observation that

FIST-mediated STATS and PI3K activation levels are similar to the one induced by

equimolar concentration of IL-2, since these transcription factors are implicated in T cell

proliferation and survival356' 357 FIST dependent-SMAD7 overexpression was only

detected in the presence of active TGF|3, which can be explained by the reported SMAD binding elements (SEE) in the SMAD7 promoter required for TGF|3-dependent transcriptional activation316.

167 FIST-mediated hyperagonist signal transduction downstream of the IL-2 receptor coupled to TGF|3 blockade leads to resistance to TGF|3-mediated suppression and to a potent anti tumor effect in vivo. Consequently, melanoma B16 FIST and PANC02 FIST cells fail to form tumors in immunocompetent mice. Since it is not possible to modify all pre-existing tumor cells with suicide or proinflammatory cytokine genes in situ by any contemporary gene transfer technology, an important feature to consider for cancer immunotherapy is the bystander effect285. The secretion of FIST by genetically modified B16 cells promotes a bystander anti-cancer effect that protected 60% of mice bearing non-genetically

modified cancer cells from tumor development. Although FIST promoted significant

recruitment of IL-2 receptor-expressing immune cells such as NK, MKT, CDS T cells

and B cells to the tumor site, FIST still prevents tumor formation in CD4 KO, CDS KO

and B cell deficient mice, which indicate that these immune cell types, although may

contribute, are not essential mediators of FIST anti-tumor activity. In contrast, B16 FIST

cells were tumorigenic in NOD-SCID and Rag2/yc KO mice, which implicate by exclusion NK cells as the major immune mediator of FIST effects. The necrotic

appearance of tumors secreting FIST from NOD-SCID mice also suggests a profound

defect of tumor-driven angiogenesis. FIST not only antagonizes TGF|3, an essential

regulator of both phases of angiogenesis193, but also stimulates virtually all lymphoid

subsets, including NK cells to secrete substantial amounts of IFNy, which may act as an

angiostatic by suppressing the expression of genes required for VEGF response via

STATI activation358. However, B16 cells expressing FIST fail to form tumors in IFNy

and IFNy receptor KO mice, suggesting that additional effector molecules are at play.

FIST-stimulated NK cells produced significant amounts of CXCL10 and CXCL9.

168 CXCL10 and CXCL9 as well as CXCL11 are chemokines induced by IFNy via STATI

activation, and are CXCR3 ligands that share potent angiostatic and chemoattractant

properties359"361. In particular, high level of CXCL10 in the tumor is correlated with high

amounts of tumor-infiltrating NK cells and better survival362. Interestingly, FIST-

stimulated NK cells from IFNy KO mice display a significant decrease of CXCL10

production, which support the role of IFNy as main inducer of CXCL10 expression.

However, FIST-stimulated NK cells from IFNy KO mice still produce elevated levels of

CXCL10 indicating that upon binding to IL-2 receptor, FIST directly induces CXCL10

production via STATI hyperactivation. As expected, CXCL10 secretion was

significantly reduced in FIST-stimulated NK cells from NOD-SCID mice which display

severe defect in NK cell functions, and was completely abrogated in FIST-stimulated NK

cells from STATI KO mice. The specific angiostatic effect of FIST on tumor-derived

angiogenesis is likely due to the transient CXCR3 expression restricted to newly formed

blood vessels in vivo and in vitro363. In concordance with previous studies, we found that

CXCL10 behaves as an inhibitor of endothelial cell migration and differentiation into

branching networks of tubular structures in vitro, a process that requires the interaction of

endothelial cells with extracellular matrix components360'364' 365. However, the ability of

CXCL10 to inhibit endothelial cell proliferation is controversial360'366. Despite this potent

angiostatic effect observed in vitro and in vivo in immunocompetent mice, B16 FIST

cells fail to form tumors in CXCR3 knockout mice, which indicate that other additive

FIST-mediated anti-cancer mechanisms are at play. Indeed, FIST still induces a potent

Thl cell mediated immunity via STATI activation in CXCR3 knockout mice. In contrast,

FIST-dependent CXCL10 induction via STATI activation may prevent tumor growth in

169 IFNy knockout mice. These two FIST properties are not mutually exclusive and operate

contemporaneously to prevent cancer progression.

Based on these results, we propose a working model to describe the molecular

mechanism of FIST: upon binding to IL-2 receptor, FIST not only induce the activation

of STATS and STAT3, but also promotes a potent activation of STATI and consequently

upregulates the expression of STATI target genes essential for an effective Thl cell

mediated cell immunity such as T-bet, SMAD7 and CXCL10. In turn, T-bet upregulates the expression of its target genes, CXCR3 and IL-2R|3. Through Smad7 expression, FIST inhibits active TGF|3, which exerts dramatic suppression of proinflammatory cytokine

mediated-IFNy production indirectly by downregulating T-bet expression via SMAD

dependent mechanism, and directly by T-bet-independent negative regulatory effect on the IFNG promoter367. IFNy secreted may amplify FIST dependent activation by binding to IFNy receptor and creating a positive autocrine loop that enhance the expression of

STATI target genes (Figure 26).

Consequently, FIST-treated mice develop a robust anti-cancer adaptive immunity that

protects 50% of mice from high-burden tumor challenge. Interestingly, FIST-stimulated

NK cells from STATI knockout mice still produce significant higher amounts of IFNy than equimolar concentrations of controls. This suggests that the contemporaneous blockade of active TGF|3 combined with an immune stimulation significant enhances the

activation level of NK cells, which constitutively produce active TGFJ31, as an

autocrine/negative regulator of IFNy367

170 By targeting several immune cell types expressing the IL-2 receptor, inhibiting active

TGF|3 and upregulating the expression of STATI target genes, FIST conveys host's

angiogenic and immune response against cancer cells. Therefore, cancer cells expressing

FIST only form tumors in NOD-SCID and Rag2/yc double knockout mice, which lack

most of these immune system components. Although other TGF|3 antagonists like

Fc:T|3RII may neutralize large amounts of active TGF|3368, Fc:T|3RII treatment does not

alter primary tumor growth of transplantable models of breast cancer metastases, which

suggests that the antimetastatic effects of Fc:T|3RII in vivo are independent of tumor cell

proliferation. In addition, inhibition of tumor angiogenesis in vivo by Fc:T|3RII treatment is controversial. Muraoka et al. observed no reduction of vascular density in endogenously arising tumors128.

FIST treatment can be applied in humans systemically or intratumoral as recombinant

protein since low doses of the fusion (0.002 pmols) are effective in the treatment of

carcinoma. Alternatively, FIST can be delivered by vehicle cells such as genetically

modified stem cells or autologous immune cells or as irradiated FIST-tumor cell vaccine.

In conclusion, FIST is a novel biopharmaceutical characterized by inhibiting TGF|3

canonical pathway simultaneously with a distinctive STATI hyperactivation via IL-2

receptor on immune cells, which conveys a robust upregulation of STATI target genes including key factors essential for an effective anti-tumor response. This first-in-class

biological agent may represent a new paradigm in cancer immunotherapy

contemporaneously modulating pro and anti-inflammatory immune checkpoints operative

on immune effector cells.

171 Figure 26

FIST mechanism of action. Upon binding to IL-2 receptor, FIST not only induce the

activation of STAT5 and STAT3, but also promotes a potent activation of STATI and

consequently upregulates the expression of STATI target genes essential for an effective

Thl cell mediated immunity such as IFNy, T-bet, SMAD7, CXCR3, IL-2RJ3 and

CXCL10. T-bet upregulates the expression of its target genes IFNy, CXCR3 and IL-2R|3

and via Smad7, FIST inhibits the suppressive effects of active TGF|3 on T-bet gene expression and IFNy production. In turns, IFNy amplify FIST-dependent activation by binding to IFNy receptor and creating a positive autocrine loop that enhance the expression of STATI target genes.

172 Figure 26

FIST

TGFR »»»»»»»»»»»

ill; STATS STAT3

Smad? CXCL10

IL-2RP IFN'/ CXCR3

m CL

173 4.6 ACKNOWLEDGMENTS

This work was supported by a Canadian Institute for Health Research operating grant

MOP-15017. C P. is a recipient of US army pre-doctoral training award and JG is a

Fonds de recherche en sante du Quebec chercheur-boursier senior. We thank Naciba

Benlimame at the George and Olga Minarik Research Pathology Facility, Catherine

Lemarie and Joan Durbin for technical advice and materials.

174 CHAPTER 5

FIST-activated B cells act as potent antigen presenting cells. Manuscript in preparation

Preface to Chapter 5: We have previously shown that FIST, as a Afunctional protein,

not only inhibits TGF|3 signaling pathway but also exerts potent immunostimulatory

activity on IL-2 expressing cells. In Chapter 5, we aim to characterize and evaluate FIST-

activated B cells in their ability to act as APCs and induce protective immunity against tumor challenge.

175 5.1 ABSTRACT

We have previously shown that the fusion between IL-2 and the ectodomain of TGF|3

receptor II (aka FIST) acts as an effective antagonist of TGF|3 canonical pathway and

promotes potent immune stimulation of IL-2 expressing cells. In particular, FIST-

stimulated B cells secrete significant greater amounts of IL-2 and IFNy. In the present

study, we characterize and evaluate FIST-stimulated B cells as cell-based platform for therapy of cancer. FIST upregulates co-stimulatory molecule, activation marker and

MHC class II molecule expression, which is supported by robust hyperactivation of

STAT3 and STATS downstream of IL-2 receptor. In addition, FIST-stimulated B cells

maintain B cell identity based on the expression of PAX5 and CD 19. As antigen

presenting cells, FIST-stimulated B cells induce the activation and cell proliferation of

antigen-specific CD4+ and CDS T cells. Interestingly, FIST-stimulated B cells confer

complete protective immunity to EG.7 tumor challenge in vivo. In conclusion, FIST-

stimulated B cells constitute an alternative source of effective APC useful for anti-cancer therapy.

5.2 INTRODUCTION

Antigen presentation is essentially required for the development of effective cell

mediated immunity312. Dendritic cells (DCs) have been extensively used as cellular

"7/TQ Q"7A adjuvants to present antigen in vivo ' . Although highly effective in their ability to induce T cell mediated immunity, DCs clinical applicability has encountered several

disadvantages. First, they are relatively rare in peripheral blood (<1% of leukocytes) and

are therefore usually isolated from apheresis or marrow sources371. Secondly, they are

comprised by a heterogeneous population with distinctive functions and third, they are

176 difficult to expand in vitro from a non-stem cell source372. As alternative source of APC,

B cells are reliably expandable from non-stem cell source with small amounts of

peripheral blood and as DCs, B cells can process and present antigens in the context of

MHC class II molecules, which recruits specific CD4+ T cell help and stimulates B cell

proliferation and differentiation26'373. In addition, B cells express high levels of MHC

class I and therefore act as APC for CDS T cells, which stimulates IL-2 production and

CTL activity304' 374 B cells are divided in two lineages B1 and B2 cells. B1 cells

constitute the antibody producers in response to thymus-independent antigens indicating their role in the innate arm of immune response. In contrast, B2 cells or conventional B

cell response to most protein antigens or thymus-dependent antigens requires the

activation of dendritic cells and the recruitment of antigens-specific T helper cells, therefore comprises the adaptive arm the immune system 312' 312. Once naive B2 cells internalize antigens through their surface immunoglobulins (IgM and IgD), process, and

present antigenic peptides-MHC class II complexes to CD4+ helper T cells, they

differentiate into IgM-producing short-lived plasma cells or undergo hypersomatic

mutations and class switch recombination within the germinal centers, thereby producing long-lived plasma cells and memory B cells312. The combination of antigen stimulation

and polarized effector Thl and Th2 cells dictate the differentiation of naive B cells toward IFNy or IL-4 producing cells capable of triggering a Thl or Th2 differentiation

O 1 r\ T »7c O "7/T program in naive T cells respectively ' ' . Hence, activated B cells behave as

classical APC that regulate the fate of the immune response. At the molecular level, B

cell commitment is regulated by the transcriptional regulator B cell lymphoma 6 (BCL-6)

that has been shown to control the fate, proliferation or death of germinal center (GC) B

cells. BCL-6 inhibits the differentiation of GC B cells into plasma cells through

177 transcriptional repression of Prdml (positive regulatory-domain containing 1) gene, which encodes BLIMP1 (B-lymphocyte-induced maturation protein 1), a master regulator

of terminal B cell development312'312'377'378. BLIMP 1 triggers plasma cell differentiation

by inhibiting MHC CUT A, c-myc, and PAX5 expression127' 312 These genetic changes

result in suppression of MHC class II expression, cell proliferation and loss of B cell identity312. In addition, BLIMP1 induces the expression X-box binding protein 1 (XBP1),

a transcriptional factor required for the secretory phenotype of plasma cells. In contrast,

PAX5 mediates the blockade of plasma cell differentiation by repressing several genes

required for plasma cell differentiation including Prdml254' 255. Interestingly, the

Jak/STAT pathway plays an essential role in establishing the balance between BCL6 and

BLIMP1 gene expression in B cells. Specifically, STAT3 activation up-regulates

BLIMP 1 gene expression379, whereas STAT5 activation leads to self-renewal and inhibition of plasma B cell differentiation due to upregulation of BCL6 expression380. We

have previously shown that a fusion of IL-2 and the ectodomain of TGF|3 receptor II (aka

FIST) not only act as an effective inhibitor of TGF|3 canonical pathway, but also FIST

possesses potent immune stimulatory properties that relies on robust activation of

Jak/STAT signaling pathway downstream of IL-2 receptor. In particular, FIST-activated

B cells secrete substantial amount of Thl cytokines such as IFNy and TNFa (Chapter 4).

In the present study, we characterized and evaluated FIST-activated B cells in their

ability to act as effective antigen presenting cells promoting a protective immunity

against tumor challenge.

178 5.3 MATERIALS AND METHODS

5.3.1 Mice, reagents and antibodies

All experimental C57BL/6 and transgenic mice were females 6 to 8 weeks old (Jackson

Laboratory, Bar Harbor, ME). Recombinant mouse IL-2 and soluble TGF|3 receptor II

(TJ3RII) were obtained from R&D Systems, Minneapolis, MN; Anti-phosphorylated

STAT3 and STAT5 antibodies were obtained from Cell Signalling Technology, Danvers,

MA; a-tubulin antibody and anti-SMAD7 were obtained from Santa Cruz Biotechnology,

Santa Cruz, CA. Anti-mouse FcR III/II, CD79b (BCR subunit), MHC class I and II,

CD 19, CD45R (B220), CD24, CD22.2, CD43, CD279, CD138, CD27, CD80, CD86,

CD25, CD69, CD23, CD95 and CD40 and the isotype control antibodies for flow

cytometry were obtained from BD Biosciences, San Diego, CA.

5.3.2 B cell isolation, generation and characterization of FIST-stimulated B cells

Enriched B cell population was obtained from splenocytes of immunocompetent or transgenic mice by magnetic separation according to the manufacturer's recommendation

(Stem Cell Technology, Vancouver). B cell population purity assessed by flow cytometry was 94%. FIST-stimulated B cells were generated by culturing purified B cells in the

presence of 2 pmols/ml of FIST for 5 days at 37°C. For cell surface marker staining,

FIST- or control-stimulated B cells were resuspended in phosphate-buffered saline (PBS) with 2% FBS, incubated with anti-mouse FcR III/II for 30 mins and labelled with

conjugated antibodies specific for CD79b (BCR subunit), MHC class I and II, CD 19,

CD45R (B220), CD24, CD22.2, CD43, CD279, CD138, CD27, CD80, CD86, CD25,

CD69, CD23, CD95 and CD40. The expression of these cell surface markers was

179 determined by FACS Calibur cytometer (BD) and analyzed using Cellquest software

(BD).

5.3.3 Intracellular signaling

For signaling analysis, FIST- or control-stimulated B cell lysates were probed by Western

blot (WB) with rabbit anti-phosphorylated or anti-total STAT3, STATS, SMAD7 and

PAX5 antibodies. Anti a-tubulin antibodies were used as loading controls.

5.3.4 In vitro antigen presentation assay (APC)

To assess the antigen presentation ability of FIST-stimulated B cells, lxlO4 FIST-or

control-stimulated B cells were co-cultured with lxlO5 CD4+ or CDS T cells. After 48

hours, the supematants were collected to quantify the concentration of IL-2 and IFNy by using the enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-2 and IFN- y respectively (BD Biosciences). To determine the cell proliferation rate of antigen-

specific CD4+ and CDS T cells. These cells were pre-stained with Carboxyfluorescein

succinimidyl ester (CFSE) before culturing them with FIST- or control- activated B cells

and the percent of proliferating cells was quantified by flow cytometry. The data were

analyzed using Cellquest software (Becton & Dickinson).

5.3.5 In vivo antigen presentation assay (APC)

C57BL/6 mice were intravenously injected with lxlO5 FIST-stimulated and OVA pulsed

B cells and three weeks later, mice were boosted with 4xl05 FIST-stimulated and OVA

pulsed B cells. One week after the second boost, mice were challenged with SxlO5 EG.7-

180 OVA tumor cells. To determine FIST-stimulated B cells protective anti-tumor immunity, we measured the tumor volume and percent of survival over time.

5.3.6 Statistic evaluation

P values were calculated by paired student T test. P<0.05 was considered statistically

significant.

5.4 RESULTS

5.4.1 FIST induces potent B cell activation and proliferation

B cells cultured with mFIST for five days undergo a differentiation process characterized

by phenotypic changes including an increase in size and granularity defined by side and forward scattergram (SSC and FSC, Figure 26A), and H&N staining (Figure 26B). For

phenotypic analysis of FIST-activated B cells, we measured the expression levels of

several B cell receptors or co-stimulatory molecules including CD79b (BCR subunit),

MHC class I and II, CD 19, CD45R (B220), CD24, CD22.2, CD43, CD279, CD138,

CD27, CD80, CD86, CD25, CD69, CD23, CD95 and CD40. In comparison with B cells

stimulated with equimolar concentrations of controls (2 pmols/ml of IL-2, sT|3RII or IL2

plus sT|3RII), we observed that FIST-stimulated B cells significantly upregulate MHC

class II and costimulatory molecules (CD86) expression, as well as markers of activation

(CD69) and IL-2 receptor alpha subunit (CD25) (Figure 26C). In addition, FIST-

stimulated B cells maintain their identity based on CD 19 expression (data not shown).

We also observed that FIST promotes a robust B cell proliferation (Figure 26D).

181 5.4.2 FIST promote potent B cell activation via Jak/STAT signaling pathway

We determined the activation status of the transcription factors STAT3 and STATS

downstream of IL-2 receptor since B cells express the receptor for IL-2 and therefore are

responsive to FIST. We observed that B cells stimulated with FIST for 30 minutes do not

show a significant activation of these transcription factors compared to equimolar

concentrations of controls (data not shown), whereas B cells cultured with FIST for 5

days display a significant hyperactivation of both STAT3 and STAT5 (Figure 27A). The

high levels of MHC class II expression as well as CD19 expression, suggest that FIST-

stimulated B cells do not undergo a differentiation process to plasma cells, which require

Pax5 downregulation. As result, we determined that Pax5 is highly expressed in all the

conditioned tested without differences between FIST or control activated B cells. As

previously observed for IL-2 expressing cells (Figure 17, Chapter^), SMAD7 expression was also significant upregulated in FIST-activated B cells (Figure 27B).

5.4.3 FIST-stimulated B cells promote potent CD4 and CD8+ T cell activation in an antigen specific manner

OT1 and OT2 T cells were designed to recognize OVA 257-264 residues in the context

of H2Kb or OVA 323-339 residues in the context of I-Ab respectively. FIST-stimulated B

cells only induce OT2 CD4+ T cell activation when they were previously pulsed with

OVA antigens, whereas no IL-2 or IFNy amounts were detected in the absence of OVA

antigens (Figure 28AB). As expected, FIST-stimulated and OVA pulsed B cells promote

significantly greater CD4+ and CDS T cell activation and therefore cytokine production than OVA pulsed B cells previously stimulated with cytokine controls (Figure 28CD).

182 Similarly, we observed in vitro that both CD4+ T cells and CDS T cells proliferate in

response to OVA specific antigens presented by FIST-stimulated B cells (Figure 28EF).

5.4.4 FIST-stimulated and OVA pulsed B cells induce protective immunity against tumor challenge

FIST-stimulated and OVA pulsed B cells offered complete protection to mice from E.G7-

OVA tumor cell challenge. In contrast, the majority of mice (60-80%) previously treated with B cells previously stimulated with IL-2, sT|3RII or both combined developed tumors

and were sacrificed (Figure 29A). Interestingly, we observed that a small percentage

(20%) of mice treated with non-activated and OVA pulsed B cells developed protective immunity (Figure 29B).

183 Figure 26: FIST-stimulated B cell phenotype

(A) Side and forward scattergram of FIST- or control-stimulated B cells. (B) H&N

stainning of FIST- or control-stimulated B cells. (C) Phenotypic characterization of

FIST-or control-stimulated B cells. (D) Proliferation of B cells stimulated with FIST for 5

days.

184 Figure 26

8 IL-2 FIST RPMI IL-2 7& • :-r y.f . : A *

sTpRll lL-2+sTBRll FIST r ' - r r \ >V • mm* {• * \

RPMI IL-2 STPRII lL-2+sTpRII FIST H- 504

ot 623 613 624 610 <0 Ml 1 | Ml 1 , Q V J u

C0a6bioivS>..PE.-rvr CM6tWM*SA-PECvT Ct-WtkMSA-fttyt I 1?2 I ^

.. I 251

Cti;3bk4*S>.-PECy7 C&ShwtSA.PECyr

185 Figure 26 cont.

Media IL-2 sTpRII IL2+sTfjRII FIST

1 '"'H| 1 i mi ll 2 3 4 lllW| I IIM| . , . I ""T J iq^ ao io to 10 io° aq1 scr io3 101 1ft2 103 10' aq' w* IQ 10' CFSE CFSE CFSE CFSE CFSE

186 Figure 27: FIST-stimulated B cells display potent activation of STATS and STATS

5xl06 FIST-stimulated B cell lysates were probed for (A) Phosphorylated STAT3 and

STATS, as well as total STAT3 and STAT5 antibodies. (B) PAX5 and SMAD7 antibodies. Antibodies anti full length proteins and a-tubulin were used as loading controls.

187 Figure 27

P-STAT 3

T-STAT 3

P-STAT 5

T-STAT 5

Pax5

SMAD7

p-Tubulin

188 Figure 28: FIST-stimulated B cells act as effective antigen presenting cells

(A) and (B) IL-2 and IFNy secreted by 0T2 CD4+ T cells previously activated by FIST-

stimulated B cells in the presence or absence of OVA specific antigen. (C) and (D) IL-2

and IFNy secreted by OT1 CDS and OT2 CD4+ T cells previously activated by FIST- or

control-stimulated and OVA pulsed B cells. (E) and (F) Percent and mean fluorescence intensity (MFI) of proliferating OT1 CDS and OT2 CD4+ T cells previously activated by

FIST- or control-stimulated and OVA pulsed B cells. For each figure n=3; data are shown as means + s.d. Asterisks represent P values (P<0.05).

189 Figure 28

mnBtfU FISTBctU FIST B eel RPMIBcdl HSTSMIIOVA FISTBcdl OVA RPMIB cdl OVA - mn 3 cell OVA 0 100 200 300 400 $00 600 0 500 1000 1500 2000 2500 3000

IL-2 pg ml IFN gamma pg ml

OT : • OH 2

• OTI1 aOTIl •L-2-rsTbSn iL-:-^TbRn

STDRII iTbRIl

F' me

0 100 200 300 400 500 600 700 S00 0 100 200 300 400 500 600 700 800 IFN gamma pg ml IL-2 pgml

Media IL-2 sTpRII 11.21 s'lpRll FIST 4% it 7% 15% O ML RI-RM io" 1(3 io2 id3 to"* io° to1 io2 i:i" to^ I'1- to1 to2 m3 101- to1 io2 io5 io4 io° 1- :o2 1:1" ^ CFSE CFSE CFSE CFSE CFSE Mf 1=2196 MFN2122 MFI=2096 MFI=2005 MFI=1699

D**,0iQ g 4% Ml

1 2 to" to 10 10^ 0 I0l to2 io3 to4 10' to2 103 10' 10° to1 CFSE CFSE CFSE CFSE MFI=2168 MF 1=2195 MFI=2065 MFN2002 MFI=1536

190 Figure 29: FIST-stimulated and OVA pulsed B cells promote complete protective

immunity against tumor challenge

(A) EG.7-OVA tumor growth over time in C57BL/6 mice pre-treated with FIST- or

control-stimulated and OVA pulsed B cells. (B) Percent of survival over time of

C57BL/6 mice bearing EG.7-OVA tumors. For each figure n=5; data are shown as means

+ s.d.

191 Figure 29

-0-LL2 slbRII

Days post tumor inaptantation Days post tumor implantation

192 5.4 DISCUSSION

B cells cultured in the presence of FIST show a dramatic morphological change

characterized by a granular appearance and an increase in cell size. As indicative of B

cell activation, we observed that FIST induces significant upregulation of co-stimulatory

molecule CD86, activation marker CD69 as well as IL-2 receptor a (CD25) expression, which suggest that FIST-stimulated B cells become highly responsive to IL-2.

Experiments to define the secretome of FIST-stimulated B cells are in progress. At the

molecular level, FIST-stimulated B cells display potent activation of STAT3 and STATS

downstream of IL-2 receptor. STAT3 and STAT5 play opposite roles in the regulation of

plasma cell differentiation and their activation status dictates the fate of B cells380'381.

Several cytokine stimuli that signal through Jak/STAT pathway can promote proliferation

or differentiation of B cells into plasma cells. For instance, STAT3 activating cytokine

such as IL-10, IL-6 and IL-21 have been implicated in plasma cell differentiation259, while STAT5 activating cytokine such as IL-2 and IL-4 have shown to promote B cell

proliferation and long-term growth256"258. In addition, STATS binding to the class II transactivator (CUT A) promoter induces epigenetic modifications that favor CUT A

expression and therefore MHC class II upregulation294. Our results indicate that FIST

maintains B cell identity based on the expression of CD19, MHC class II, and PAX5.

Currently we are investigating the level of expression of BCL6 and BLIMP1 in FIST-

stimulated B cells. This phenotype indicates that FIST-stimulated B cells behave as

effective antigen presenting cells (APC). Indeed, in vitro FIST-stimulated and OVA-

pulsed B cells activate CD4+ T and CDS T cells to secrete significant greater amounts of

IL-2 and IFNy. Similarly, FIST-stimulated and OVA-pulsed B cells protect mice implanted with E.G7 cells from tumor development. These new features acquired by

193 FIST-stimulated B cells suggest that FIST is suppressing TGF|3 pathway in B cells.

Previous studies report that TGF|3 can inhibit the proliferation of both murine and human

IQ'I QQQ B cells activated by a variety of stimulus such as mitogen and ligation of CD40 '

These growth inhibitory properties are associated with a decrease in c-Myc expression288.

In addition, TGF|3 increases the rate of apoptosis of normal resting B cells via caspase

3289, inhibits class II MHC transactivator (CIITA), class II MHC expression384 and class

switching for the majority of isotype except for IgA. We are currently investigating the

anti-tumor effect of FIST-stimulated B cells in the therapeutic setting. Basically, mice

bearing E.G7-OVA tumors are being treated with FIST-stimulated and OVA- pulsed B

cells and tumor growth and survival is being monitored over time. Our preliminary

results indicate that FIST-stimulated B cells promote tumor regression in mice with pre-

established tumors may be by inducing adaptive tumor immunity. However, future

experiments to determine and quantify tumor-specific CD8+ T cells are required. As

dendritic cells, FIST-activated B cells display a robust APC phenotype that take up and

process whole protein antigens for presentation to naive CD4+ T cells, a requirement for the induction of long-lasting immunity. In addition, FIST-stimulated B cells are effective

at inducing antigen-specific CDS T cell activation in vitro. The ability of FIST to expand

B cells from small blood volumes may be relevant in the clinic, in particular for

paediatric patients. Based on the potent APC properties acquired, FIST-stimulated B cells

could be used as a cellular pharmaceutical for the immunotherapy of cancer and infectious diseases.

194 5.5 ACKNOWLEDGMENTS

This work was supported by Canadian Institute for Health Research operating grant

MOP-15017. CP is recipient of Montreal Centre for Experimental Therapeutics in

Cancer Scholarship and US Army Graduate study Scholarship and JG is a Fonds de recherche en sante du Quebec chercheur-boursier senior.

195 CHAPTER 6: CONCLUSIONS

196 Chapter 6: Conclusions

John Stagg et al. in 2004 generated and characterized the murine fusion protein between

IL-2 and GM-CSF (aka GIFT2). GIFT2 is endowed with distinctive pharmacological

properties compared to the combination of each of its components (GM-CSF and IL-2).

As a new biochemical feature, murine GIFT2 induces greater recruitment of macrophages

and NK cells to the tumor site, which promote potent antitumor response. Consequently, live B16 melanoma cells genetically modified to express GIFT2 (B16GIFT2) were unable to form tumors in immunocompetent mice. In addition, irradiated B16GIFT2 also induced protective immunity against tumor challenge by live B16 cells. This was the first report that a fusion between two cytokines can invoke greater antitumor effect than both

cytokines in combination, thus representing a new class of biopharmaceuticals for cancer immunotherapy. Fusion proteins have several advantages over combinatory cytokine based therapy; first, the optimization of the activity of two agents with different

pharmacological properties; second, fusion proteins have been shown to recapitulate

synergistic effects while eliminating the need for dual delivery and more important the fusion proteins may display unheralded biopharmaceutical properties that induce more

effective antitumor responses297. As continuation of that work, we investigated the

potency of the murine GIFT2 to induce an effective antitumor response against non- genetically modified tumor cells in vivo, a phenomenon termed "bystander effect". The bystander effect of a therapeutic agent is an important feature to consider for cancer immunotherapy, since it is not possible to modify all pre-existing tumor cells by any

contemporary gene transfer technology 285. As results, we observed that GIFT2 promoted

a bystander effect in vivo that was mainly mediated by recruited NK cells in the tumor

197 site. However, GIFT2 mediated bystander effect is lost as tumor burden increases.

Interestingly, we found that B16-derived TGF|3 was responsible of this acquired

refractoriness by its direct effect on innate effector cells despite local production of a

potent pro-inflammatory fusokine. Based on the potent stimulatory properties of GIFT2

on NK cells, we suggest that GIFT2 may serve as mean to generate oncolytic NK cells for cancer therapy. Indeed, several in vitro as well as in vivo studies indicate that tumor

cells can be recognized and killed by activated NK cells. In addition, NK cells not only

directly eliminate tumor cells, but also constitute a bridge between innate and adaptive immunity by inducing the subsequent development of tumor-specific T cell responses. To address this hypothesis, we successfully generated and characterized the human ortholog

of the GM-CSF/IL-2 fusion protein (hGIFT2), as well as evaluated its immunostimulatory properties on human NK cells. As the murine GIFT2, the human

ortholog GIFT2 is endowed with unheralded properties that overcome GM-CSF mediated

NK cell suppression and promote potent NK cell activation. We found that human GIFT2 upregulated the expression of natural cytotoxicity receptors, promoted NK maturation

and pro-inflammatory cytokine production. Consequently, GIFT2-activated NK cells

display effective cytotoxicity against both NK-resistant and NK-sensitive tumor cells.

These potent stimulatory properties are supported by a robust hyperactivation of STAT1,

STAT3, STAT5 transcription factors downstream of the IL-2 receptor. Therefore, the use

of GIFT2-activated NK cells may be of interest in personalized cell therapy of cancer.

As previously demonstrated, tumor-derived TGF|3 is an extremely potent suppressive factor that inhibits the effector functions of several pro-inflammatory cytokines such as

IL-2, IFNy and IL-12 385'386, while promoting T-cell production of IL-10 likely through

198 direct activation of the IL-10 promoter via Co-Smad4298. TGFP potently suppresses the

effector functions of activated T cells and thus their differentiation into Thl or Th2

effector cells by inhibiting the expression of T-bet and Gata-3299' 305. Thl polarizing

condition promotes CD 122 (IL-2 receptor P chain) expression through T-bet, thereby

enhancing the clonal expansion and survival of Thl cells. TGFP suppressed CD 122

upregulation under Thl-skewing conditions. Therefore, TGFP also limits Thl effector

cell numbers through inhibiting the upregulation of CD122306. TGFP directly suppresses

T cell proliferation by inhibiting the production of IL-2, a lymphokine that potently

activate T cells, NK cells and other immune cell types322' 323. In addition, TGF-P

suppresses T cell proliferation through controlling the expression of cell cycle regulators.

Upon TGF-P treatment, cyclin-dependent kinase inhibitors (CKIs), such as pl5, p21, and

p27, are upregulated, while cell cycle promoting factors, such as c-myc, cyclin D2,

CDK2, and cyclin E, are downregulated387-389. Futhermore, TGFP induces epithelial to

mesenchymal transition that has been associated with increased tumor cell motility, invasion and metastasis177' 178. Active TGFP, particularly TGFpi is secreted by many

human cancers. High levels of active TGFP in the circulation are correlated with

enhanced invasion and metastases130. Therefore, the blockade of TGFP oncogenic

properties without affecting its homeostatic role in healthy tissues has great relevance for therapy of cancer. Several TGFP antagonists have been developed to neutralize tumor-

derived active TGFp131'390-394 For instance, the use of type I TGFP receptor (TPRI)

kinase inhibitors and TGFP neutralizing antibodies enhance tumor immunity and reduce tumor angiogenesis and metastasis. In particular, soluble type II TGFP receptor (TPRII) treatment in mice bearing tumors has shown to significant decrease tumor metastasis and

199 induces anti-tumor immunity131'392'395. Transgenic mice expressing soluble TJ3RII fused to Fc fusion protein class (Fc:T|3RII), under the regulation of the mammary-selective

MMTV-LTR promoter/enhancer were protected to the development of metastases arise from either an endogenous primary tumor or from injection of metastatic cells.

Importantly, the metastases from endogenous primary tumors were suppressed without inducing life threatening pathology observed in TGFJ31 null mice, which develop severe immune system defects characterized by lethal multifocal inflammatory syndrome 396.

When TGFJ31 null mice were crossed onto a Rag2 null background to prevent inflammatory syndrome, TGFJ31 null mice developed colon carcinoma with high incidence397. The analysis of aged transgenic mice expressing soluble TJ3RII indicate that lifetime exposure to this form of TGF|3 antagonist did not provoke any increase of

spontaneous tumorigenesis, which suggest that soluble TJ3RII antagonist is able to

discriminate between TGF|3 required for the maintenance of normal homeostasis and the

undesired amount involved in disease pathogenesis. On the other hand, all normal cells

potently produce TGF|3 exclusively in its latent form and the activation of latent TGF|3

occurs very locally on a cell-by-cell basis. In contrast, tumor cells and more remarkable

metastatic cells produce significant amounts of active TGF|3. An antagonist like soluble

TJ3RII may neutralize the large amounts of active TGF|3 that are present in pathological

states without affecting the physiological levels of cell-associated active TGF|3 in normal tissues or early stage tumors368. However, Fc:T|3RII treatment did not alter primary tumor growth of transplantable models of breast cancer metastases, which suggest that the

antimetastatic effects of Fc:T|3RII in vivo are independent of tumor cell proliferation128'

398' 399. In addition, inhibition of tumor angiogenesis in vivo by Fc:T|3RII treatment is

200 controversial. Muraoka et al. observed no reduction of vascular density in endogenously

arising tumors131, whose findings are in contrast to other studies using TGF|3 inhibitors

127,390,400 §jmjjarjy^ t|3RI kinase inhibitors (SD-093 and SD-208) and TGF|3 neutralizing

antibodies therapies although blocks TGF|3-induced epithelial to mesenchymal transition

(EMT) and reduce lung metastases, they fail to prevent or completely inhibit primary tumor growth of mammary carcinomas. In addition, the observed antitumor activity was

modest in magnitude, which could be justified by the role of TGF|3 as tumor promoter at

relatively late stages of tumor progression, when solely blocking TGF|3 pathway is

unlikely to be sufficient to reverse the established tumor-induced immune suppression127,

401. Although, the blockade of TGF|3 signaling has strong therapeutic potential, a potent immune stimulation is required to shift the balance of the immune response in favor of a

robust anti-tumor immunity. To address this hypothesis, we generated a new chimeric

protein that comprises IL-2 fused to the ectodomain of TGF|3 receptor II (aka FIST).

Mechanistically, FIST not only acts as decoy receptor for active TGF|3 but also inhibits

TGF|3 canonical pathway by inducing SMAD7 expression due to hyperactivation of

STAT1 downstream of the IL-2 receptor. Interestingly, the hyperactivation of STAT1 by immune cells leads to resistance to TGF|3 mediated suppression and therefore secretion of

substantial amounts of Thl cytokines and chemokines. In particular, FIST-activated NK

cells secrete high amounts of IFNy and the angiostatic chemokine CXCL10. Moreover,

FIST-activated NK cells display significant STAT1 mediated T-bet upregulation, a

"master regulator" of commitment to T helper type 1 lineage and positive regulator of

IFNy expression. As a result, FIST secreted by genetically modified B16 melanoma cells

prevented tumor formation in immunocompetent mice and in several immunodeficient

201 mice including CD4 KO, CDS KO, B-cell deficient (|iMT) and NK-defective beige mice, whereas mice with NK defective functions such as nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice and Rag2/yc KO mice developed tumors and were

sacrificed. We also observed a robust FIST-mediated bystander effect in vivo and the

development of tumor immunity that protect mice against tumor challenge with live B16 tumor cells. These spectacular antitumor effects are due not only to FIST-mediated

stimulatory properties in IL2-receptor expressing cells but also are due to FIST-mediated

angiostatic properties in NK cells. Due to the fact that FIST targets all the immune cell types that express the IL-2 receptor, we investigated the effect of FIST on B cells, and we

observed that FIST-stimulated B cells display enhanced APC features, such as

upregulation of co-stimulatory molecules, activation marker and MHC class II

expression. In vitro, FIST-activated B cells induce a robust activation of antigen-specific

CD4+ and CDS T cells, which is essentially required for the development of an effective

antitumor immunity. Indeed, FIST-stimulated B cells induce complete protective immunity against EG.7 tumor challenge. Consequently, FIST-stimulated B cells

constitute an alternative source of potent APC that can be pulsed with tumor-associated

antigens to generate personalized cellular pharmaceuticals for the immunotherapy of

cancer. The innovative aspect of our fusokines is based on the therapeutic potential of

new chimeric proteins that not only recapitulate the immunostimulatory properties of

each component but also display novel immunopharmacologic features. GIFT2 induces a

significant NK cell activation and cytotoxic activity while overcomes GM-CSF mediated

NK cell suppression. On the other hand, FIST acts specifically on IL-2 receptor

expressing cells (i.e T cells, NK and NKT cells), triggering signal transduction that made them hyper-activated and resistant to TGF|3 dependent immunosuppression.

202 Consequently, FIST promotes an effective antitumor response that completely blocks tumor metastasis, inhibits tumor growth and prolongs survival. In summary, these

chimeric proteins can be delivered as recombinant proteins in vivo since have shown no toxicity or they can be used as a mean to generate activated immune cells such as NK or

B cells as cellular pharmaceuticals. The anti-cancer strategy here developed is entirely

novel and complementary to current standard of care (chemotherapy, hormonal therapy,

surgery and radiotherapy). Indeed, the harnessing of the immune system as a means to

eradicated minimal residual disease and/or treating advanced chemorefractory disease is a

venue which would offer interesting options for patients with otherwise incurable disease.

Furthermore, this pre-clinical strategy here examined in experimental mice can be readily translated to a human pharmaceutical by generating the human ortholog of our mouse fusokine, and serve as the basis of future phase I/II studies of patients suffering from

cancer (therapy) or at high risk of relapse following treatment (prophylaxis).

203 CHAPTER 7: Contribution to original knowledge

204 Chapter 7: Contribution to original knowledge

I. My main contribution to the original knowledge is the development and

characterization of a new chimeric protein, the fusion of IL-2 and the ectodomain of

TGF|3 receptor II (aka FIST). FIST is the first bi-functional protein endowed with the

ability to antagonize TGF|3 canonical signaling pathway coupled to a potent immune

stimulation. FIST promotes a robust anti-tumor bystander effect and protective

immunity against tumor challenge. The most important feature is that FIST blocks

tumor-derived angiogenesis, which makes FIST a potential pharmaceutical for all

types of carcinomas that by nature require blood supply for growth and progression

into metastases.

II. I have characterized and evaluated FIST-stimulated B cells as a novel APC for cell-

based therapy of cancer. I have demonstrated that FIST-stimulated and antigen pulsed

B cells confer complete protective immunity against tumor challenge.

III. I have generated and characterized the human ortholog of GIFT2, as well as evaluate

its effect on NK cells, which could be used for the generation of oncolytic NK cells as

cell-based therapy of cancer.

IV. As continuation of Jonh Stagg et al. work, I have assessed the bystander effect of the

murine GIFT2 and determined that GIFT2-mediated bystander effect can be

attenuated by tumor-derived active TGF|3 in a doses dependent manner.

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