Re-programming Immunity Against Glioblastoma via RNA Nanoparticle Vaccines

by

Elias Joseph Sayour

Department of Pathology Duke University

Date:______Approved:

______Duane Mitchell, Supervisor

______Soman Abraham

______Matthias Gromeier

______Oren Becher

______Salvatore Pizzo, Chair

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology in the Graduate School of Duke University

2015

i

v

ABSTRACT

Re-programming Immunity Against Glioblastoma via RNA Nanoparticle Vaccines

by

Elias Joseph Sayour

Department of Pathology Duke University

Date:______Approved:

______Duane Mitchell, Supervisor

______Soman Abraham

______Matthias Gromeier

______Oren Becher

______Salvatore Pizzo, Chair

An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology in the Graduate School of Duke University

2015

Copyright by Elias Joseph Sayour 2015

Abstract

Despite aggressive surgical resection, cytotoxic chemotherapy, and external beam radiotherapy, most cases of glioblastoma (GBM) remain recalcitrant. These outcomes necessitate novel developmental therapeutics that spare normal tissue.

Immunotherapy is a promising novel adjuvant treatment that can harness the cytotoxic capacity of the immune system against tumor-associated antigens with exquisite specificity. To circumvent the challenges associated with the advancement of adoptive cellular immunotherapy, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor derived RNA to peripherally activate T cells against GBM antigens. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a NP vaccine formulation that can reshape a host’s immune profile through combinatorial delivery of RNAs encoding for tumor antigens and RNAs encoding for immunomodulatory molecules to mediate long-lived T cell persistence.

We sought to assess if vaccination with amplified tumor derived RNA encapsulated in lipophilic NPs could be assembled to transfect antigen presenting cells

(APCs) in vivo and induce therapeutic anti-tumor immunity in pre-clinical murine tumor models. We hypothesized that RNA encapsulated nanoliposomes would localize to reticuloendothelial organs such as the spleen and liver, transfect APCs therein and

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induce peripheral antigen specific T cell immunity against GBM. Since activated T cells can cross the blood brain barrier and exert their effector functions against GBM antigens, peripheral transfection of APCs by RNA-NPs represents an attractive vaccination approach for priming endogenous immunity against refractory brain tumors.

We screened several translatable NP formulations for their ability to transfect dendritic cells (DCs) in vitro with GFP mRNA. We demonstrated that the NP DOTAP was the most promising translatable formulation compared to alternative cationic liposomal preparations and linear polyethylenimine NPs with and without DC targeting mannose receptors. RNA-NP vaccines formulated in DOTAP were shown to induce in vivo gene expression and preserve RNA stability over time. We determined that intravenous (IV) injection of RNA-NPs was requisite for inducing functional antigen specific immunity, which was superior to standard peptide vaccines formulated in complete Freund’s adjuvant (CFA). IV administered RNA-NPs localized to splenic and hepatic white blood cells (WBCs); these cells expanded antigen specific T cells when transferred to naïve immunocompetent mice. RNA-NPs induced increased percentages of B7 co-stimulatory molecules, but also elicited compensatory PD-L1 expression. We enhanced the immunogenicity and anti-tumor efficacy of RNA-NP vaccines by combining RNA-NPs with immune checkpoint blockade against PD-L1. We also enhanced the immunogenicity and efficacy of this platform by simply combining mRNAs encoding for immunomodulatory cytokines (i.e. GM-CSF). Finally, we

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demonstrated that RNA-NP vaccines mediate anti-tumor efficacy against intracranial and subcutaneous melanomas and engender therapeutic anti-tumor efficacy in a cellular immunotherapy model against a radiation/temozolomide resistant invasive murine high-grade glioma.

GBM remains invariably associated with poor patient outcomes thus necessitating development of more targeted therapeutics. Clinically translatable RNA-

NPs form stable complexes making them amenable to overnight shipping. They induce potent immune responses when administered systemically and mediate robust anti- tumor efficacy that can be enhanced through co-delivery of immunomodulatory RNAs.

This technology can simultaneously bypass the complexity of cellular therapeutics while cutting down the time to generation of personalized vaccines. Since RNA-NP vaccines can be made within days from a tumor biopsy, providing near immediate immune induction against GBM, these formulations can provide a more feasible and effective therapy with a wide range of applicability for all malignancies that can be targeted using

RNA obtained from surgical resection of solid tumors.

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Dedication

To my parents Zakieh and Antoine Sayour, you’re unconditional love has been the life force of my intended path.

To Dr. Pierre Zalzal, whose life was cut all too short by this terrible tumor, you inspired me to question and nourished my curiosity.

To my darling wife Tanya, it was not until meeting you that I discovered my wants in life.

And, to my precious children Luke and Lilah, you above all motivate me to create a better tomorrow.

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Contents

Abstract ...... iv

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations ...... xvii

Acknowledgements ...... xxii

1. Glioblastoma and Analysis of Intratumoral T cell Infiltrates ...... 1

1.1 Glioblastoma and Immunotherapy ...... 1

1.2 Analysis of Intratumoral T cell Infiltration in GBM ...... 2

1.3 Conclusion ...... 15

2. Enhancing Effector TILs via RNA Vaccines ...... 17

2.1 Adoptive Cellular Therapy ...... 17

2.2 Cancer Vaccines ...... 18

2.3 ‘Naked’ RNA Vaccines ...... 20

2.4 RNA Loaded Dendritic Cell Vaccines ...... 24

2.5 RNA Encapsulated Liposomes ...... 25

2.6 Harnessing RNA-loaded Nanoliposomes against GBM ...... 27

3. Materials and Methods...... 30

3.1 Isolation of RNA ...... 30

3.2 Nanoparticles ...... 30

3.3 RNA-NP Complex Preparation ...... 31

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3.4 Cryo-electron Microscopy ...... 31

3.5 Dynamic Light Scatter and Zeta-Potential Analysis ...... 32

3.6 Mice ...... 32

3.7 Bioluminescent Imaging ...... 33

3.8 OT-I Transfer and RNA-NP Vaccination ...... 33

3.9 Flow Cytometric Analysis ...... 34

3.10 Functional Stimulation Assay ...... 34

3.11 Anti-PD-L1 Monoclonal Antibodies ...... 35

3.12 Tumor Cells ...... 35

3.13 Tumor Implantations ...... 36

3.14 Dendritic Cell Vaccines ...... 36

3.15 Generation of Tumor Specific T cells ...... 37

3.16 Cellular Immunotherapy High Grade Glioma Model ...... 38

3.17 Statistical Analysis ...... 39

4. RNA Nanoparticle Vaccines: Characterization Studies ...... 40

4.1 Introduction ...... 40

4.2 Identifying a Target RNA-NP Formulation ...... 41

4.3 Size and Charge of RNA-NPs encapsulated in DOTAP ...... 45

4.4 RNA-NP “Off the Shelf” Stability and Translation Efficiency ...... 46

4.5 Discussion ...... 49

5. Immune Responses from RNA-Nanoparticle Vaccines ...... 52

5.1 Introduction ...... 52

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5.2 Antigen Specific Immune Responses...... 53

5.3 Serum Cytokine Levels Post-Vaccination ...... 59

5.4 Discussion ...... 60

6. Localizing Effects of RNA-Nanoparticle Vaccines on the Spleen and Liver ...... 62

6.1 Introduction ...... 62

6.2 Gene Expression from RNA-NPs in the Spleen and Liver ...... 62

6.3 Characterization of CD11c expressing Splenocytes and Hepatic WBCs ...... 64

6.4 Discussion ...... 72

7. Effects of RNA-Nanoparticles on Co-stimulatory Molecules and Immune Checkpoints ...... 74

7.1 Introduction ...... 74

7.2 Expression of B7 Co-stimulatory Molecules following RNA-NP Vaccination ...... 75

7.3 Expression of Immune Checkpoints following RNA-NP Vaccination ...... 77

7.4 Discussion ...... 86

8. Tumor Efficacy Induced by RNA-Nanoparticle Vaccines and Capacity for Immunomodulatory Enhancement ...... 88

8.1 Introduction ...... 88

8.2 Anti-tumor Efficacy ...... 89

8.3 Capacity for Enhancement ...... 90

8.4 Alternative Strategies for Enhancement ...... 96

8.5 Enhancing RNA-NP Efficacy with Combinatorial Checkpoint Blockade ...... 102

8.6 Discussion ...... 105

9. Supplanting DC vaccines with RNA-Nanoparticles targeting GBM ...... 109

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9.1 Introduction ...... 109

9.2 DC Vaccines against Intracranial Tumors...... 111

9.3 Replacing DCs with RNA-NP Vaccines ...... 112

9.4 RNA-NPs versus DC vaccines ...... 113

9.5 Discussion ...... 118

10. Conclusions and Future Directions ...... 120

10.1 Summary ...... 120

10.2 Future Directions ...... 122

10.3 Conclusion ...... 123

References ...... 125

Biography ...... 147

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List of Tables

No table of figures entries found.

xii

List of Figures

Figure 1: IHC of GBM pathology specimen ...... 7

Figure 2: Manuel counting of GBM pathology specimen by IHC versus Aperio analysis 8

Figure 3: Concordance between FoxP3 expression alone and FoxP3+CD3+ cells ...... 10

Figure 4: Concordance between FoxP3 expression alone and FoxP3+CD4+ cells ...... 11

Figure 5: Concordance between FoxP3 expression and FoxP3+CD8+ cells...... 12

Figure 6: CD4/FoxP3 in primary versus recurrent GBM ...... 13

Figure 7: CD3+/ FoxP3+ TILs versus survival ...... 14

Figure 8: CD8+/ FoxP3+ TILs versus survival ...... 15

Figure 9: Comparison of commercially available NPs on DC transfection in vitro ...... 42

Figure 10: Comparison of commercially available NPs on OVA specific immunity in vivo ...... 43

Figure 11: Ratio of mRNA to DOTAP ...... 44

Figure 12: Cryo-electron microscopy of NPs and RNA encapsulated NPs ...... 45

Figure 13: Dynamic Light Scatter of RNA-NPs ...... 46

Figure 14: RNA-NP stability over time ...... 48

Figure 15: RNA-NP in vivo expression ...... 49

Figure 16: Comparison of antigen specific immunity from RNA-NP vaccines by different routes of administration ...... 54

Figure 17: Gating strategy of antigen specific immune responses ...... 55

Figure 18: OVA Specific T cell expansion by CFSE Dilution ...... 56

Figure 19: Comparison of OVA specific T cell immunity from murine splenocytes following RNA-NPs and OVA peptide vaccines in CFA ...... 57

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Figure 20: Comparison of antigen specific immunity in lymph nodes from RNA-NP vaccines versus CFA-OVA...... 58

Figure 21: Functional T cell assays from splenocytes of mice vaccinated with RNA-NPs ...... 59

Figure 22: IL-6, TNF-alpha and CCL2 levels post-vaccination ...... 60

Figure 23: GFP RNA-NP localization to the spleen and liver ...... 63

Figure 24: GFP RNA-NP localization to CD11c cells in the spleen and liver ...... 64

Figure 25: Percentage of CD11c+ cells in the spleen and liver post RNA ...... 65

Figure 26: MHC II expression on CD11c+ cells in the spleen and liver ...... 66

Figure 27: CD40 expression on CD11c+ cells in the spleen and liver post RNA-NP vaccination ...... 67

Figure 28: F4-80 expression in the spleen and liver post RNA-NP vaccination...... 68

Figure 29: Gr-1 expression in spleen and liver post RNA-NP vaccination ...... 69

Figure 30: CD11b+ expression on Gr-1+ cells in the spleen and liver post RNA-NP vaccination ...... 70

Figure 31: Gr-1 expression on CD11c+ cells in the spleen and liver post RNA-NP vaccination ...... 71

Figure 32: Antigen specific immunity induced in naïve mice from adoptively transferred splenic and hepatic WBCs harvested from RNA-NP vaccinated mice ...... 72

Figure 33: CD80 expression on CD11c+ cells in the spleen and liver post RNA-NP vaccination ...... 76

Figure 34: CD86 expression on CD11c+ cells in the spleen and liver post RNA-NP vaccination ...... 77

Figure 35: PD-L1 expression in the spleen and liver post RNA-NP vaccination ...... 78

Figure 36: PD-L1 expression on CD11c+ cells in the spleen and liver post RNA-NP vaccination ...... 79

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Figure 37: PD-L1 expression in the spleen and liver after combinatorial administration of RNA-NPs and anti-PD-L1 mAbs ...... 80

Figure 38: PD-L1 expression on CD11c expressing cells in the spleen and liver after combinatorial administration of RNA-NPs and anti-PD-L1 mAbs ...... 81

Figure 39: MHC II+ splenocytes and CD11c+ liver WBCs expressing CD86 after combinatorial administration of RNA-NPs and anti-PD-L1 mAbs ...... 82

Figure 40: PD-L2 expression on CD11c+ cells in the spleen and liver post RNA-NP vaccination ...... 83

Figure 41: PD-L2 expression on primary ex vivo expanded DCs ...... 84

Figure 42: PD-1 expression on CD8+ splenocytes from mice vaccinated with RNA-NPs 85

Figure 43: RNA-NP efficacy against intracranial tumors ...... 89

Figure 44: GM-CSF ELISA after GM-CSF RNA-NP transfection of DC2.4s ...... 91

Figure 45: Comparison of antigen specific T cell immunity from RNA-NPs co- encapsulated with or without GM-CSF mRNA...... 92

Figure 46: Gating strategy of antigen specific immune responses in liver ...... 93

Figure 47: Comparison of efficacy in mice vaccinated with RNA-NPs co-encapsulated with or without GM-CSF mRNA ...... 94

Figure 48: Tumor volumes of B16F10-OVA in mice receiving RNA-NPs co-encapsulated with or without GM-CSF mRNA ...... 95

Figure 49: Comparison of efficacy in mice vaccinated with RNA-NPs co-encapsulated with or without CpG oligonucleotides ...... 97

Figure 50: Comparison of efficacy in mice vaccinated with RNA-NPs co-encapsulated with or without polyI:C ...... 98

Figure 51: HSVsr39tk mRNA transfected DCs in vitro ...... 99

Figure 52: Comparison of efficacy in mice vaccinated with RNA-NPs co-encapsulated with or without HSVsr39tk mRNA ...... 100

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Figure 53: Comparison of efficacy in mice vaccinated with RNA-NPs co-encapsulated with or without HSP mRNA ...... 101

Figure 54: Comparison of efficacy in mice vaccinated with RNA-NPs co-encapsulated with or without CD40L mRNA ...... 102

Figure 55: Tumor volumes of B16F10 in untreated mice versus mice receiving TTRNA- NP vaccines ...... 103

Figure 56: Tumor volumes of B16F10 in mice receiving TTRNA-NPs with or without co- administered anti-PD-L1 mAb ...... 104

Figure 57: Tumor volumes of B16F10 in mice receiving anti-PD-L1 mAbs with or without co-administered TTRNA-NPs ...... 105

Figure 58: Comparison of OVA specific T cell immunity from RNA-NP vaccines versus DC vaccines ...... 114

Figure 59: Representative flow plots comparing expansion of T cells specific against KR158B-luc from RNA-NP vaccines versus DC vaccines in murine splenocytes and lymph nodes ...... 115

Figure 60: Expansion of T cells specific against KR158B-luc from RNA-NP vaccines versus DC vaccines in murine splenocytes and lymph nodes...... 116

Figure 61: Survival curve of mice vaccinated with TTRNA-NP vaccines versus TTRNA pulsed DC vaccines in a cellular immunotherapy model for high grade invasive glioma ...... 117

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List of Abbreviations

APC Antigen Presenting Cell

ALT Adoptive Lymphocyte Transfer

AP1 Activator Protein

BBB Blood Brain Barrier

CAR Chimeric Antigen Receptor

CCL2 Monocyte Chemoattractant Protein 1

CDCM Complete DC Media

CEA Carcinoembryonic Antigen

Chol Cholesterol

CNS Central Nervous System

CFA Complete Freund’s adjuvant

CFSE Carboxyfluorescein succinimidyl

CTL Cytotoxic T Lymphocyte

CSF Cerebrospinal Fluid

DC Dendritic Cell

DLS Dynamic Light Scatter

DNA Deoxyribonucleic acid

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DOPE dioleoyl-L-a-phosphatidylethanolamine

DOTAP 1,2-dioleoyloxypropyl-3-N,N,N-trimethylammonium

EGFRvIII Epidermal Growth Factor variant III

FBS Fetal Bovine Serum

Fig Figure

GBM Glioblastoma

GM-CSF Granulocyte macrophage colony stimulating factor

GMP Good Manufacturing Practice

GCV Ganciclovir

GFP Green Fluorescent Protein

Gy Gray

H and E Hematoxylin and Eosin

HCV Hepatitis C Virus

HSC Hematopoietic stem cell

HSP Heat Shock Protein

HSV-TK Herpes Simplex Virus- Thymidine Kinase i.d. Intradermal i.p. Intraperitoneal

IKK IκB kinase

IHC Immunohistochemistry

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IFN Interferon

IRF Interferon Regulatory Factor isRNA immunostimulatory RNA i.v. Intravenous

IVT in vitro transcription

JETPEI linear polyethylenimine

Lin- Lineage negative mAb monoclonal Antibody

MAPK Mitogen-Activated Protein Kinases

MDSC Myeloid Derived Suppressor cells

MHC Major Histocompatibility Complex mRNA messenger RNA

MYD88 Myeloid Differentiation primary-response protein 88

NF-κB Nuclear Factor-κB nm nanometer ns not significant

NSCLC Non-Small Cell Lung Cancer

NP Nanoparticle

OVA Ovalbumin

OT-I Ovalbumin-specific TCR transgenic

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PAMP Pathogen Associated Molecular Pattern

PRR Pathogen Recognition Receptor

PBMCs Peripheral Blood Mononuclear Cell

PD-1 Programmed Death-1

PD-L1 Programmed Death Ligand-1

PEG Polyethylene Glycol

PPCv9 Positive Pixel Count v9

RCC Renal Cell Carcinoma

RES Reticuloendothelial System

RNA Ribonucleic Acid s.c. Subcutaneous

ScFv single chain antibody fragments

TAA Tumor Associated Antigen

TAM Tumor Associated Macrophage

TANK TRAF family member–associated NF-κB activator

TBI Total body irradiation

TBK TANK binding kinase

TCR T cell Receptor

TIL Tumor Infiltrating Lymphocyte

TLR Toll Like Receptor

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TRAF TNF receptor–associated factor

Tregs Regulatory T cells

TTRNA Total Tumor derived RNA

UFBTIP University of Florida Brain Tumor Immunotherapy Program

UTR Untranslated Region

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Acknowledgements

I want to thank the members of the Duke Brain Tumor Immunotherapy Program for their tutelage and support. I am especially indebted to Luis Sanchez-Perez, who taught me each day how to be a better scientist.

I want to express my gratitude to Dean John Klingensmith for supporting my vision as a physician-scientist, and my candidacy as a Duke graduate student. Your advocacy opened doors that were otherwise closed thereby fortifying my educational experience and potentiating my career goals.

I want to thank Dr. Soman Abraham who has tirelessly advocated on my behalf.

Your support never wavered and provided a calm light during graduate school’s tumultuous times.

I am grateful to all the members of the University of Florida Brain Tumor

Immunotherapy Program, especially Gabe De Leon, Christina Pham and Catherine

Flores. Your guidance and camaraderie have been invaluable. Thank you for always being there.

Lastly, I want to thank my mentor Duane Mitchell. I look up to you in more ways than I can articulate. You have allowed me to dream the impossible and nourished every one of my ambitions. You have instilled in me a belief in myself and my abilities I never knew existed. You have spent endless hours mentoring me not only in science but also in life. Your mentorship, counsel and friendship have made inroads

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that have shaped every aspect of my career and molded my vision of the future. Thank you, ever so much, for believing in me.

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1. Glioblastoma and Analysis of Intratumoral T cell Infiltrates

1.1 Glioblastoma and Immunotherapy

Glioblastoma (GBM) is the most common malignant brain tumor in adults but despite aggressive surgical resection, cytotoxic chemotherapy, and external beam radiotherapy, most GBMs remain recalcitrant with median overall survivals less than 12-

15 months (Stupp et al., 2009; Stupp et al., 2005). Due to the severe, non-specific damaging effects of radiation and chemotherapy, targeted therapies capable of selectively killing tumor cells in patients with GBM are desperately needed (D. A.

Mitchell, Fecci, & Sampson, 2008; Stupp et al., 2009; Stupp et al., 2005).

A major limiting factor for the development of novel therapeutics against GBM remains the blood brain barrier (BBB). Since peripherally activated T cells have been shown to traverse the BBB and suppress tumor growth, tumor-specific immunotherapy holds the promise of eradicating malignant brain tumors with exquisite precision without additional toxicity to standard treatments (Miao et al., 2014; D. A. Mitchell et al.,

2008; Paller & Antonarakis, 2012; Ransohoff, Kivisakk, & Kidd, 2003; Ribas, 2007;

Sampson et al., 2011; Schuster et al., 2011; Sayour, Sanchez-Perez, et al, 2015).

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1.2 Analysis of Intratumoral T cell Infiltration in GBM

While T cells can cross the BBB and infiltrate GBMs, endogenous tumor infiltrating lymphocytes (TILs) may contain FoxP3 regulatory T cells that can stymie an inflammatory effector response (Sayour, McLendon, et al., 2015). Through immunohistochemical staining, we have shown that T cell infiltrates are found in primary and recurrent GBM specimens (Fig. 1) (Sayour, McLendon, et al., 2015). To more thoroughly quantify the presence of effector versus regulatory TILs, we analyzed

GBM specimens using a novel automated software (Aperio) (Sayour, McLendon, et al.,

2015). Aperio avoids evaluator dependent bias of cell counting in high power fields

(Sayour, McLendon, et al., 2015). This analysis allowed for accurate and expeditious detection of lymphocytic infiltrates from entire tissue sections enabling us to more fully analyze whole GBM slides for TIL presence (Sayour, McLendon, et al., 2015).

1.2.1 Methods

The methods, figures and excerpts below contained in this chapter are taken directly from our publication by Sayour and Mclendon et al. (Cancer Immunology,

Immunotherapy, 2015). Relevant permissions and a license agreement with Springer were provided by the Copyright Clearance Center on November 13, 2015. Briefly, De- identified archival GBM tissue samples were obtained with IRB approval from the

Preston Robert Tisch Brain Tumor Center Biorepository (Sayour, McLendon, et al., 2015).

GBMs were selected from archived resected tumor blocks preserved in cryogenic storage

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(Sayour, McLendon, et al., 2015). Serial section slides from each tumor were fixed in formalin, embedded in paraffin and stained for CD3, CD4, CD8 or FoxP3 using standard immunohistochemistry (IHC) techniques (Sayour, McLendon, et al., 2015). Tonsil sections were utilized to determine efficiency of each stain and overall technique

(Sayour, McLendon, et al., 2015).

1.2.1.1 Study Population

The material consisted of representative formalin-fixed, paraffin embedded GBM sections taken from 39 patients, 21 undergoing primary surgery for untreated GBM

(primary group) and an additional 18 patients undergoing surgery for recurrent GBM

(recurrent group), at Duke University Medical Center (Sayour, McLendon, et al., 2015).

The study population consisted of 33 males and 24 females with an age range from 26 to

79 years of age at the time of tumor resection/biopsy (Sayour, McLendon, et al., 2015).

The study population had an average age of 52, and was primarily made up of individuals of Caucasian descent (n=55) along with two individuals of African American descent, one individual of Hispanic descent and one individual of Indian descent

(Sayour, McLendon, et al., 2015). Representative blocks of tissue were chosen by a neuropathologist at Duke University Medical Center to represent nearly 100% viable tumor in the selected block and over 1 cm2 of tissue section by light microscopic examination of Hematoxylin and Eosin (H&E) stained sections (Sayour, McLendon, et

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al., 2015). Serial unstained sections were cut from these blocks and submitted for IHC staining by FoxP3, CD3, CD4, and CD8 subset analysis (Sayour, McLendon, et al., 2015).

1.2.1.2 Immunohistochemistry After staining for the T-cell markers of interest, slides were scanned with a high- resolution scanner (ScanScope CS; Aperio) at 40x magnification and analyzed using image software (Aperio ScanScope) (Sayour, McLendon, et al., 2015). Pixel counts were gated to strongly positive pixel counts using the ScanScope software and the Positive

Pixel Count v9 (PPCv9) algorithm within the program (Sayour, McLendon, et al., 2015).

Evaluation of T-cell marker density was carried out blinded to clinicopathologic information (Sayour, McLendon, et al., 2015). To assure that identical regions of tumor were being examined, serially stained sections of individual biopsies stained for the T cell markers of interest were examined by one observer using the multiple imaging modality of the software (Sayour, McLendon, et al., 2015). To determine the possibility that cell size in a histologic section could result in a variation of cell counts via a highly variable pixel count per cell, a manual count of individually stained cells identified on the monitor was performed on a randomly selected subset of 3 tumor samples (Sayour,

McLendon, et al., 2015). For each stain within these samples, four loci of highest T-cell density were identified and manually assessed for positively stained cells (Sayour,

McLendon, et al., 2015). These same loci were evaluated through the PPCv9 algorithm

(Sayour, McLendon, et al., 2015). The pixel counts and raw cell counts for each loci were

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entered into an excel file and evaluated for concordance and statistical relationship

(Sayour, McLendon, et al., 2015). We additionally configured the PPCv9 algorithm to produce a hyperpigmented digital color overlay of each tissue area analyzed to allow clear identification and pathological classification of each positively stained cell (Sayour,

McLendon, et al., 2015).

Once the chosen methodology was successfully evaluated and the stained tumor slides were digitized, whole tissue area analysis was performed on each slide file with the PPCv9 algorithm (Sayour, McLendon, et al., 2015). Individual data from each slide was recorded in an excel file for statistical analysis (Sayour, McLendon, et al., 2015).

1.2.1.3 Statistical Analysis

Absolute counts were divided by surface area of each specimen to standardize measurements, and the ratio of CD4, CD8, and FoxP3+ cells was measured over CD3+ cells (Sayour, McLendon, et al., 2015). Analysis of these values was obtained via unpaired t tests with a significant result limited to p-values of less than 0.05 (Sayour,

McLendon, et al., 2015). Proportions of CD3, CD4, and CD8+ cells over FoxP3 expressing cells was also measured with unpaired t tests and significant results were limited to p-values of less than 0.05 (Sayour, McLendon, et al., 2015). Correlations of primary GBM survival with CD3, CD4, and CD8+ over FoxP3+ cell ratios were evaluated using standard linear regression models (Sayour, McLendon, et al., 2015).

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1.2.2 Immunohistochemistry of intratumoral T cells by manual cell counting versus Aperio automated analysis

We analyzed CD3, CD4, CD8 and FoxP3+ cells from three different intratumoral pathology specimens by conventional cell counting and via Aperio analysis (Sayour,

McLendon, et al., 2015). Figure 1 shows a primary GBM IHC specimen stained for

FoxP3 next to Aperio software analysis of the same specimen (Sayour, McLendon, et al.,

2015). Strongly positive pixel counts (shown in Fig. 1 as cells colored in red, with background stained in blue) represent the quantitative measure of detecting FoxP3+ cells by Aperio analysis (Sayour, McLendon, et al., 2015). Manual cell counting by a trained observer correlates well with Aperio analysis of identical sections (as measured by strongly positive pixel counts with a correlation coefficient of 0.9353; p < 0.0001, Fig. 2)

(Sayour, McLendon, et al., 2015). While manual counting was limited to selected high- powered fields and subject to observer bias, automated Aperio analysis indiscriminately scanned selected fields and demonstrated strong concordance with manual cell counting

(Sayour, McLendon, et al., 2015).

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Figure 1: IHC of GBM pathology specimen

GBM specimen stained for FoxP3 by IHC (top) and Aperio analysis by strong positive pixels in red (bottom) (Sayour, McLendon, et al., 2015).

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Figure 2: Manuel counting of GBM pathology specimen by IHC versus Aperio analysis

Overlay of GBM specimen stained for FoxP3 by IHC with Aperio analysis (top). Correlation of manual cell counting by trained observer versus strong positive pixel count by Aperio automated analysis (bottom) (****p < 0.0001; R value 0.9353) (Sayour, McLendon, et al., 2015).

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1.2.3 Intratumoral T cell proportions in Glioblastoma and survival outcome

Several studies have assessed the prognostic significance of TILs in gliomas, but the reported results have been mixed possibly due to the effects of FoxP3+ T cells (El

Andaloussi, Han, & Lesniak, 2006; A. B. Heimberger et al., 2008; Jacobs et al., 2010; Lohr et al., 2011; Sayour, McLendon, et al., 2015; Sonabend, Rolle, & Lesniak, 2008; Yang et al.,

2010; Yue et al., 2014). Despite having overall depleted lymphocyte numbers in the periphery, the proportion of FoxP3 expressing regulatory T cells (Tregs) is elevated within peripheral blood mononuclear cells (PBMCs) of GBM patients suggesting that these cells contribute significantly to the profound systemic cell-mediated immune deficits observed in this disease (Fecci et al., 2007; P. E. Fecci et al., 2006; Learn et al.,

2006; D. A. Mitchell et al., 2011; Sayour, McLendon, et al., 2015). However, the proportion of FoxP3+ cells in GBM TILs and effect on prognosis and outcome remains indeterminate.

1.2.3.1 FoxP3 Validation Studies

Since FoxP3 is not an exquisitely specific marker for regulatory T cells and can be transiently expressed in subsets of activated T effector cells, we sought to assess whether

FoxP3 expression within GBM TILs delineated CD4+FoxP3+ T cells from other cell populations. Therefore, 7 separate GBM samples were dual-stained for FoxP3 by CD3,

CD4 or CD8 (Sayour, McLendon, et al., 2015). Concordance testing for FoxP3 expression versus FoxP3+CD3+, FoxP3+CD4+, and FoxP3+CD8+ cells demonstrate very strong

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correlations with FoxP3 expression and FoxP3+CD3+ and FoxP3+CD4+ cells by double staining (Fig. 3-4); however, there were no identified FoxP3+CD8+ cells and thus no correlation with FoxP3 and FoxP3+CD8+ lymphocytes (Fig. 5) (Sayour, McLendon, et al.,

2015). These data demonstrate strong concordance between FoxP3 expression alone and double staining for FoxP3 by CD4 or CD3 and support the utility of FoxP3 single staining as a marker for CD4+FoxP3+ regulatory T cells within GBMs (Sayour,

McLendon, et al., 2015).

Figure 3: Concordance between FoxP3 expression alone and FoxP3+CD3+ cells

GBM samples were dual stained for FoxP3 by CD3 and compared for expression levels of FoxP3 alone versus dual staining. There is a very strong positive correlation between FoxP3 expression alone and FoxP3+CD3+ double staining (****p value < 0.0001; correlation coefficient = 0.9968) (Sayour, McLendon, et al., 2015).

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Figure 4: Concordance between FoxP3 expression alone and FoxP3+CD4+ cells

GBM samples were dual stained for FoxP3 by CD4 and compared for expression levels of FoxP3 alone versus dual staining. There is a very strong positive correlation between FoxP3 expression alone and FoxP3+CD4+ double staining (****p value < 0.0001; correlation coefficient = 0.9975) (Sayour, McLendon, et al., 2015).

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Figure 5: Concordance between FoxP3 expression and FoxP3+CD8+ cells.

GBM samples were dual stained for FoxP3 by CD8 and compared for expression levels of FoxP3 alone versus dual staining. There was no correlation between FoxP3 expression alone and FoxP3 expression by CD8+ double staining (Sayour, McLendon, et al., 2015).

1.2.3.2 FoxP3 proportions in primary versus recurrent GBM

Since we validated that FoxP3 single staining could delineate Tregs, and since

Aperio software could analyze whole IHC slides with concordance to manual cell counting, we analyzed the proportion of FoxP3+ TILs in primary versus recurrent GBMs

(Sayour, McLendon, et al., 2015). It was determined that there was a decreased proportion of CD4/FoxP3+ cells in recurrent GBM (Fig. 6) suggesting that as a GBM recurs there is an increased percentage of FoxP3+ CD4 + TILs (Sayour, McLendon, et al.,

2015).

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Figure 6: CD4/FoxP3 in primary versus recurrent GBM

There is an increased proportion of CD4/FoxP3+ cells in primary versus recurrent GBM by strong positive pixilation. To normalize slides across specimens, absolute counts by strong positive pixilations were divided by their respective slide’s surface area (*p = 0.0137, unpaired t test) (Sayour, McLendon, et al., 2015).

1.2.3.3 FoxP3 proportions versus survival outcomes in primary GBM

Since effector TILs that enhance anti-tumor activity may be suppressed by Tregs expressing FoxP3, the ratio of these compartments in the intratumoral microenvironment may have stronger prognostic implications than a single T cell subset on the overall anti-tumor response (Bennett et al., 2001; Brunkow et al., 2001; Khattri,

Cox, Yasayko, & Ramsdell, 2003; Sayour, McLendon, et al., 2015). Therefore, we compared the proportion of CD3+ or CD8+ cells over FoxP3+ cells versus survival in patients with primary GBM. We found that an increased TIL proportion of CD3 or

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CD8+ cells over FoxP3+ TILs correlated directly with survival outcomes in our study population (Fig. 7-8) (Sayour, McLendon, et al., 2015).

Figure 7: CD3+/ FoxP3+ TILs versus survival

Primary GBM pathology specimens were stained for CD3 and FoxP3+ markers and quantified using Aperio software. Survival (in days) of patients was plotted versus ratios of CD3 over FoxP3+ cells. Patients still alive at the time of data analysis are represented by a clear circle. There is a positive correlation between survival and increased CD3+ to FoxP3+ ratios (***p = 0.0005; R value = 0.7091) (Sayour, McLendon, et al., 2015).

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Figure 8: CD8+/ FoxP3+ TILs versus survival

Primary GBM pathology specimens were stained for CD8 and FoxP3+ markers and quantified using Aperio software. Survival (in days) of patients was plotted versus ratios of CD8+ cells over FoxP3+ cells. Patients still alive at time of data analysis are represented by a clear circle. There is a positive correlation between survival and increased CD8+ to Foxp3+ ratios (**p = 0.0027; R value = 0.6343) (Sayour, McLendon, et al., 2015).

1.3 Conclusion

Since proportions of intratumoral CD3+ and CD8+ T cells (relative to FoxP3+ cells) correlate with GBM survival, enhancing the precursor frequencies of these cells may overcome the tumor’s immunosuppressive niche (D. A. Mitchell et al., 2008). This niche is prototypically characterized by secretion of regulatory cytokines such as IL-10 and TGF-beta, upregulation of regulatory checkpoints (i.e. programmed death ligand) and infiltration of immunosuppressive cell subsets including tumor associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs), and FoxP3 expressing

15

Tregs (D. A. Mitchell et al., 2008). To unlock and re-program anti-tumor immunity against GBMs, tipping the balance of TILs in favor of effector T cells mediating tumor lysis is essential (Sayour, McLendon, et al., 2015). This can be accomplished using adoptive cellular immunotherapy or through endogenous activation of T cells.

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2. Enhancing Effector TILs via RNA Vaccines

Excerpts from this chapter are taken directly from our publication by Sayour et al.

(Journal for Immunotherapy of Cancer, 2015).

2.1 Adoptive Cellular Therapy

Strategies have emerged to increase the relative proportions of activated CD8 effector T cells against malignancies through ex vivo activation and expansion before re- introduction (Dudley et al., 2002; Gattinoni et al., 2005; Gros et al., 2014; Restifo, Dudley,

& Rosenberg, 2012; Rosenberg, 2012; Rosenberg & Dudley, 2004; Wrzesinski et al., 2007).

TILs can be harvested from easily accessible tumors, cultured in the presence of high dose IL-2 and adoptively transferred to patients after conditioning chemotherapy

(i.e. cyclophosphamide and fludarabine) (Rosenberg, 2012). While TILs have been shown to mediate regression in cases of refractory tumors, they are ideally generated from easily accessible malignances such as melanomas; however, ample tissue is more difficult to procure in central nervous system (CNS) tumors. Alternatively, chimeric antigen receptors (CARs) have been used to imbue peripherally collected T cells with the specificity of an antibody’s variable region fragments adjoined to the CD3 zeta signaling motif (Garfall et al., 2015; Kalos et al., 2011; Maude et al., 2014; Porter et al., 2015; Porter,

Levine, Kalos, Bagg, & June, 2011; Rapoport et al., 2015). CARs bypass major histocompatibility complex (MHC) class restriction (requisite for antigen presentation

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and T cell activation) and have been used to induce efficacy in human trials for chronic lymphocytic leukemia, acute lymphoblastic leukemia and multiple myeloma (Garfall et al., 2015; Grupp et al., 2013; Maude et al., 2014; Porter et al., 2015; Rapoport et al., 2015).

However, while these adoptive cellular immunotherapeutic strategies hold significant potential to meet the need for more effective and less toxic treatment options for patients with refractory malignancies, they remain limited by cost and complexity.

2.2 Cancer Vaccines

Alternatively, cancer vaccines can be used to endogenously activate T cells against malignancies. Monoclonal antibodies (mAbs) have been developed to passively immunize patients; examples include anti-CD30 mAbs in Hodgkin’s lymphoma

(brentuximab), and anti-CD20 mAbs in Non-Hodgkin’s lymphoma (rituximab). While mAbs are amenable to commercialization, passive immunity with mAbs is not expected to stimulate endogenous immunologic memory. Active cancer immunizations, however, have been employed to engender long-term anti-tumor immunity via tumor peptides, whole tumor lysate, or nucleic acids encoding for tumor associated antigens.

While peptide vaccines are dependent on MHC restriction limiting their availability to the population at large, nucleic acid vaccine bypass this restriction. In addition, nucleic acids are recognizable by pathogen-associated molecular patterns

(PAMPs), which stimulate pathogen recognition receptors (PRRs) such as toll like

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receptors (TLR) and intracellular sensors thereby activating dendritic cells (DCs) and antigen presenting cells (APCs) (Fotin-Mleczek et al., 2011; S. K. Nair et al., 2000; Sayour,

Sanchez-Perez, Flores, & Mitchell, 2015; Scheel et al., 2005; Schlake, Thess, Fotin-

Mleczek, & Kallen, 2012). Through PAMP recognition by PRRs, APCs become activated and release type I interferon (IFN) inducing Th1 type immune responses (Desmet &

Ishii, 2012; Sayour, Sanchez-Perez, et al., 2015). Given their penchant for stimulating innate immunity juxtaposed to their propensity for inducing T cell responses, nucleic acids encoding for cancer antigens are an attractive immunotherapeutic platform for

APC transfection (Sayour, Sanchez-Perez, et al., 2015; Ulmer, Mason, Geall, & Mandl,

2012). The magnitude of these responses has been lower in DNA vaccines, which are hampered by inadequate delivery mechanisms that are mired with constraints of crossing both cell and nuclear membranes (Liu, 2010; Sayour, Sanchez-Perez, et al., 2015;

Ulmer et al., 2012, Coban et al., 2011; Guo et al., 2013). These concerns, along with the potential for oncogenesis through genomic integration, have ignited RNA vaccine research as a promising alternative to DNA vaccines (Sayour, Sanchez-Perez, et al., 2015;

Ulmer et al., 2012). RNA vaccines require only cytoplasm for entry, cannot be integrated into the genome, and are easy to produce and store (Kreiter, Diken, Selmi, Tureci, &

Sahin, 2011; Sayour, Sanchez-Perez, et al., 2015). RNA is also advantageous since it can be derived from limited tumor specimens, amplified to generate copious amounts of patient-specific tumor antigens, and be delivered to patients using ex vivo priming of

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autologous DCs or in vivo delivery to target APCs (Ancliff, Gale, Liesner, Hann, & Linch,

2001; Kasper, Tidow, Grothues, & Welte, 2000; D. A. Mitchell & Nair, 2000b; Sayour,

Sanchez-Perez, et al., 2015).

2.3 ‘Naked’ RNA Vaccines

The initial experiments with RNA vaccines demonstrated that direct injection of messenger RNA (mRNA) into murine skeletal muscle could induce in vivo gene expression (Sayour, Sanchez-Perez, et al., 2015; Wolff et al., 1990). These initial observations provided the groundwork for later experiments exploring the anti-tumor immunity elicited from mRNA expression of cancer antigens (Conry et al., 1995; Sayour,

Sanchez-Perez, et al., 2015). One of the first mRNA polynucleotide vaccines was constructed using mRNA encoding for the human carcinoembryonic antigen (CEA), which conferred protective immunity in mice challenged with CEA expressing tumor cells (Conry et al., 1995; Sayour, Sanchez-Perez, et al., 2015). In these experiments, RNA transcripts were stabilized through 5’ end capping, 3’ end polyadenylation and through incorporation of the human beta-globin 5' and 3' untranslated regions (UTRs) (Conry et al., 1995; Sayour, Sanchez-Perez, et al., 2015). Direct injection of naked mRNA stabilized with beta-globin UTRs has also been shown to prime Th2 responses that can be shifted to Th1 responses after administration of GM-CSF in murine models (Carralot et al., 2004;

Sayour, Sanchez-Perez, et al., 2015). Since the in vivo half-life of RNA is very narrow,

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other studies have focused on direct injection of RNA into target organs (Kreiter et al.,

2010; Qiu, Ziegelhoffer, Sun, & Yang, 1996; Sayour, Sanchez-Perez, et al., 2015; Steitz,

Britten, Wolfel, & Tuting, 2006). Using gene gun treatment, direct injection of human alpha-1 antitrypsin mRNA into the epidermis of mouse tissue elicited potent antibody responses with increasing titers after repeated vaccinations (Qiu et al., 1996; Sayour,

Sanchez-Perez, et al., 2015). In addition, gene gun-based immunization using RNA encoding for TRP2 linked to EGFP demonstrated induction of anti-tumor immunity in mice bearing melanomas (Sayour, Sanchez-Perez, et al., 2015; Steitz et al., 2006). Other methods of direct injection have included intranodal administration of RNA directly into lymphoid organs, which was shown to induce anti-tumor immunity in a murine melanoma model (Kreiter et al., 2010; Sayour, Sanchez-Perez, et al., 2015). Interestingly, systemic administration of FLT3 ligand prior to intranodal RNA injection was shown to augment activation and expansion of CD8+ antigen specific T cells, promote T-cell trafficking to melanomas, and increase anti-tumor efficacy (Kreiter, Diken, Selmi,

Diekmann, et al., 2011; Kreiter et al., 2010; Sayour, Sanchez-Perez, et al., 2015).

Much of this pre-clinical data has been used to translate naked RNA vaccination into phase I trials for refractory malignancies (Rittig et al., 2011; Sayour, Sanchez-Perez, et al., 2015; Weide et al., 2008). In a phase I/II trial, total tumor-derived RNA from melanomas, administered intradermally with GM-CSF was safe but of uncertain efficacy

(Sayour, Sanchez-Perez, et al., 2015; Weide et al., 2008). These patients were vaccinated

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with amplified autologous tumor mRNA that could be produced in copious quantities from a personalized library representing a tumor specific transcriptome (Carralot et al.,

2005; Sayour, Sanchez-Perez, et al., 2015). In a phase I/II trial for patients with renal cell carcinoma (RCC), naked RNA encoding for RCC antigens was co-administered with

GM-CSF and precipitated anti-tumor immunity (Rittig et al., 2011). These vaccines generated CD8+/CD4+ T cell responses with no significant adverse effects and induced fifteen stable disease responses out of thirty evaluable patients with RCC (Rittig et al.,

2011; Sayour, Sanchez-Perez, et al., 2015).

Alternatively, strategies have focused on making self-replicating RNA vaccines

(Geall et al., 2012; Rodriguez-Gascon, del Pozo-Rodriguez, & Solinis, 2014; Sayour,

Sanchez-Perez, et al., 2015; Ying et al., 1999). In these strategies, the virus’ structural genes are exchanged for mRNAs coding for cancer antigens, which can then be produced and replicated by the virus’ non-structural machinery (Jackson, Messinger,

Palmer, Peduzzi, & Morrow, 2003; Kofler et al., 2004; Rodriguez-Gascon et al., 2014;

Sayour, Sanchez-Perez, et al., 2015; Vignuzzi, Gerbaud, van der Werf, & Escriou, 2001;

Ying et al., 1999). Immunizations with a self-replicating RNA immunogen (derived in part via Semliki forest virus) elicited humoral and CD8+ T-cell responses against model antigens and lengthened survival in tumor-bearing mice (Sayour, Sanchez-Perez, et al.,

2015; Ying et al., 1999). In a phase I trial against colorectal metastases, tumor RNA embedded into alphaviral vectors transfected DCs and elicited clinically meaningful T

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cell and humoral immune responses (Morse et al., 2010; Sayour, Sanchez-Perez, et al.,

2015). However, virally engineered RNA vaccines require targeted approaches and contain safety and feasibility issues such as the induction of vector-specific neutralizing antibodies (Morse et al., 2010; Sayour, Sanchez-Perez, et al., 2015).

Alternative approaches have focused on condensing naked RNA to enhance its stability and immunogenicity. Polypeptide cations such as protamines, which function to condense nucleic acid, can stabilize RNA (Sayour, Sanchez-Perez, et al., 2015; Scheel et al., 2005). These mRNA-protamine complexes can serve as a potent immune stimulus activating murine cells through a myeloid differentiation primary-response protein 88

(MYD88)/TLR7 dependent pathway and were shown to induce anti-tumor immunity in a murine glioma model after direct intratumoral injection (Kallen et al., 2013; Sayour,

Sanchez-Perez, et al., 2015; Scheel et al., 2006; Scheel et al., 2004; Scheel et al., 2005).

These complexes can also provide balanced cell mediated and humoral immunity when co-delivered with naked uncomplexed mRNA (Fotin-Mleczek et al., 2011; Fotin-Mleczek et al., 2012; Sayour, Sanchez-Perez, et al., 2015). In a phase I/II trial, intradermal injection of protamine-stabilized mRNAs coding for melanoma antigens was shown to be safe and feasible with one complete remission in patients with metastatic melanoma (Sayour,

Sanchez-Perez, et al., 2015; Weide et al., 2009). The use of protamine condensed RNA has also allowed for the development of novel self-adjuvanted vaccines containing nucleotide modifications to mRNA transcripts (Fotin-Mleczek et al., 2011; Kallen et al.,

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2013; Sayour, Sanchez-Perez, et al., 2015). While protein and peptide-based tumor vaccines require strong adjuvants to induce immunity, non-coding long chain RNA- based adjuvants have been shown to enhance immunostimulatory effects over poly(I:C) and may further improve the immunogenicity of mRNA cancer vaccines (Heidenreich et al., 2014; Sayour, Sanchez-Perez, et al., 2015). Currently, self-adjuvanted mRNA cancer vaccines encoding for commonly expressed tumor associated antigens are in trials for patients with prostate cancer and stage IV non-small cell lung cancer (NSCLC) (Rausch,

Schwentner, Stenzl, & Bedke, 2014; Sayour, Sanchez-Perez, et al., 2015; Sebastian et al.,

2014).

2.4 RNA Loaded Dendritic Cell Vaccines

Since ‘naked’ RNA can degrade very quickly, and protamine encapsulated RNA cannot be targeted to APCs, alternate strategies have been developed. One alternative involves ex vivo loading of DCs with RNA (i.e. electroporation) before in vivo host re- introduction (Edlich, Hogdal, Rehermann, & Behrens, 2010; Ponsaerts, Van Tendeloo, &

Berneman, 2003; Sayour, Sanchez-Perez, et al., 2015). These RNA loaded DCs have been shown to stimulate polyclonal T cell responses against antigens expressed in metastatic colon cancer and in RCC (Aytekin et al., 2010; Heiser et al., 2001; Kasper et al., 2000;

Sayour, Sanchez-Perez, et al., 2015). In a phase IB study in previously treated advanced melanoma patients, monocyte-derived DC vaccines electroporated with mRNA coding

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for melanoma-associated antigens combined with immunostimulatory RNAs (isRNAs)

(i.e. CD40 ligand, TLR 4 and CD70), induced CD8+ T cell immunity against tumor associated antigens (TAAs) (Benteyn et al., 2013; Sayour, Sanchez-Perez, et al., 2015;

Wilgenhof et al., 2013). In these melanoma patients, two of fifteen patients experienced a complete response while two had a partial response (Sayour, Sanchez-Perez, et al.,

2015; Wilgenhof et al., 2013). While these findings are encouraging, the advancement of ex vivo generated cellular vaccines through multi-institutional clinical trials and eventual distribution for widespread clinical utility has proven to be fraught with developmental challenges prompting the advancement of RNA-loaded nanoparticle (NP) vaccines as an attractive, “off-the-shelf” therapeutic platform (Sayour, Sanchez-Perez, et al., 2015).

2.5 RNA Encapsulated Liposomes

Since, ex vivo generation of RNA-pulsed DCs is fraught with developmental challenges, RNA encapsulated NPs can be used as more feasible alternative (Farrell,

Ptak, Panaro, & Grodzinski, 2011; D. A. Mitchell & Sampson, 2009; Sayour, Sanchez-

Perez, et al., 2015). To protect and deliver RNA to target cells, the NP delivery mechanism should be safe, feasible, clinically translatable, and amenable to ‘off the shelf’ manufacturing/distribution to the population at large (Sayour, Sanchez-Perez, et al.,

2015). Nanoliposomes can meet this need and have been studied as vehicles for RNA vaccines to elicit antigen-specific cytotoxic T lymphocytes in vivo (Martinon et al., 1993; 25

Sayour, Sanchez-Perez, et al., 2015). Liposomes deliver their contents into cellular compartments via receptor-mediated endocytosis, but endosomal contents are frequently shuttled into lysosomal degradation pathways prompting the development of pH sensitive liposomes, which can trigger the release of antigens for MHC processing at low pH levels (Joshi, Unger, Storm, van Kooyk, & Mastrobattista, 2012; Sayour,

Sanchez-Perez, et al., 2015). These specialized liposomes can load DCs in vitro with mRNA coding for tumor associated antigens, elicit cytotoxic T cell responses in murine models, and induce suppression of metastatic spread in a murine melanoma model

(Chikh & Schutze-Redelmeier, 2002; Hess, Boczkowski, Nair, Snyder, & Gilboa, 2006;

Markov et al., 2012; S. Nair, Zhou, Reddy, Huang, & Rouse, 1992; Sayour, Sanchez-

Perez, et al., 2015). Liposomes also have significant capacity for enhancement. They can be combined with polymers to form lipopolyplexes and have been formulated with polyethylene glycol (PEG)ylated derivatives of histidylated polylysine to encapsulate

MART1 antigens inducing protective anti-tumor immunity against murine melanomas

(Mockey et al., 2007; Rodriguez-Gascon et al., 2014; Sayour, Sanchez-Perez, et al., 2015).

Liposomes may be combined with self-amplifying RNA (Geall et al., 2012; Ransohoff et al., 2003; Sayour, Sanchez-Perez, et al., 2015). Self-amplifying RNA liposomes elicited antigen specific CD4+ and CD8+ T-cells capable of producing interferon-gamma in a murine model for respiratory syncytial infection (Geall et al., 2012; Sayour, Sanchez-

Perez, et al., 2015). In addition to encapsulating novel RNA designs, liposomes can be

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combined with viral envelopes to form virosomes (liposomes containing viral envelopes) and further augment delivery of tumor antigens (Bungener et al., 2002;

Sayour, Sanchez-Perez, et al., 2015). Fusion-active virosomes can deliver picomolar concentrations of antigen (i.e. protein encapsulated ovalbumin (OVA)) to DCs for MHC class presentation (Bungener et al., 2002; Sayour, Sanchez-Perez, et al., 2015). Liposomes may be additionally engineered with targeting ligands and moieties to transfect desired cells (Sayour, Sanchez-Perez, et al., 2015). IgG-coated liposomes were effectively internalized by DCs via Ig FcR while mannosylated liposomes augmented DC activation

(Copland et al., 2003; Sayour, Sanchez-Perez, et al., 2015; Serre et al., 1998). Meanwhile, single chain antibody fragments (scFv) attached to liposome surfaces have been developed against DC receptors CD11c or DEC-205 and demonstrated efficient DC targeting and anti-tumor immunity (Sayour, Sanchez-Perez, et al., 2015; van

Broekhoven, Parish, Demangel, Britton, & Altin, 2004).

2.6 Harnessing RNA-loaded Nanoliposomes against GBM

The safety and stability profile of liposomes is attractive, and the immunogenicity of RNA is appealing for initiating prolific immune responses (Sayour,

Sanchez-Perez, et al., 2015). To bypass the complexity of cellular therapeutics and unlock the potency of RNA cancer vaccines against GBM, we have investigated the immunologic effects of RNA encapsulated in commercially available cationic liposomes.

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Cationic liposomes condense RNA into ~100-200 nanometer (nm) particles that are stable in solution, protecting nucleic acids from degradation while simultaneously delivering them to APCs (Hoekstra, Rejman, Wasungu, Shi, & Zuhorn, 2007). These APCs can then induce peripheral T cell immunity, which can traffic to the CNS shifting the balance of intratumoral TILs toward cytolytic effector cells.

Cationic nanoliposomes such as 1,2-dioleoyloxypropyl-3-N,N,N- trimethylammonium (DOTAP) have been shown to be safe in several clinical trials and are readily available in cGMP formulation (Fasol et al., 2012; Lu et al., 2012; Porteous et al., 1997; Simberg, Weisman, Talmon, & Barenholz, 2004; Strieth et al., 2013). Although

DOTAP has been used in the context of drug delivery and gene therapy with a promising safety profile, it remains limited by localization to the reticuloendothelial system (RES) (Fasol et al., 2012; Porteous et al., 1997; Simberg et al., 2004; Strieth et al.,

2013). This system contains mononuclear phagocytes requisite to eliminate foreign material, but is also imbued with APC functions necessary to present infectious material to T cells activating them against virulent etiologies (Crispe, 2009). Although RES deposition is unattractive for drug and gene delivery, it may the ideal location for a cancer immunotherapeutic vaccine to induce APC activation and T cell priming. We thus hypothesized that intravenously administered tumor mRNA encapsulated in

DOTAP nanoliposomes would localize to RES sites such as the spleen and liver, transfect APCs therein, and induce CD8+ T cell responses against intracranial tumors.

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Since RNA-NP vaccines can be made within days of tumor resection, they can provide near immediate immune induction against inciting malignancies without the delay that hampers cellular therapeutics such as DC vaccines.

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3. Materials and Methods

The methods below are directly based on our publication by Flores et al.

(Oncoimmunology, 2015) and our group’s publication by Sanchez-Perez et al. (Plos One

2013).

3.1 Isolation of RNA

Total RNA derived from tumor cells was isolated using commercially available

RNeasy mini kits (Qiagen) per manufacturer instructions (Flores et al., 2015). To generate model mRNA templates (Green Fluorescent Protein (GFP), luciferase, and

OVA) for in vitro transcription (IVT), plasmids were linearized with restriction enzymes

(i.e. SpeI) and purified with Qiagen PCR MiniElute kits. Linearized DNA was then transcribed using the mMessenger RNA IVT kit (Life technologies, Invitrogen) and cleaned up using RNA Maxi kits (Qiagen).

3.2 Nanoparticles

Cationic liposomes (DOTAP, DOTAP-DOPE, DOTAP-Cholesterol) were acquired from Avanti, Polar Lipids Inc. (Alabaster, AL, USA). For preparation, chloroform was added to re-suspend 25-100 mg of cationic liposome. Chloroform was evaporated off until a thin lipid layer remained. The mixture was re-suspended in 5-20 mL of PBS (Gibco, Phosphate-Buffered Saline pH 7.4, 1x) before being placed in a 50° water bath for two hours and vortexed intermittently. One day later, 5-20 mL of PBS

30

was added to the mixture, vortexed and placed in a bath sonicator for 5 minutes before being filtered through a 0.43 m syringe and subsequently a 0.22 m syringe (PALL

Acrodisc syringe filter with Supor membrane). The prepared NP solution was then stored at 4°C until use. Ratio of mRNA to filtered DOTAP was based on pre-filtration DOTAP concentration (2.5 µg/uL). Polyethylenimine NPs (JETPEI, and JETPEI mannose) were obtained from Polyplus transfection and prepared using manufacturer’s instructions.

3.3 RNA-NP Complex Preparation

For in vivo preparations, 375 µg of DOTAP was added to 25 µg of mRNA (per mouse) in HBS or PBS buffer. For RNA-NPs encapsulated with GM-CSF mRNA, 375 µg of DOTAP was added to a mixture of 25 µg of OVA mRNA and 25 µg of GM-CSF mRNA (per mouse). For in vitro preparations 25 µg of DOTAP was added to 1.67 µg of

RNA (per 1 x 105 cells) in HBS or PBS buffer. This mixture was kept at room temperature for 15-20 minutes to allow RNA-DOTAP complexes to form. For in vivo IV injections,

200 L of the RNA-DOTAP vaccine was injected into the tail vein of each C57 BL/6 mouse.

3.4 Cryo-electron Microscopy

Methods for cryo-electron microscopy were prepared in collaboration with Paul

Chipman at the University of Florida Interdisciplinary Center of Biotechnology

Research. Briefly, for preparation of thin films, 3 L of the sample suspension were placed of onto holey carbon grids (C-Flats; Protochips, Inc.) and vitrified by plunging

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into nitrogen cooled ethane, using a Vitrobot™ Mark IV freezing device (FEI

Co.). Subsequently, using a 16-megapixel CCD camera (Gatan, Inc.) in a Tecnai G2 F20-

TWIN Transmission Electron Microscope (FEI), operated at a voltage of 200 kV using low dose conditions (~20 e/Å2), the frozen grids were imaged.

3.5 Dynamic Light Scatter and Zeta-Potential Analysis

Methods for dynamic light scatter and zeta-potential analysis were prepared in collaboration with Gary Scheiffele at the University of Florida Particle Engineering

Research Center. Briefly, the mean particle diameter and size distribution were determined by dynamic light scattering using the Microtrac Nanotrac DLS. The surface charge of RNA-NP complexes was determined by analysis of the ζ-potential using the

Brookhaven Zeta Plus instrument. Measurements were performed at 25°C using deionized water and repeated for each sample. 10 runs of 5 cycles were performed with pH assumed to be 7. Measurements were obtained using the Smoluchowski calculation.

3.6 Mice

Four to eight week old C57Bl/6 mice were obtained from the Jackson Laboratory

(Bar Harbor, ME, stock#000664) (Flores et al., 2015). CD8+ OVA-specific TCR (OT-I) transgenic mice were all on the C57Bl/6 background and purchased from Jackson

Laboratories. The investigators adhered to the “Guide for the Care and Use of

Laboratory Animals” as proposed by the committee on care of Laboratory Animal

Resources Commission on Life Sciences, National Research Council (Flores et al., 2015).

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The facilities are fully accredited by the American Association for Accreditation of

Laboratory Animal Care, and all studies were approved by the Institutional Animal

Care and Use Committee (Flores et al., 2015).

3.7 Bioluminescent Imaging

Transfected cells in vitro and animals vaccinated with luciferase RNA-NPs were imaged in vivo using an IVIS Kinetic (Perkin Elmer) (Flores et al., 2015). Mice were injected with 100 uL luciferin substrate and imaged after 6 hrs following injection for optimal signal output (Flores et al., 2015). Human embryonic kidney cells were transfected with luciferase RNA-NPs and bioluminescence was assessed 6 hrs later after luciferin substrate addition to the cell media.

3.8 OT-I Transfer and RNA-NP Vaccination

Naïve C57Bl/6 mice received white blood cells (WBCs) harvested from the spleens of OT-I transgenic mice (C57Bl/6-Tg(TcraTcrb)1100Mjb/J) (0.5-1×107 cells total). WBCs from OT-I were administered either i.v./i.p. and labeled with CFSE

(Celltrace) in select experiments. Immediately following cell transfer, mice were vaccinated with RNA-NPs or intradermally (i.d.) with 100 µg OVA class I peptide

(SIINFEKL; American Peptide Company, Inc., CA) in 10% DMSO with an equal volume of complete Freund’s adjuvant (CFA) (DIFCO Laboratories, MI) (100 µl/mouse) (L. A.

Sanchez-Perez et al., 2013). Fresh spleens were harvested from vaccinated mice one week later before undergoing RBC lysis. Splenocytes were then washed and co-stained

33

with anti-CD8 APC antibody (ebioscience) and a Ly5.1 antibody (BD Pharmingen) or tetramer specific for H-2Kb-restricted SIINFEKL (MBI). Samples were analyzed by flow cytometry.

3.9 Flow Cytometric Analysis

Liver and spleens were harvested from mice one day after injection of RNA-NP vaccines. Organs underwent RBC lysis before being washed and stained for surface expression of either CD11c, MHCII, CD40, Gr-1, CD11b, CD80, CD86, or PD-L1

(Affymetrix and BD Pharmingen). After incubation, cells were fixed and analyzed using a BD Bioscience FACS Calibur.

3.10 Functional Stimulation Assay

To analyze memory recall responses, spleens were harvested, lysed, and washed before being stimulated with 1 µM SIINFEKL chicken OVA peptide or negative control peptide in complete T-cell media (RPMI 10% FBS 100 µM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µg/ml gentamycin sulfate, 100 IU/ml penicillin/Streptomycin and 55 µM 2-mercaptoethanol) (L. A. Sanchez-Perez et al., 2013).

Cells were incubated at 37°C with 5% CO2, and supernatants were collected after 24-48 hours (L. A. Sanchez-Perez et al., 2013). A 50 µl culture supernatant was mixed with 50

µl capture beads and 50 µl detection reagent for Th1/Th2 cytokine release (BD

Bioscience) and incubated for 2 hours at room temperature (L. A. Sanchez-Perez et al.,

34

2013). Cells were washed and analyzed by flow cytometry per manufacturer instructions

(L. A. Sanchez-Perez et al., 2013).

3.11 Anti-PD-L1 Monoclonal Antibodies

Anti-PD-L1 mAbs were purchased from BioXcell and 200-400 g/dose was administered in the peritoneum of C57Bl/6 mice or mixed within RNA-NP vaccine formulations 1-2 times weekly for immunogenicity and efficacy experiments.

3.12 Tumor Cells

The tumor cell lines B16F10 and B16F10-OVA were obtained as a kind gift from

Dr. Richard G. Vile, PhD, at Mayo Clinic (Daniels et al., 2004; Sanchez-Perez et al., 2005).

Culture media consist of DMEM with sodium pyruvate, supplemented with 10% heat inactivated fetal bovine serum (FBS), and 5.5 mL Penicillin/Streptomycin; cells were kept in a 37˚C incubator with 5% CO2 levels (L. A. Sanchez-Perez et al., 2013). KR158B-luc cells were a kind gift from Dr. Tyler Jacks (Massachusetts Institute of Technology,

Boston, MA) (Flores et al., 2015; Reilly, Loisel, Bronson, McLaughlin, & Jacks, 2000). This tumor cell line was originally isolated from a spontaneously arising astrocytoma in an

Nf1;Trp53 mutant mouse on a C57Bl/6 background (Flores et al., 2015). Culture media consist of DMEM without sodium pyruvate (LifeTechnologies), supplemented with 10% heat inactivated fetal bovine serum (FBS)(LifeTechnologies), 5.5 mL Penicillin/

Streptomycin (LifeTechnologies); cells were kept in a 37C incubator with 5% CO2 levels

(Flores et al., 2015).

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3.13 Tumor Implantations

B16F10-OVA, B16F10, and KR158B-luc cells were harvested with trypsin (Gibco) and washed once in serum-containing medium, before being washed in phosphate- buffered saline (PBS) (Flores et al., 2015). Cell pellets were re-suspended in PBS at the appropriate concentration of viable cells as determined by trypan blue dye exclusion

(Flores et al., 2015). B16F10 and B16F10-OVA tumors were injected subcutaneously in the left flank of C57Bl/6 mice anesthetized with isoflurane. For intracranial implantations, B16F10-OVA and KR158B-luc cells were mixed with an equal volume of

10% methylcellulose in PBS and loaded into a 250-µl syringe (Hamilton, Reno, NV) with an attached 25-gauge needle (L. A. Sanchez-Perez et al., 2013). In C57Bl/6 mice, the tip of the needle was positioned at the bregma and 2 mm to the right of the cranial midline suture and 4 mm below the surface of the cranium using a Kopf stereotactic frame

(David Kopf Instruments, Tujunga, CA) (Flores et al., 2015).

3.14 Dendritic Cell Vaccines

The methods below describing development of dendritic cell vaccines are obtained directly from our publication by Flores et al. (Oncoimmunology, 2015). Briefly, femurs and tibias of C57Bl/6 mice were harvested and bone marrow flushed with RPMI

(LifeTechnologies) in 10% FBS (LifeTechnologies) (Flores et al., 2015). Red cells were lysed with 10 mL Pharmlyse (BD Bioscience) and mononuclear cells were re-suspended in CDCM (RPMI-1640, 5% FBS, 1 M HEPES (LifeTechnologies), 50 mM, 55 mM

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bmercaptoethanol (LifeTechnologies), 100 mM Sodium pyruvate (LifeTechnologies), 10 mM Nonessential amino acids (LifeTechnologies), 200 mM L-glutamine

(LifeTechnologies), 10 mg GM-CSF (R&D Systems), 10 mg IL- 4 (R&D Systems), 5.5 mL

Penicillin/Streptomycin (LifeTechnologies)) and plated into tissue culture treated six well plates at a density of 106 cells/mL in a total volume of 3 mL/well (Flores et al., 2015).

Nonadherent cells were discarded at day 3; at day 7, non-adherent cells were collected and re-plated onto 100 mm tissue treated culture dishes at a density of 106 cells/mL in a total volume of 5 mL/dish (Flores et al., 2015). Twenty four hours later, resulting cells were electroporated with 25 g of total RNA isolated from KR158B-luc cells, or OVA mRNA (RNeasy, Qiagen) (Flores et al., 2015). RNA-pulsed DCs were collected the following day and suspended in PBS at a final concentration of 1.25 x 106 cells/mL and

200 L was administered via intradermal or intraperitoneal injection (Flores et al., 2015).

3.15 Generation of Tumor Specific T cells

The methods below describing development of tumor-specific T cells are obtained directly from our publication by Flores et al. (Oncoimmunology, 2015). Briefly, total RNA was isolated from KR158B-luc tumors and electroporated into bone marrow derived DCs using BTX Single Waveform Electroporation System (Harvard Apparatus)

(Flores et al., 2015). Naıve mice received intradermal vaccination with total tumor RNA- pulsed DCs, their spleens harvested 7 days later, and the splenocytes expanded ex vivo using RNA-pulsed DCs and 100IU IL-2 (R&D Systems) for 7 days (Flores et al., 2015). T

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cells were expanded from primed spleens of either wildtype C57Bl/6 mice (Jackson

Laboratories, Bar Harbor, ME, stock #000664), or DsRed transgenic mice on a C57Bl/6 background (Jackson Laboratories, stock #006051) (Flores et al., 2015). Tumor-reactive T cells were adoptively transferred intravenously after 5–7 days of in vitro activation

(Flores et al., 2015).

3.16 Cellular Immunotherapy High Grade Glioma Model

The methods below describing development of a cellular immunotherapy high grade glioma model are obtained directly from our publication by Flores et al.

(Oncoimmunology, 2015). Briefly, C57Bl/6 mice (Jackson Laboratories) were stereotactically implanted with 104 KR158B-luc astrocytoma cells into the right caudate nucleus on Day 0 (Flores et al., 2015). Mice then received a single dose of 9 Gray (Gy) total body irradiation (TBI) on Day 4 (Flores et al., 2015). Mice then received hematopoietic stem cell (HSC) rescue through intravenous injection of 5 x104 lineage negative (lin-) bone marrow derived stem cells within 6 hours of TBI (Flores et al., 2015).

HSCs were derived from the bone marrow of C57Bl/6 mice and magnetically depleted for lin- bone marrow stem cells (Miltenyi Biotec) (Flores et al., 2015). Intravenous injection of 107 tumor specific T lymphocytes was administered between 16 and 24 hours after TBI (Flores et al., 2015). This was immediately followed by an intradermal vaccination of 2.5 x 105 total tumor RNA pulsed DCs or RNA-NP vaccines (Flores et al.,

2015). The second and third DC and RNA-NP vaccines were administered at weekly

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intervals. The mice were then followed for survival and sacrificed when moribund

(Flores et al., 2015).

3.17 Statistical Analysis

Survival data from the animal studies were analyzed by the Gehan-Breslow-

Wilcoxon test. Unmatched two-way ANOVA was used to determine statistical significance of subcutaneous tumor growth curves. Unless otherwise specified, Mann-

Whitney non-parametric test was used to determine statistical significance for in vivo experiments and student’s t test was used to determine statistical significance for in vitro experiments. Statistical significance was determined using p values less than 0.05.

Analyses were conducted using GraphPad Prism and Microsoft Excel.

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4. RNA Nanoparticle Vaccines: Characterization Studies

Excerpts from the introduction below are taken directly from our publication by Sayour et al. (Journal for Immunotherapy of Cancer, 2015).

4.1 Introduction

Nanomaterials can deliver drugs directly to malignant tissues, bypassing their systemic toxicity (Farrell et al., 2011; Sayour, Sanchez-Perez, et al., 2015). Based on the physical properties of NPs (i.e. size, shape, charge), they can be engineered to boost circulation half-life, deposition, and drug-payload inside tumors (Farrell et al., 2011;

Sayour, Sanchez-Perez, et al., 2015). Nanoscale drugs can be made with different targeting ligands and encapsulation methods to prevent payload degradation (Farrell et al., 2011; Ferrari, 2005; Heath & Davis, 2008; Peer et al., 2007; Sayour, Sanchez-Perez, et al., 2015; Zhang et al., 2008). They may also be developed into multi-functional delivery systems with tumor-specific targeting moieties (Farrell et al., 2011; Ferrari, 2005; Heath &

Davis, 2008; Peer et al., 2007; Sayour, Sanchez-Perez, et al., 2015; Zhang et al., 2008).

These nanomaterials have a multitude of various advantages in cancer therapeutics including bypassing multi-drug resistance mechanisms, accessing solid tumors, and engineering the intratumoral immune matrix (Farrell et al., 2011; Sayour, Sanchez-Perez, et al., 2015).

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Despite this active area of research, the emergence of new nanoscale drugs to the clinic has been stagnant with only a handful of FDA-approved agents (Farrell et al.,

2011; Harries, Ellis, & Harper, 2005; Sayour, Sanchez-Perez, et al., 2015). Meanwhile, biodistribution and toxicity are poorly understood in a complex in vivo system, which contains plasma proteins that could alter a nanomaterial’s surface property and affect its biocompatibility (Choi J, 2011; Sayour, Sanchez-Perez, et al., 2015). These challenges make each NP and its toxicity unique and difficult to investigate since each falls under a diffuse realm of categories (i.e. drugs, devices) (Adiseshaiah, Hall, & McNeil, 2010;

Bawa, 2008; Choi J, 2011; McNeil, 2009; Sayour, Sanchez-Perez, et al., 2015). Based on these inherent complications, FDA approval of NPs has slowed, and most approved agents have been used only in the context of drug delivery (Bawa, 2008; Fasol et al.,

2012; Sayour, Sanchez-Perez, et al., 2015).

Since the advancement of novel NP platforms into clinical evaluation has been encumbered by substantial costs and preclinical toxicity evaluations of nanocarriers with unknown reactivity in humans, we chose to evaluate readily available and clinical-grade nanoliposomal formulations for encapsulating and protecting mRNA transcripts encoding for bioactive proteins (Ferrari, 2005; Peer et al., 2007; Sayour, Sanchez-Perez, et al., 2015).

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4.2 Identifying a Target RNA-NP Formulation

We screened several translatable NP formulations for their ability to transfect

DCs in vitro with GFP mRNA (Demeneix & Behr, 2005; Simberg et al., 2004). We demonstrated that the NP DOTAP is a superior formulation compared to alternative cationic liposomal preparations embedded with pH buffers (DOTAP:DOPE) or membrane fluidity buffers (DOTAP:Cholesterol). In addition, DOTAP was superior to linear polyethylenimine NPs with (JETPEI-Mannose) and without DC targeting mannose receptors (JETPEI) (Fig. 9); we corroborated DOTAP’s superiority based on induction of antigen specific CD8+ immunity (Fig. 10).

Figure 9: Comparison of commercially available NPs on DC transfection in vitro

An immortalized murine bone marrow-derived DC line (DC2.4) was transfected with GFP mRNA using several translatable NP formulations and cells were screened one day later for assessment of GFP expression via flow cytometry. Transfection was best using the NP DOTAP (*p <0.05; **p<0.01; ***p<0.001, unpaired t test).

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Figure 10: Comparison of commercially available NPs on OVA specific immunity in vivo

C57Bl/6 mice spiked with a low precursor frequency of OT-Is were vaccinated intravenously with OVA mRNA encapsulated in either DOTAP, cationic liposomal preparations embedded with pH buffers (DOTAP:DOPE), or linear polyethylenimine NPs with DC targeting mannose receptors (JETPEI-Mannose); OVA specific T cell immunity was assessed from spleens of vaccinated mice 1 week later by flow cytometry. Induction of OVA specific CD8+ T cells appeared best with DOTAP encapsulated RNA- NPs.

Later, we investigated the optimal ratio of mRNA to DOTAP based on DC transfection efficiency in vitro and determined that ratios ranging from 1 µg of mRNA to

10-20 µg of DOTAP achieved peak transfection efficiencies (Fig. 11). We have thus identified an optimal translatable RNA-NP formulation consisting of mRNA encapsulated in DOTAP at a ratio of 1:15 µg.

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Figure 11: Ratio of mRNA to DOTAP

Different ratios of GFP mRNA to NP DOTAP were encapsulated and assessed for % transfection efficiency of DC2.4s in vitro. Cells were grown in vitro and harvested 1 day after addition of RNA-NPs to culture media. Ratios of mRNA to DOTAP of 1:15 µg achieved optimal transfections.

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4.3 Size and Charge of RNA-NPs Encapsulated in DOTAP

Uncomplexed NPs and RNA encapsulated NPs were determined to have sizes ranging above and below 100 nm by cryo-electron microscopy (Fig. 12); this was corroborated by dynamic light scatter (Fig. 13), which showed a distribution of sizes ranging from 70-200 nm.

Figure 12: Cryo-electron microscopy of NPs and RNA encapsulated NPs

DOTAP NPs were left uncomplexed (Left) or complexed (Right) with luciferase mRNA and measured using cryo-electron microscopy. There was a heterogeneous distribution of individual particle sizes ranging above and below 100 nms.

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Figure 13: Dynamic Light Scatter of RNA-NPs

DOTAP NPs were complexed with total RNA derived from a glioma cell line and particle size was measured using dynamic light scattering. There was a spectrum of size distributions with most falling between 70-200 nm.

To determine the zeta potential of RNA-NP vaccines total tumor derived RNA was encapsulated with DOTAP and successive runs were performed on the Brookhaven

ZetaPlus instrument. The average zeta potential of these runs measured at 27.28 mV.

4.4 RNA-NP “Off the Shelf” Stability and Translation Efficiency

To develop and translate personalized RNA-NP vaccines, total RNA must be extracted from a surgical biopsy specimen and reverse transcribed into a cDNA template

(D. A. Mitchell & Nair, 2000a, 2000b). Tumor mRNA must then be amplified for complexation into our target NP formulation before being shipped back to a patient for

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administration (Heiser et al., 2001; D. A. Mitchell & Nair, 2000a, 2000b). To evaluate the viability and shelf-life of our formulation, we determined the translation efficiency of luciferase RNA-NPs at different time points after encapsulation. We transfected human embryonic kidney (HEK) cells in vitro with luciferase RNA-NPs and compared the transfection efficiency at these time points versus the baseline transfection efficiency.

Based on bioluminescent imaging, our data demonstrates maintenance of optimal translation efficiency in vitro for nearly 60 hrs after vaccine formulation at room temperature (Fig. 14).

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Figure 14: RNA-NP stability over time

RNA-NP complexes encoding for luciferase were used to transfect HEK cells at different time points. Before transfection of HEK cells, RNA-NPs were left at either room temperature (blue) or 37oC (red) after encapsulation of luciferase mRNA with DOTAP NPs. RNA-NPs still elicited optimal in vitro transfection even 1-2 days after encapsulation at room temperature.

We also corroborated that our NP formulation could induce luciferase mRNA expression in vivo after local injection (Fig. 15).

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Figure 15: RNA-NP in vivo expression

NPs containing luciferase mRNA were injected locally into the peritoneum of albino C57Bl/6 mice with peritoneal luciferin injection 6 hrs post-vaccine. RNA-NP induced luciferase expression in areas of local vaccination.

4.5 Discussion

Compared with other translatable formulations, the cationic lipid DOTAP achieved optimal transfection efficiency of encapsulated mRNA. DOTAP addition to

RNA appears to generate multilamellar vesicles (Fig. 12) as opposed to the prototypical bilamellar vesicles characteristic of uncomplexed DOTAP. For multilamellar vesicles to be generated, it is expected that RNA adheres to the outside of the bilamellar uncomplexed DOTAP particles (Simberg et al., 2004). This attachment may lead to interactions with other bilamellar DOTAP particles, which could engulf the initial 49

complex, leading to development of multilamellar vesicles and protection of RNA between the bilayers of two DOTAP particles (Simberg et al., 2004).

Although the zeta potential is positive, as expected for cationic liposomes, it is within a range suggestive of individual particle aggregation. Fifteen minutes after addition of DOTAP to RNA, cloudy precipitates appear to form, but homogenize within solution immediately after mixing. This suggests that interactions between individual

RNA-NPs are loose and likely ionic in nature. If these interactions were stronger (i.e. covalent bonds), larger aggregates would be expected to form that would not be as easily dissociated. While these in vitro interactions between individual RNA-NPs may not be representative of what happens in vivo, these particles could develop ionic interactions with endogenous plasma proteins, which may affect their distribution and uptake by target cells; however, these interactions are likely mitigated by systemic vascular flow.

After characterizing the size and zeta potential of a target RNA-NP formulation, we demonstrated that RNA can remain stable for several days after encapsulation. This will allow for total RNA to be extracted from individual tumors for formulation into a personalized NP vaccine before being shipped back to the patient for administration.

Lastly, we showed that luciferase RNA-NPs mediate efficient expression in vivo after local injection, suggesting that our formulation may introduce genes of interest through direct injection. For example, to increase T cell trafficking to the CNS, RNA-

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NPs encoding for chemokines can be injected directly into brain tumors for recruitment of effector T cells. Since this is an invasive approach, we prioritized development of systemically administered RNA-NPs that localize to peripheral APCs for induction of peripheral T cell responses that can then traffic to the CNS.

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5. Immune Responses from RNA-Nanoparticle Vaccines

5.1 Introduction

Although cancer vaccines have suffered from poor in vivo localization and inadequate antigenicity, mRNA encoding for tumor antigens may represent a superior source of antigens for induction of innate and antigen specific tumor immunity (Kreiter et al., 2010; Kreiter et al., 2015; Sayour, Sanchez-Perez, et al., 2015; Ulmer et al., 2012).

RNA can serve as a TLR agonist for receptors 7 and 8, bypasses MHC class restriction, and does not require translocation across the nuclear membrane (Napolitani, Rinaldi,

Bertoni, Sallusto, & Lanzavecchia, 2005; Sayour, Sanchez-Perez, et al., 2015). While naked mRNA is inherently unstable, we have shown that the nanoliposome DOTAP preserves mRNA integrity (Fig. 14) (Napolitani et al., 2005; Sayour, Sanchez-Perez, et al.,

2015). Since cationic nanoliposomes have been used in clinical drug delivery trials and localize to reticuloendothelial organs when administered intravenously, we have advanced a RNA-DOTAP formulation that can be harnessed to precipitate activated T cell frequencies necessary to engender long-term anti-tumor efficacy against malignancies (Fasol et al., 2012; Simberg et al., 2004). Based on our hypothesis, we sought to assess if intravenous (i.v.) administered RNA-NPs could generate functional antigen specific immunity.

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5.2 Antigen Specific Immune Responses

In previous studies RNA encapsulated in cationic liposomes have been shown to elicit immune responses in vivo when delivered intravenously (Hess et al., 2006). To identify the optimum route of administration, OVA RNA-NP complexes were injected i.v. into naïve immunocompetent C57Bl/6 mice that had been spiked with a low frequency of OVA-specific CD8+ T cells (OT-I) obtained from transgenic mice

(C57Bl/6-Tg(TcraTcrb)1100Mjb/J) and compared with other routes of injection.

Spleens were harvested after one week for assessment of antigen specific CD8 T cell immunity. I.V. RNA-NP complexes showed superior expansion of OVA-specific T cells compared with intradermal (i.d.) vaccines, subcutaneous (s.c.) vaccines and unvaccinated mice receiving adoptive lymphocyte transfer (ALT) with OT-Is alone (Fig,

16). Gating strategy of OVA specific CD8+ T cells is shown in Fig. 17.

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Figure 16: Comparison of antigen specific immunity from RNA-NP vaccines by different routes of administration

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single OVA RNA-NP vaccine before splenocytes were harvested one week later. Compared with i.d. vaccines, s.c. vaccines, and mice receiving ALT with OT-Is without vaccination, i.v. administration of RNA-NPs was the optimal delivery route for inducing OVA+ specific T cell immunity (*p <0.05; **p <0.01, Mann-Whitney Test).

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Figure 17: Gating strategy of antigen specific immune responses

RNA-NP complexes were injected though different routes of administration. Lymphocytes were gated off by forward and side scatter, followed by gating of CD8+ (APC) T cells by side scatter. OVA specific T cells were then identified by %Ly5.1 positive (PercP) of CD8+ (APC) T cells.

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To assess proliferation of antigen specific T cells, we labeled splenocytes from

OT-I transgenic mice with carboxyfluorescein succinimidyl (CFSE), an intracellular dye that dilutes over time with cellular proliferation. We vaccinated mice with RNA-NPs, spiked with CFSE labeled cells, and measured OT-I proliferation based on CFSE dilution. OT-I T cells preferentially expand in the presence RNA-NP vaccines (Fig. 18).

These results demonstrate that RNA-NP vaccines are capable of inducing T cell proliferation in vivo in an antigen-specific manner.

Figure 18: OVA Specific T cell expansion by CFSE Dilution

Expansion of CFSE labeled splenocytes from OT-I transgenic mice in the presence of OVA mRNA-NPs, NPs alone, or GFP mRNA NP vaccines. Compared with empty NPs and control RNA-NPs (GFP), there was increased CFSE dilution after OVA mRNA-NP vaccines.

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To compare the immunogenicity of RNA-NPs with peptide vaccines, we emulsified the class I SINFEKL epitope of OVA peptide in CFA, which is a composite of mineral oil and mycobacterium that is too toxic for clinical use, but has been shown to induce potent immune responses in mice (L. A. Sanchez-Perez et al., 2013). Compared with OVA SINFEKL peptide emulsified in CFA OVA, mRNA encapsulated in NP vaccines elicited a greater expansion of OVA specific CD8+ T cells in the spleens (Fig. 19) and lymph nodes (Fig. 20) of immunocompetent mice.

Figure 19: Comparison of OVA specific T cell immunity from murine splenocytes following RNA-NPs and OVA peptide vaccines in CFA

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single OVA RNA-NP vaccine, RNA alone, or CFA-OVA peptide before splenocytes were harvested one week later. Compared with OVA peptide vaccines in CFA and OVA mRNA alone, RNA-NPs induced an increased percentage of OVA specific CD8+ T cells in the spleens of vaccinated mice (**p <0.01, Mann-Whitney Test).

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Figure 20: Comparison of antigen specific immunity in lymph nodes from RNA-NP vaccines versus CFA-OVA.

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single OVA RNA-NP vaccine, control RNA-NPs, NPs alone, or CFA-OVA peptide before lymph nodes were harvested one week later. Compared with OVA peptide vaccines in CFA, NPs alone and control GFP RNA-NPs, RNA-NPs induced an increased percentage of OVA specific CD8+ T cells in lymph nodes of vaccinated mice (*p <0.05; **p <0.01, unpaired t test).

To determine T cell functionality, splenocytes from mice vaccinated with RNA-

NPs were harvested and co-cultured with OVA peptide for assessment of IFN-gamma production. Compared to peptide vaccines in CFA, C57Bl/6 mice vaccinated with RNA-

NPs had a significant increase in IFN-gamma production and an increased percentage of antigen specific T cells secreting IFN-gamma (Fig. 21).

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Figure 21: Functional T cell assays from splenocytes of mice vaccinated with RNA-NPs

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single OVA RNA-NP vaccine before splenocytes were harvested one week later. Splenocytes were then co- cultured with OVA peptide for 1-2 days, before cells were spun down for assessment of functional antigen specific T cells by intracellular staining while supernatants were separated and simultaneously assessed for IFN-gamma production. Compared with mice receiving mRNA alone or ALT from OT-Is without vaccination, there is increased IFN gamma production in the supernatants of co-cultured splenocytes from mice that received RNA-NPs (Left). Compared to CFA-OVA, RNA alone or ALT from OT-Is without vaccination, there is increased percentage of antigen specific T cells secreting IFN-gamma in splenocytes from mice that received RNA-NPs (Right) (*p <0.05, Mann- Whitney Test).

5.3 Serum Cytokine Levels Post-Vaccination

To sample the systemic cytokine milieu post RNA-NP vaccination, we performed a cytokine bead array for inflammatory cytokines IL-6, TNF-alpha, IFN-gamma, IL-10,

IL-12 and the chemokine CCL2. While there was a mild increase in the levels of IL-6 and

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TNF-alpha, there was no change in the levels of IL-10, IFN-gamma, and IL-12p70; however there was a dramatic rise in the chemokine CCL2 (monocyte chemoattractant protein-1 (MCP-1)) (Fig. 22).

Figure 22: IL-6, TNF-alpha and CCL2 levels post-vaccination

C57Bl/6 mice were vaccinated with a single RNA-NP vaccine and serum was collected from mice 1 day later for assessment of cytokine production by bead array. Compared with mice receiving either RNA or NPs alone, RNA-NPs elicited a mild increase in IL-6 and TNF-alpha, and a significant increase in CCL2 (MCP-1) (*p <0.05, Mann-Whitney Test).

5.4 Discussion

Since CFA is composed of a composite of mycobacterium and mineral oil, it is too toxic to use in humans, but has been shown to be effective at inducing Th1 responses in mice (L. A. Sanchez-Perez et al., 2013). Our translatable RNA-NP vaccines were

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superior to CFA-OVA peptide vaccines at inducing antigen specific CD8+ immunity, and are expected to have a promising safety profile relative to CFA.

Interestingly, RNA-NPs only mediated antigen specific immune responses when administered intravenously. It was expected that intradermal and subcutaneous injections could mediate responses, but these routes may have suffered from lack of adjuvant effect at local injection sites. Intradermal and subcutaneous injection of RNA-

NPs may also immediately transfect cells under the skin; by the time DCs or resident

APCs traffic to site of RNA-NPs, there may be minimal antigen left for uptake.

Alternatively, i.v. injection of RNA-NPs may localize to reticuloendothelial organs such as the spleen and liver, which have copious amounts of APCs. Following i.v. RNA-NP vaccination, the lung is the first-pass organ where transfection occurs initially (Simberg et al., 2004). The remaining RNA-NPs that traverse across the lung capillaries then make their way to the left side of the heart where they are systemically delivered (Simberg et al., 2004). Since there is a systemic surge in CCL2, monocytes and CD11c expressing

APCs may be drawn toward areas of vaccine localization, abrogating need for engineering RNA-NPs to target APCs. Since RNA-NPs are expected to localize to RES sites such as the liver and spleen, we studied the localization and immune effects of our vaccine on CD11c expressing cells in these organs (Fasol et al., 2012; Simberg et al.,

2004).

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6. Localizing Effects of RNA-Nanoparticle Vaccines on the Spleen and Liver

6.1 Introduction

NPs such as DOTAP have been well tolerated in human clinical trials for drug and gene delivery without toxicity, but remain limited in these contexts by decreased targeting efficiency and reticuloendothelial deposition (Fasol et al., 2012; Porteous et al.,

1997; Simberg et al., 2004; Strieth et al., 2013). Although hepatic and splenic uptake is a clear impediment in the context of standard liposomal drug delivery, we hypothesized that these organs are critical and advantageous areas of localization for an RNA nanoliposomal vaccine to induce APC transfection and T cell priming (Simberg et al.,

2004). We thus focused on the gene expression and immune effects of RNA-NPs in the spleen and liver and sought to elucidate the cellular compartments that are critical for vaccine-mediated immunity.

6.2 Gene Expression from RNA-NPs in the Spleen and Liver

To assess gene expression induced by RNA-NPs, GFP encoding RNA-NPs were systemically administered to immunocompetent mice and spleens and livers were harvested 1 day later for GFP expression by flow cytometry. Compared to untreated mice and mice receiving uncomplexed NPs, i.v. administered RNA-NPs induced GFP expression within the spleens and livers of immunocompetent mice (Fig. 23). 62

Figure 23: GFP RNA-NP localization to the spleen and liver

NPs were complexed with GFP mRNA and injected i.v. into C57Bl/6 mice. Spleens and livers were harvested 1 day after injection for assessment of GFP+ cells by flow cytometry. Compared with NPs alone and untreated mice, there is increased GFP expression in the spleen and liver post RNA-NP vaccination (*p <0.05; **p <0.01, Mann- Whitney Test).

Since CD11c is a marker on DCs and APCs, we analyzed the percentage of GFP expressing CD11c+ cells in the spleen and liver. Compared with untreated mice and mice receiving uncomplexed NPs, the percentage of GFP expressing CD11c+ cells was increased in the spleen and liver post RNA-NP vaccination (Fig. 24).

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Figure 24: GFP RNA-NP localization to CD11c cells in the spleen and liver

NPs were complexed with GFP mRNA and injected i.v. into C57Bl/6 mice. Spleens and livers were harvested 1 day after injection for assessment of CD11c+ cells expressing GFP by flow cytometry. Compared with NPs alone and untreated mice, there is an increased percentage of CD11c+ cells expressing GFP in the spleen and liver post RNA-NP vaccination (*p <0.05; **p <0.01, Mann-Whitney Test).

6.3 Characterization of CD11c expressing Splenocytes and Hepatic WBCs

Since RNA-NPs localize to CD11c expressing cells in the spleen and liver, we attempted to further characterize these cell populations and changes therein following vaccination. Compared with untreated mice and mice receiving uncomplexed NPs, we observed that RNA-NP vaccines induced an increased percentage of CD11c+ cells in the liver that was not appreciated in the spleen (Fig. 25).

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Figure 25: Percentage of CD11c+ cells in the spleen and liver post RNA NP vaccination

RNA-NPs were systemically administered to C57Bl/6 mice and spleens and livers were harvested 1 day later. Compared with NPs alone, and untreated mice, RNA- NP vaccines increased the percentage of CD11c+ cells in the liver but not the spleen (*p <0.05; **p <0.01, Mann-Whitney Test).

After observing an increase in the percentage of hepatic CD11c+ cells, we assessed them for concurrent expression of MHC II and CD40, as would be expected of prototypical APCs. While the percentage of MHCII and CD40 was increased on

CD11c+ cells in the spleen post RNA-NP vaccination, the percentage of MHCII and

CD40 on CD11c+ hepatic WBCs was decreased and unchanged respectively (Fig. 26-27).

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Figure 26: MHC II expression on CD11c+ cells in the spleen and liver post RNA-NP vaccination

RNA-NPs, encapsulating either GFP or OVA mRNA, were administered to C57Bl/6 mice and spleens and livers were harvested 1 day after i.v. administration. After RNA-NP vaccination, the percentage of CD11c+ cells expressing MHCII was increased in the spleen but decreased in the liver (**p <0.01, Mann-Whitney Test).

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Figure 27: CD40 expression on CD11c+ cells in the spleen and liver post RNA- NP vaccination

RNA-NPs were administered to C57Bl/6 mice and spleens and livers were harvested 1 day after i.v. administration. Compared with NPs alone, and untreated mice, RNA-NP vaccines increased the percentage of CD11c+ cells expressing CD40 in the spleen but not the liver (**p <0.01, Mann-Whitney Test).

Since transfected CD11c+ splenocytes express MHC class II, these are expected to be more traditional APCs such as DCs; however, since the liver WBCs that accumulate are predominantly MHC class II negative, these cells could be more immature APCs, kuppfer cells, or monocytes/macrophages (Crispe, 2009, 2011; Klein et al., 2007;

Klugewitz et al., 2002). To assess if there was an increased percentage of macrophage markers after RNA-NP administration, we screened splenocytes and hepatic WBCs for expression of F4-80 and observed a slight decrease in the percentage of F4-80+ cells in the spleen and a trend towards slight decrease in the liver (Fig. 28).

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Figure 28: F4-80 expression in the spleen and liver post RNA-NP vaccination

RNA-NPs were administered to C57Bl/6 mice and spleens and livers were harvested 1 day after i.v. administration. After RNA-NP vaccination, the percentage of F4-80+ cells was decreased in the spleen but unchanged in the liver (*p <0.05, Mann- Whitney Test).

While there was not an increase in macrophage markers on splenocytes or hepatic WBCs, there was an increased percentage of Gr-1+ cells in both the spleen and the liver (Fig. 29).

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Figure 29: Gr-1 expression in spleen and liver post RNA-NP vaccination

RNA-NPs were administered to C57Bl/6 mice and spleens and livers harvested 1 day after IV administration. Compared with NPs alone and untreated mice, RNA-NP vaccines increased the percentage of Gr-1 + cells in both the spleen and liver (*p <0.05, Mann-Whitney Test).

Since the percentage of Gr-1 was elevated in the spleen and liver, these cells could represent neutrophils, monocytes or plasmacytoid DCs. To help determine if these cells were monocytes or neutrophils, we dual stained harvested splenocytes and hepatic WBCs for Gr-1 and CD11b, an integrin responsible for innate immune cell trafficking. We observed that there were no changes in the percentages of CD11b expressing Gr-1 cells in the spleen and liver post RNA-NP vaccination (Fig. 30).

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Figure 30: CD11b+ expression on Gr-1+ cells in the spleen and liver post RNA- NP vaccination

RNA-NPs were administered to C57Bl/6 mice and spleens and livers were harvested 1 day after i.v. administration. Compared with NPs alone and untreated mice, RNA-NP vaccines did not change the percentage of Gr-1+ CD11b+ cells in the spleen and Gr-1+ cells expressing CD11b in the liver.

Since there was no change in the expression of CD11b+ Gr-1+ cells in the spleen and the liver, the increase in Gr-1+ cells observed in these organs is not expected to be myeloid derived suppressor cells.

To assess whether there was an increase in CD11c+ cells expressing Gr-1 post

RNA-NP vaccines, we dual stained harvested splenocytes and hepatic WBCs for both markers. Compared with untreated mice and mice receiving NPs alone, there was an increase in the percentage of CD11c+ cells expressing Gr-1 after RNA-NP vaccination

(Fig. 31).

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Figure 31: Gr-1 expression on CD11c+ cells in the spleen and liver post RNA- NP vaccination

RNA-NPs were administered to C57Bl/6 mice and spleens and livers were harvested 1 day after i.v. administration. Compared with NPs alone and untreated mice, RNA-NP vaccines increased the percentage of CD11c+ cells expressing Gr-1 in both the spleen and liver (**p <0.01, Mann-Whitney Test).

To determine if WBCs from the organs of mice vaccinated with RNA-NPs could expand antigen specific T cell responses in naïve mice, OVA RNA-NPs were administered to immunocompetent C57Bl/6 mice with spleens and livers harvested one day later; harvested splenic and liver WBCs were then administered to naïve C57Bl/6 mice in conjunction with OT-I cell transfer (Fig. 32). Compared with untreated mice, splenic and hepatic WBCs from mice vaccinated with OVA RNA-NPs were able to expand OVA specific CD8+ T cells when transferred to naïve mice (Fig. 32).

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Figure 32: Antigen specific immunity induced in naïve mice from adoptively transferred splenic and hepatic WBCs harvested from RNA-NP vaccinated mice

NPs encapsulating OVA mRNA were injected i.v. into immunocompetent C57Bl/6 mice with spleens and livers harvested one day later. A single cell suspension was generated from the spleens and livers of vaccinated mice, and 5 x 105 WBCs from each organ was re-administered to naïve C57Bl/6 mice in conjunction with OT-I cell transfer. Spleens were harvested one week later from these previously naïve mice for comparison of antigen specific T cell immunity. Compared with untreated mice and mice receiving GFP-RNA-NPs/OT-Is, liver WBCs from mice vaccinated with OVA RNA- NPs were able to expand OVA specific CD8+ T cells when transferred to naïve mice (Left). Compared with untreated mice, liver and splenic WBCs from mice vaccinated with OVA RNA-NPs expanded OVA specific CD8+ T cells in naïve mice (Right) (*p <0.05, Mann-Whitney Test).

6.4 Discussion

Since RNA-nanoliposomes cannot hone to targeted cells, engineering targeting ligands and moieties may be utilized to enhance localization of our vaccine formulation

(Sayour, Sanchez-Perez, et al., 2015). While modification of the DOTAP cationic lipid

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backbone may enhance RNA uptake, protection, and delivery in vivo, we have demonstrated that systemic administration of unmodified RNA-NPs transfect CD11c expressing cells in the spleen and liver (D. A. Mitchell et al., 2008; Sayour, Sanchez-

Perez, et al., 2015). While CD11c expressing splenocytes have increased percentages of

CD40 and MHC II, CD11c+ hepatic WBCs have an unchanged percentage of CD40 and a decreased percentage of MHC II; however, both organs have an increased percentage of

CD11c+ cells expressing Gr-1 consistent with a plasmacytoid DC phenotype (Nakano,

Yanagita, & Gunn, 2001). WBCs harvested from spleens and livers of mice vaccinated with RNA-NPs expanded antigen specific T cells in naïve immunocompetent mice

(Nakano, Yanagita, & Gunn, 2001). Based on these data, we expect that RNA-NPs do not need to be further engineered to target APCs. Since the localizing effects of RNA-

NPs increase the percentage of CD11c+ cells at target sites, and WBCs from these organs expanded antigen specific immunity, we further evaluated these CD11c+ cells for expression of co-stimulatory molecules and immune checkpoints.

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7. Effects of RNA-Nanoparticles on Co-stimulatory Molecules and Immune Checkpoints

7.1 Introduction

For a T cell to become activated, not only must there be MHC class presentation of antigen to the T cell receptor (TCR), but there must be a second co-stimulatory signal.

While the B7 molecules such as CD80 and CD86 on APCs have canonically been associated as co-stimulatory markers requisite for T cells activation, there are regulatory checkpoints such as programmed death ligand-1 (PD-L1) that can turn off the engagement of APCs attempting to activate T cells (Karyampudi et al., 2014; Okazaki,

Chikuma, Iwai, Fagarasan, & Honjo, 2013; West et al., 2013). Re-directing the immune system to target tumor cells and establish sustained immunity hinges on preserving the safety profile of our translatable NP vaccines while understanding the regulatory pathways complicit in vaccine response. While we identified increased percentages of

CD11c+ cells expressing Gr-1 in the spleen and liver, there was differential expression of

MHC II and CD40 on CD11c+ cells. Although WBCs from both organs (following RNA-

NP vaccination) were shown to induce immunologic response in naïve mice, we sought to further delineate the splenic and hepatic CD11c+ subsets based on their expression of

B7 co-stimulatory molecules and regulatory checkpoints.

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7.2 Expression of B7 Co-stimulatory Molecules following RNA- NP Vaccination

While RNA-NP localize to CD11c+ cells, localization and antigen presentation alone are not enough to re-direct host-immunity; a second co-stimulatory signal is requisite to activate and sustain a robust T cell response. Therefore, we assessed the effects of RNA-NP vaccines on the expression of B7 co-stimulatory molecules by CD11c+ splenocytes and hepatic WBC. Within twenty four hours of vaccination, RNA-NP vaccines induced increased percent expression of CD80 on CD11c+ cells in the spleen and liver (Fig. 33). To evaluate whether the upregulation of CD80 was mRNA species specific, we vaccinated mice with either GFP or OVA RNA-NPs and demonstrated that both formulations increased the percentage of CD11c+ cells expressing CD80 in the spleen and liver (Fig. 33).

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Figure 33: CD80 expression on CD11c+ cells in the spleen and liver post RNA- NP vaccination

I.V. RNA-NPs were administered to C57Bl/6 mice and spleens and livers were harvested 1 day later. Compared with untreated mice, RNA-NPs encapsulating either GFP or OVA mRNA increased the percentage of CD80 on CD11c expressing cells in the spleen and CD80+CD11c+ cells in the liver (*p < 0.05; **p <0.01, Mann-Whitney Test).

We also assessed the effects of RNA-NP vaccines on CD86 expression by CD11c+ splenocytes and hepatic WBCs. To evaluate whether the increase in B7 co-stimulatory molecules was observed in other vaccine formulations, we compared expression levels of CD86 following RNA-NPs versus peptide vaccines formulated in CFA. I.V. RNA-NP vaccines induced increased percent expression of CD86 on CD11c+ cells in the spleen and liver, which was not observed following peptide vaccines formulated in CFA (Fig.

34)

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Figure 34: CD86 expression on CD11c+ cells in the spleen and liver post RNA- NP vaccination

I.V. RNA-NP vaccines were administered to C57Bl/6 mice and spleens and livers were harvested 1 day later. Compared with untreated mice and mice receiving NPs alone or peptide vaccines in CFA, RNA-NP vaccines increased the percentage of CD86 on CD11c expressing cells in both the spleen and liver (*p <0.05, Mann-Whitney Test).

7.3 Expression of Immune Checkpoints following RNA-NP Vaccination

To determine the effects of RNA-NP vaccines on regulatory checkpoint expression, we vaccinated mice with RNA-NPs and harvested splenocytes and hepatic

WBCs for assessment of PD-L1 expression. NPs encapsulating GFP or OVA mRNA were injected i.v. into immunocompetent C57Bl/6 mice with spleens and livers harvested one day later. RNA-NP vaccines induced upregulation of PD-L1 in the spleen and liver (Fig.

35).

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Figure 35: PD-L1 expression in the spleen and liver post RNA-NP vaccination

NPs encapsulating either GFP or OVA mRNA were injected i.v. into immunocompetent C57Bl/6 mice with spleens and livers harvested one day later for assessment of PD-L1 expression by flow cytometry. Both GFP and OVA RNA-NP vaccines increased the percentage of PD-L1+ cells in the spleen and liver (**p <0.01, Mann-Whitney Test).

We also assessed the effects of RNA-NP vaccines on PD-L1 expression by

CD11c+ splenocytes and hepatic WBCs. RNA-NP vaccines induced an increase in percent PD-L1 expression on CD11c+ splenocytes and hepatic WBCs (Fig. 36).

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Figure 36: PD-L1 expression on CD11c+ cells in the spleen and liver post RNA- NP vaccination

NPs encapsulating either GFP or OVA mRNA were injected i.v. into immunocompetent C57Bl/6 mice with spleens and livers harvested 24 hrs later for assessment of PD-L1 expression on CD11c+ cells by flow cytometry. Both GFP and OVA RNA-NP vaccines increased the percentage of CD11c+ cells expressing PD-L1 in the spleen and liver (**p <0.01, Mann-Whitney Test).

Since PD-L1 is an immunoregulatory ligand likely mitigating the potency of the immune response generated by RNA-NPs, we sought to assess if we could block this axis with anti-PD-L1 mAbs. We also sought to determine if increased percent PD-L1 was observed in other vaccine formulations. Compared with peptide vaccines formulated in CFA, i.v. RNA-NPs with and without anti-PD-L1 mAbs were administered to immunocompetent C57Bl/6 mice. Percent PD-L1 was only increased following RNA-NPs and not observed following peptide vaccines in CFA (Fig. 37). This increase was abrogated with anti-PD-L1 mAbs administered one day prior to RNA-NP

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vaccination (Fig. 37). We also corroborated these effects in the CD11c compartment (Fig.

38). Finally, we observed that addition of anti PD-L1 mAb increases MHC II expression on murine splenocytes and CD86 on CD11c+ hepatic WBCs (Fig. 39).

Figure 37: PD-L1 expression in the spleen and liver after combinatorial administration of RNA-NPs and anti-PD-L1 mAbs

C57Bl/6 mice were vaccinated with GFP RNA-NPs, CFA-OVA peptide, NPs alone or GFP RNA-NPs + anti-PD-L1 mAbs. Anti-PD-L1 mAbs were administered i.p. one day prior to vaccines and spleens and livers were harvested one day after vaccine administrations. Compared to NPs alone and peptide vaccines formulated in CFA, RNA-NPs increased percent PD-L1 expression in splenocytes and hepatic WBCs. Administration of anti PD-L1 mAbs prior to RNA-NPs abrogated percent increase in PD-L1 expression in both the spleen and liver (*p <0.05, Mann-Whitney Test).

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Figure 38: PD-L1 expression on CD11c expressing cells in the spleen and liver after combinatorial administration of RNA-NPs and anti-PD-L1 mAbs

C57Bl/6 mice were vaccinated with GFP RNA-NPs, CFA-OVA peptide, NPs alone or GFP RNA-NPs + anti-PD-L1 mAbs. Anti-PD-L1 mAbs were administered i.p. one day prior to vaccines and spleens and livers were harvested one day after vaccine administrations. Compared to NPs alone and peptide vaccines formulated in CFA, RNA-NPs increased percent PD-L1 expression on CD11c expressing splenocytes and hepatic WBCs. Administration of anti PD-L1 mAbs prior to RNA-NPs abrogated percent increase in PD-L1 expression on CD11c expressing splenocytes and hepatic WBCs. (*p <0.05, Mann-Whitney Test).

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Figure 39: MHC II+ splenocytes and CD11c+ liver WBCs expressing CD86 after combinatorial administration of RNA-NPs and anti-PD-L1 mAbs

MHCII expression on splenocytes and CD86 expression on CD11c+ WBCs was assessed one day after GFP RNA-NP vaccines with and without concomitant administration of anti-PD-L1 mAbs. Co-administration of anti-PD-L1 mAbs increased the percentage of MHCII on splenocytes and CD86 on CD11c+ hepatic WBCs (*p <0.05, Mann-Whitney Test).

To determine if other regulatory immune ligands are upregulated by RNA-NPs, we stained CD11c+ splenocytes and hepatic WBCs for expression of PD-L2 one day post- vaccination (Latchman et al., 2001). Compared with NPs alone, RNA-NPs did not increase the percentage of PD-L2 expression on CD11c+ splenocytes and hepatic WBCs

(Fig. 40).

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Figure 40: PD-L2 expression on CD11c+ cells in the spleen and liver post RNA- NP vaccination

NPs encapsulating GFP were injected i.v. into immunocompetent C57Bl/6 mice with spleens and livers harvested one day later for assessment of PD-L2 expression on CD11c+ cells by flow cytometry. PD-L2 expression on CD11c+ cells in the spleen and liver was unchanged following RNA-NP vaccines.

To ensure viability of PD-L2 antibody staining, DCs were ex vivo matured and expanded before PD-L2 staining and analysis by flow cytometry. PD-L2 was expressed by nearly a third of ex vivo matured and expanded DCs (Fig. 41).

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Figure 41: PD-L2 expression on primary ex vivo expanded DCs

Bone marrow was harvested from C57Bl/6 mice and grown in DC maturation media. After one week in culture, DCs were stained for PD-L2, fixed and assessed for expression by flow cytometry. Primary bone marrow derived DCs from C57Bl/6 mice demonstrated an increase in PD-L2 expression by flow cytometry (****p <0.0001, unpaired t test).

The absence of PD-L2 expression on CD11c+ splenocytes and liver WBCs indicates that the increased percentage of PD-L1 on CD11c+ cells may be a unique compensatory ligand following RNA-NP vaccination. To determine if there was an increase in PD-1 expressing T cells after RNA-NP vaccination, C57Bl/6 mice were given

OVA RNA-NP vaccines in conjunction with anti-PD-L1 mAbs. Splenocytes harvested from mice one week after vaccination were assessed for antigen specific T cell immunity and percentage of PD-1+ CD8 T cells. Mice vaccinated with RNA-NP vaccines showed

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an increase in the percentage of PD-1+ CD8+ splenocytes, which was further increased in mice receiving RNA-NP vaccines with concomitant PD-L1 mAbs (Fig. 42).

Figure 42: PD-1 expression on CD8+ splenocytes from mice vaccinated with RNA-NPs

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single OVA RNA-NP vaccine before splenocytes were harvested one week later. Anti-PD-L1 mAbs were administered one day prior to vaccines and every 3-4 days thereafter for a total of three administrations until organ harvest. Splenocytes harvested from mice were assessed for the percentage CD8+ T cells expressing PD-1. Compared with untreated mice, RNA-NP vaccines precipitated an increase in the percentage CD8+ T cells expressing PD-1. Compared with RNA-NP vaccines, RNA-NPs with combinatorial anti-PD-L1 mAbs increased the percentage of CD8+ T cells expressing PD-1 (*p <0.05; **p <0.01, Mann- Whitney Test).

Although counterintuitive, RNA-NPs in conjunction with blockade of PD-L1 increased the percentage of CD8+ T cells expressing PD-1. Since PD-1 has been shown to be expressed as an early activation marker on cytotoxic T cells, our data demonstrates 85

that combinatorial blockade of regulatory checkpoints with RNA-NP vaccines increases the percentage of activated T cells (Gros et al., 2014).

7.4 Discussion

By harnessing the natural depots of nanoliposomal RNA-NPs localizing to APCs residing in the spleen and liver, we have demonstrated the ability to transfect CD11c+ cells in these organs and established that our formulation can mediate effective antigen specific immune responses. Although RNA-NP vaccines hold the promise of redirecting immunity against intracranial malignancies, they remain limited by regulatory immune pathways that can stymie an effective host response (Okazaki et al., 2013; Rosenberg,

2012; Sonabend et al., 2012). While RNA-NPs increased the percentage of B7 co- stimulatory molecules on CD11c+ cells, we identified compensatory expression of PD-

L1. However, addition of mAbs abrogated PD-L1 expression and increased the percentage of activated T cells post RNA-NP vaccination.

Since the immunogenicity of this versatile platform can be enhanced when co- delivering checkpoint blocking antibodies, these clinically translatable nanoliposomes may be employed to simultaneously target immune regulatory pathways pervasive in malignant propagation. Future nanoliposomal vaccines can be built to deliver immunomodulatory RNAs encoding for PD-L1 blockade during the nascent of the immune response. While vaccines have been met with tepid enthusiasm, concomitant

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checkpoint blockade in conjunction with RNA-NPs may further unlock their therapeutic potency.

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8. Tumor Efficacy Induced by RNA-Nanoparticle Vaccines and Capacity for Immunomodulatory Enhancement

8.1 Introduction

Although cancer vaccines have suffered from weak immunogenicity, we have advanced a NP vaccine formulation that can precipitate activated T cell frequencies necessary to engender long term anti-tumor efficacy against intracranial tumors

(Rosenberg, 2012). Our clinically translatable formulations can be packaged with immunostimulatory RNAs (isRNAs) to further enhance their immunogenicity. The therapeutic potency of RNA-NP vaccines can be significantly increased by encapsulating isRNAs that encode for cytokines, chemokines, TLR agonists, PAMPs or co-stimulatory molecules. In addition to co-encapsulation of isRNAs, our clinically translatable RNA-

NP formulation can be packaged with small interfering RNAs (siRNAs) or mRNAs encoding for mAbs blocking immunoregulatory pathways. Since we have shown that our vaccine upregulates PD-L1 expression, we hypothesized that combination of PD-L1 blocking antibody with RNA-NPs would be synergistic in mediating anti-tumor efficacy.

Before evaluating these enhancing strategies, we sought to assess if our RNA-NP formulation could induce anti-tumor activity. Since we have shown that our vaccine formulation can precipitate CD8+ immunity against the OVA model antigen, we further

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assessed RNA-NP efficacy and capacity for enhancement in subcutaneous and intracranial B16F10-OVA efficacy models.

8.2 Anti-tumor Efficacy

We first wanted to determine if RNA-NP vaccines could mediate anti-tumor efficacy without OT-1 transfer. We implanted B16F10-OVA tumors intra-cranially, and five days after implantation, once weekly RNA-NP complexes (x 3) were injected into naïve C57Bl/6 mice. Compared with untreated mice, RNA-NP vaccines improved the median and overall survival (Fig. 43).

Figure 43: RNA-NP efficacy against intracranial tumors

OVA RNA-NP complexes were injected once weekly for 3 weeks into naïve C57Bl/6 mice starting 5 days after intracranial implantation of B16F10-OVA (6,250 cells/mouse). Mice receiving RNA-NPs had improved median and overall survival compared with untreated mice (**p<0.01, Gehan-Breslow-Wilcoxon test).

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8.3 Capacity for Enhancement

While RNA-NPs mediate anti-tumor efficacy against intracranial tumors, not all tumor bearing animals are long-term survivors. Since this formulation has capacity for enhancement through addition of immunomodulatory RNAs, we leveraged strategies that have been employed successfully in other promising immunotherapeutic interventions such as Sipuleucel-T (Kantoff et al., 2010; Sheikh et al., 2013).

Sipuleucel-T is an immunotherapeutic agent that has proven efficacy for prostate cancer (Kantoff et al., 2010; Sheikh et al., 2013). This agent utilizes a fusion protein containing prostatic acid phosphatase conjugated to GM-CSF, which can ex vivo activate peripheral blood mononuclear cells for induction antigen-specific T cells; these cells can be adoptively transferred to patients with metastatic castration-resistant prostate cancer and have been shown to elicit effective in vivo immune responses (Kantoff et al., 2010;

Sheikh et al., 2013). However, these T cell responses were elevated predominantly in the presence of antigen conjugated to GM-CSF, and were not as robust using unconjugated antigens (Sheikh et al., 2013). Although this highlights the need for further investigation to fully delineate the immune correlates responsible for effective sipuleucel-T vaccination, we sought to embed GM-CSF encoding mRNA within our vaccine formulation to enhance the therapeutic potency of RNA-NPs.

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To determine if GM-CSF encoding mRNA could be encapsulated by NPs, and elicit production of GM-CSF protein, we transfected DC2.4 cells in vitro with GM-CSF

RNA-NPs. Transfected cells elicited GM-CSF production by ELISA (Fig. 44).

Figure 44: GM-CSF ELISA after GM-CSF RNA-NP transfection of DC2.4s

DC2.4 cells were grown in culture and transfected by GM-CSF RNA-NPs. One day later, cells were then spun down and supernatants were collected. Compared with NPs alone, ELISA on supernatants from cells transfected with RNA-NPs demonstrated elevated levels of GM-CSF (*p <0.05, unpaired t test).

We then sought to determine the effects of antigen specific T cell immunity induced by RNA-NPs co-encapsulated with or without GM-CSF mRNA. In the spleens and livers of mice vaccinated with a single OVA RNA-NP vaccine spiked with OT-I cell transfer, we determined that co-encapsulation of RNA-NPs with GM-CSF mRNA

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significantly improved their immunogenicity based on induction of OVA specific CD8+

T cells in the spleen and liver (Fig. 45). Example of gating strategy to assess OVA specific T cells in the liver is shown in Fig. 46.

Figure 45: Comparison of antigen specific T cell immunity from RNA-NPs co- encapsulated with or without GM-CSF mRNA

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single RNA-NP vaccine co-encapsulated with or without GM-CSF mRNA before spleens and livers were harvested one week later. OVA specific T cell immunity induced by RNA-NP vaccines co-encapsulated with GM-CSF mRNA was greater than RNA-NPs alone based on percentage of OVA specific T cells in murine spleens (Left) and livers (Right) (*p <0.05; **p <0.01, Mann-Whitney Test).

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Figure 46: Gating strategy of antigen specific immune responses in liver

Lymphocytes were gated off by forward and side scatter, followed by gating of CD8+ (APC) T cells by side scatter. OVA specific T cells were then identified by %OVA tetramer positive (FL-2) of CD8+ (APC) T cells.

In C57Bl/6 mice bearing intracranial (Fig. 47) and subcutaneous (Fig. 48) B16F10-

OVA tumor cells, RNA-NPs co-encapsulated with GM-CSF mRNA also significantly improved anti-tumor efficacy.

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Figure 47: Comparison of efficacy in mice vaccinated with RNA-NPs co- encapsulated with or without GM-CSF mRNA

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single RNA-NP vaccine 5 days after intracranial B16F10-OVA (6,250 cells/mouse) implantation. GFP RNA-NPs did not prolong survival (Top). RNA-NPs containing GM-CSF mRNA improved median survival by 18 days with 28% overall survival compared with standard RNA- NPs (Bottom) (*p <0.05; **p<0.01, Gehan-Breslow-Wilcoxon test).

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Figure 48: Tumor volumes of B16F10-OVA in mice receiving RNA-NPs co- encapsulated with or without GM-CSF mRNA

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single RNA-NP vaccine one day after subcutaneous B16F10-OVA tumor (1 x 106/mouse) implantation. GFP RNA-NPs did not prolong survival (Top). Mice receiving RNA-NPs co-encapsulated with GM-CSF mRNA had smaller tumor volumes compared RNA-NPs alone (***p = 0.0001; **** p <0.0001, Two-Way ANOVA).

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8.4 Alternative Strategies for Enhancement

While co-administration of GM-CSF encoding mRNA enhanced the activity of

NP vaccines, we investigated several other strategies to further potentiate the anti-tumor efficacy of our formulation. To augment the innate and adaptive immune responses, we attempted to enhance the anti-tumor efficacy of our formulations through co- administration of immunomodulatory RNAs. We screened RNA-NPs in combination with TLR agonists and suicide genes to enhance the innate response. We also screened

RNA-NPs in combination with mRNAs encoding for heat shock proteins (HSPs) and co- stimulatory ligands (i.e. CD40L) to enhance the adaptive response. In all cases, we determined success of each enhancing strategy based on heightened anti-tumor efficacy in our intra-cranial tumor models.

8.4.1 Enhancing innate immunity via TLR agonists

Since innate immunity using TLR agonists has been shown to be synergistic with immunotherapeutic vaccines, we co-delivered TLR agonists for TLR3 (polyI:C) and

TLR9 (Cpg oligonucleotides) (Chiang, Kandalaft, & Coukos, 2011; Honda et al., 2005;

Krug et al., 2001). However, addition of polyI:C and CpG oligonucleotides did not enhance the anti-tumor efficacy of RNA-NPs in a B16F10-OVA model (Fig 49-50). Since

RNA can serve as a TLR agonist for receptors 7 and 8, which promote signaling through

MYD88, there may be no additional advantage to adding agonists (i.e. TLR9 agonists) that signal through the same dependent pathway (Kawai & Akira, 2006, 2010).

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Interestingly, co-administration of TLR3 agonists abrogated the anti-tumor effect of

RNA-NP vaccines (Fig. 50). Since TLR3 signals via the TRAF3 pathway, co- encapsulation of poly(I:C) may circumvent MYD88 signaling thereby abolishing the response induced by RNA-NP vaccines (Hacker et al., 2006; Oganesyan et al., 2006).

Figure 49: Comparison of efficacy in mice vaccinated with RNA-NPs co- encapsulated with or without CpG oligonucleotides

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single RNA-NP vaccine 5 days after intracranial B16F10-OVA (6,250 cells/mouse) implantation. Compared with RNA NPs alone, there was no improvement in anti-tumor efficacy through co- administration of CpG oligonucleotides.

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Figure 50: Comparison of efficacy in mice vaccinated with RNA-NPs co- encapsulated with or without polyI:C

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single RNA-NP vaccine 5 days after intracranial B16F10-OVA (6,250 cells/mouse) implantation. Compared with RNA-NPs alone, there was no improvement in anti-tumor efficacy through co- administration of polyI:C.

8.4.2 Enhancing innate immunity using suicide gene therapy

Alternatively, we attempted to optimize the anti-tumor efficacy elicited by RNA-

NP vaccines using suicide gene therapy via herpes simplex thymidine kinase (HSV-TK) encoding mRNA. HSV-TK incorporation into the host genome can stimulate potent innate immune responses after systemic administration of ganciclovir (GCV) by inducing cross priming (Sanchez-Perez et al., 2006). GCV mediating killing releases antigens from dying cells transfected with HSV-TK and may further amplify the innate immune response mediated by RNA-NPs (Sanchez-Perez et al., 2006). We utilized a

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mutant HSV-TK mRNA with markedly enhanced kinase activity (HSVsr39tk), and demonstrated cytotoxicity in vitro after administration of GCV (Fig. 51) (Sundaram,

Harpstrite, Kao, Collins, & Sharma, 2012).

Figure 51: HSVsr39tk mRNA transfected DCs in vitro

A cell line of DC2.4s was either untransfected (Left) or transfected (Right) with RNA-NPs encoding for HSVsr39tk. Cells were then treated with GCV containing media and harvested after 72 hours in culture. HSVsr39tk mRNA transfected DCs showed GCV-mediated cytotoxicity (*p <0.05, unpaired t test).

However, co-encapsulation of RNA-NP with HSVsr39tk mRNA did not enhance the efficacy of our vaccine formulation in mice treated with GCV (Fig. 52).

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Figure 52: Comparison of efficacy in mice vaccinated with RNA-NPs co- encapsulated with or without HSVsr39tk mRNA

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single RNA-NP vaccine 5 days after intracranial B16F10-OVA (6,250 cells/mouse) implantation. Blood was drawn one week after vaccination for assessment of antigen specific T cell immunity. Compared with RNA-NPs alone, there was no improvement in antigen specific T cell immunity (Left) and anti-tumor efficacy (Right) through co-administration of HSVsr39tk mRNA followed by 5 days of i.p. GCV administration.

8.4.3 Enhancing anti-tumor immunity via co-encapsulation of heat shock proteins and CD40 ligand

We also attempted to enhance the efficacy generated by RNA-NPs through co- administration of heat shock proteins (HSPs). These proteins can serve as chaperones for tumor antigens mediating their uptake by APCs for presentation onto the surface of

MHC class molecules (Calderwood, Murshid, & Gong, 2012; Murshid, Gong, &

Calderwood, 2012). We incorporated mRNA encoding for HSP70 into our

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formulations, but the anti-tumor efficacy elicited by RNA-NPs co-encapsulating HSP70 mRNA was not significantly altered (Fig. 53).

Figure 53: Comparison of efficacy in mice vaccinated with RNA-NPs co- encapsulated with or without HSP mRNA

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single RNA-NP vaccine 5 days after intracranial B16F10-OVA (6,250 cells/mouse) implantation. Compared with RNA-NPs alone, there was no improvement in anti-tumor efficacy through co- administration of HSP mRNA.

Alternatively, we assessed if the addition of co-stimulatory molecules such as

CD40L mRNA would enhance the efficacy of our formulation. While RNA-NPs encoding for CD40L mRNA elicited increased CD40L expression in vitro, co- encapsulation of CD40L mRNA with RNA-NPs did not enhance efficacy (Fig. 54)

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Figure 54: Comparison of efficacy in mice vaccinated with RNA-NPs co- encapsulated with or without CD40L mRNA

Cell line of DC2.4s were transfected in vitro with CD40L RNA-NPs and harvested after 1 day later for assessment of CD40L expression (Left). There is increased CD40L expression on DC2.4s transfected with CD40L RNA-NPs. C57Bl/6 mice, spiked with OT- Is, were vaccinated with a single RNA-NP vaccine 5 days after intra-cranial B16F10- OVA (6,250 cells/mouse) implantation (Right). Compared with RNA-NPs alone, there is no improvement in anti-tumor efficacy through co-administration of CD40L mRNA.

Although these strategies did not enhance the efficacy of our vaccine platform, we have demonstrated proof of concept data that RNA-NP vaccines can be enhanced with immunomodulatory RNAs (i.e. GM-CSF mRNA) and have optimized a model system for testing further enhancements.

8.5 Enhancing RNA-NP Efficacy with Combinatorial Checkpoint Blockade

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To determine if anti-tumor efficacy elicited by RNA-NPs could be elicited in an

OVA independent manner, C57Bl/6 mice bearing subcutaneous melanomas were vaccinated with once weekly NP vaccines (x3) that were complexed with total tumor

RNA (TTRNA) derived from B16F10. Mice vaccinated with TTRNA-NPs had smaller tumor volumes compared with untreated animals (Fig. 55).

Figure 55: Tumor volumes of B16F10 in untreated mice versus mice receiving TTRNA-NP vaccines

C57Bl/6 mice were implanted with subcutaneous B16F10 (25,000 cells) and TTRNA-NPs co-encapsulated with GM-CSF mRNA were given one day after implantation for a total of three weekly vaccines. Mice receiving RNA-NPs had smaller tumor volumes compared with untreated mice (*p <0.05, Mann Whitney Test).

Based on upregulation of PD-L1 observed from i.v. administration of RNA-NPs, we expected that our vaccine formulation could be significantly enhanced in combination with checkpoint blockade, which we investigated in an aggressive B16F10 103

murine melanoma model. To assess if RNA-NP vaccines would be synergistic in combination with checkpoint blockade, mice were implanted with subcutaneous B16F10 and given anti-PD-L1 mAbs starting 24hrs prior to RNA-NP vaccination. Mice receiving

RNA-NPs in conjunction with anti-PD-L1 mAbs had smaller tumor volumes compared to mice receiving either RNA-NP vaccines (Fig. 56) or anti-PD-L1 mAb treatment alone

(Fig. 57).

Figure 56: Tumor volumes of B16F10 in mice receiving TTRNA-NPs with or without co-administered anti-PD-L1 mAb

C57Bl/6 mice were implanted with subcutaneous B16F10 tumors (150,000 cells) and received weekly TTRNA-NPs co-encapsulated with GM-CSF mRNA (2 days after tumor implantation) in conjunction with twice weekly anti-PD-L1 starting 1 day prior to each vaccine. Mice receiving TTRNA-NPs in conjunction with anti-PD-L1 mAbs had smaller tumor volumes compared to TTRNA-NPs alone (****p <0.0001, Two-way ANOVA).

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Figure 57: Tumor volumes of B16F10 in mice receiving anti-PD-L1 mAbs with or without co-administered TTRNA-NPs

C57Bl/6 mice were implanted with subcutaneous B16F10 tumors (100,000 cells) and received weekly TTRNA-NPs co-encapsulated with GM-CSF mRNA (2 days after tumor implantation) in conjunction with weekly anti-PD-L1 starting 1 day prior to each vaccine. Mice receiving TTRNA-NPs in conjunction with anti-PD-L1 mAbs had smaller tumor volumes compared to anti-PD-L1 mAbs alone (****p <0.0001, Two-way ANOVA).

8.6 Discussion

Tumor-specific immunotherapy holds the promise of eliminating malignant brain tumors without cytotoxic damage to normal tissue, and immunotherapeutic treatments now have proven efficacy in recent phase III clinical trials (Ansell et al., 2015;

Borghaei et al., 2015; Hodi et al., 2010; Kantoff et al., 2010; D. A. Mitchell & Nair, 2000a,

2000b; Sayour, Sanchez-Perez, et al, 2015). Although these initial efficacious findings corroborate the utility of cancer immunotherapy, greater insight is requisite to understand the immune correlates for successful vaccine approaches. Although further

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testing is necessary, innate and adaptive immunity are likely paramount for successful immunotherapeutic amelioration (Sayour, Sanchez-Perez, et al., 2015).

To enhance the innate and adaptive immune response generated by our vaccine, we investigated several immunomodulatory RNAs to be combined with our NP formulation; we measured the activity of each new enhancing strategy based on anti- tumor efficacy in an intracranial B16F10-OVA model. We utilized TLR agonists and

HSV-TK encoding mRNA to enhance the innate response, and HSP and CD40L encoding mRNA to enhance the adaptive response. While these did not show benefit,

GM-CSF enhanced both the immunogenicity and anti-tumor efficacy of RNA-NP vaccines; we also demonstrated that RNA-NPs can be potentiated in combination with immune checkpoint blockade. Co-delivery of immunomodulatory RNAs encoding for additional cytokines within our NP formulations may bypass dose limiting toxicities seen with exogenous administration and recruit multiple arms of the immune system in tandem from a single vaccination (D. A. Mitchell et al., 2008).

While these initial screens were not exhaustive, the immunogenicity and therapeutic potency of RNA-NP vaccines may be increased with additional strategies.

RNA-NP based gene expression and immunogenicity can be further enhanced through incorporation of 5’ and 3’untranslated regions (UTRs) from long-lived RNA viruses or through assimilation of nuclease-resistant nucleotides (Aviv, Voloch, Bastos, & Levy,

1976; Azzoni, Ribeiro, Monteiro, & Prazeres, 2007; Falcone & Andrews, 1991; Grudzien-

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Nogalska, Jemielity, Kowalska, Darzynkiewicz, & Rhoads, 2007; Grudzien-Nogalska,

Stepinski, et al., 2007; Holcik & Liebhaber, 1997; Kore & Shanmugasundaram, 2008;

Kozak, 1994; P. Mitchell & Tollervey, 2000; Rodgers, Wang, & Kiledjian, 2002; Wang &

Kiledjian, 2000; Yu & Russell, 2001). For example, tumor mRNA transcripts can be flanked with the 5’ internal ribosomal entry sequence and 3’ polyUUU tail UTRs derived from hepatitis C (HCV) to simultaneously enhance the gene expression and immunogenicity of mRNA cancer antigens (Bradrick, Walters, & Gromeier, 2006; Bung et al., 2010; Fang, 1999; Saito, Owen, Jiang, Marcotrigiano, & Gale, 2008). By utilizing mechanisms employed by viral infections to augment intracellular gene expression and immunogenicity, RNA-nanoliposomal vaccines can be further manipulated to engender therapeutic anti-tumor immunity against malignant brain tumors (D. A. Mitchell et al.,

2008; Sayour, Sanchez-Perez, et al., 2015).

RNA-nanoliposomes encoding for both tumor RNAs and immunomodulatory molecules, are a versatile platform for delivering combinatorial therapies through a single treatment modality, and we have shown the ability to rapidly screen various strategies to enhance the efficacy of our vaccine platform (D. A. Mitchell et al., 2008;

Sayour, Sanchez-Perez, et al., 2015). In juxtaposition with a better understanding of the immunogenicity and regulatory immune pathways implicated in GBM subgroups,

RNA-NP vaccines can be engineered with these enhancing strategies to re-program

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immunity from a regulatory state to an activated state thereby offering a more effective and specific therapy for patients with GBM without collateral toxicity.

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9. Supplanting DC vaccines with RNA-Nanoparticles targeting GBM

9.1 Introduction

DCs can prime CD4+/CD8+ T-cells against GBM antigens and have been employed to ex vivo activate T cells against intracranial tumors (D. A. Mitchell et al.,

2008; D. A. Mitchell & Nair, 2000a, 2000b; D. A. Mitchell & Sampson, 2009; Sampson &

Mitchell, 2015; Steinman, 2001a). DCs can then be simultaneously delivered to patients with ex vivo activated T cells ensuring their in vivo expansion and persistence (D. A.

Mitchell et al., 2008; D. A. Mitchell & Nair, 2000a, 2000b; Sampson & Mitchell, 2015).

To generate tumor specific T cells, DCs must be pulsed with an immunogenic tumor antigen; however, there is a relative dearth in GBM specific targets (D. A. Mitchell et al., 2008; D. A. Mitchell & Sampson, 2009; Sampson & Mitchell, 2015). While the epidermal growth factor receptor variant III (EGFRvIII) is immunogenic and unique to

GBM, it is only expressed in a subset (~ one-third) of these patients, and within this subset, only ~30-40% of malignant cells express the mutation (Babu & Adamson, 2012;

Miao et al., 2014; D. A. Mitchell et al., 2008; D. A. Mitchell & Sampson, 2009; Sampson et al., 2011; Sampson et al., 2010; L. Sanchez-Perez et al., 2013). Thus, the limited availability and dearth of widely expressed tumor specific antigens coupled with the complexity of individualized cellular vaccines targeting GBM tumor cells have limited

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immunotherapy’s advancement (D. A. Mitchell et al., 2008; D. A. Mitchell & Sampson,

2009).

Although the detection and confirmation of tumor-specific antigens in malignant brain tumors is limited, the total antigenic mRNA content of tumor cells

(total tumor derived RNA (TTRNA)) when loaded into APCs has been shown to elicit anti-tumor immune responses against predominantly uncharacterized murine and human malignancies (Boczkowski, Nair, Snyder, & Gilboa, 1996; Kim, Trivedi, Wilson,

Mahalingam, Morrison, Tsai, Chattergoon, Dang, Patel, Ahn, Boyer, Chalian,

Schoemaker, Kieber-Emmons, Agadjanyan, & Weiner, 1998; D. A. Mitchell et al., 2008;

D. A. Mitchell & Nair, 2000a, 2000b; Steinman, 2001b; Strobel et al., 2000). Since TTRNA represents a personalized cohort of tumor specific transcripts, simultaneously recruits both cytotoxic T lymphocytes (CTLs) and helper T-cells and holds substantial safety benefits over other immunotherapeutic platforms (i.e. DNA vaccines or viral vectors),

TTRNA loaded DCs presents a safe and attractive formulation to activate autologous T cells against intracranial malignancies (Kim, Trivedi, Wilson, Mahalingam, Morrison,

Tsai, Chattergoon, Dang, Patel, Ahn, Boyer, Chalian, Schoemaker, Kieber-Emmons,

Agadjanyan, & Weiner, 1998; Kim, Trivedi, Wilson, Mahalingam, Morrison, Tsai,

Chattergoon, Dang, Patel, Ahn, Boyer, Chalian, Schoemaker, Kieber-Emmons,

Agadjanyan, Weiner, et al., 1998; D. A. Mitchell et al., 2008; D. A. Mitchell and Sampson,

2009; D. A. Mitchell & Nair, 2000a, 2000b; Strobel et al., 2000).

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9.2 DC Vaccines against Intracranial Tumors

We have shown that adequate amounts of tumor RNA can be amplified from pediatric and adult brain tumor specimens using as little as 100-500 isolated tumor cells

(Gururangan et al., 2013; A. B. Heimberger, Sun, W., Hussain, S.F., Dey, M., Crutcher, L.,

Aldape, K., Gilbert, M., Hassenbusch, S.J., Sawaya, R., Schmittling, B., Archer, G.E.,

Mitchell, D.A., Bigner, D.D., Sampson, J.H 2007; D. A. Mitchell et al., 2008; D. A. Mitchell

& Nair, 2000a, 2000b; D. Mitchell, Cui, X., Chewning, T., Friedman, H., and Sampson, J.,

2007; S. K. Nair et al., 2015; Sampson, Archer, Mitchell, Heimberger, & Bigner, 2008).

Since TTRNA (prepared autologously to represent a broad repertoire of undefined and patient specific tumor antigens) can be amplified to clinical-scale from so few cells, it provides a renewable antigen resource for preparation of RNA-based immunotherapies

(Boczkowski et al., 1996; Fecci et al., 2007; P. E. Fecci et al., 2006; Peter E. Fecci et al.,

2006; Gururangan et al., 2013; Hess et al., 2006; Kim, Trivedi, Wilson, Mahalingam,

Morrison, Tsai, Chattergoon, Dang, Patel, Ahn, Boyer, Chalian, Schoemaker, Kieber-

Emmons, Agadjanyan, & Weiner, 1998; D. A. Mitchell et al., 2008; D. A. Mitchell & Nair,

2000a, 2000b; Peres et al., 2008; Steinman, 2001b; Strobel et al., 2000). In addition, we have shown the feasibility and safety of RNA-loaded DC vaccines in adults with GBM and children with recurrent medulloblastoma (MB) in ongoing phase I/II clinical trials

(FDA INDs 13630 and 14058; Duke IRB protocols 6677 and 18020; PI: Duane A. Mitchell,

M.D., Ph.D.) (Gururangan et al., 2013; A. B. Heimberger, Sun, W., Hussain, S.F., Dey, M.,

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Crutcher, L., Aldape, K., Gilbert, M., Hassenbusch, S.J., Sawaya, R., Schmittling, B.,

Archer, G.E., Mitchell, D.A., Bigner, D.D., Sampson, J.H 2007; D. A. Mitchell et al., 2008;

D. A. Mitchell & Nair, 2000a, 2000b; D. Mitchell, Cui, X., Chewning, T., Friedman, H., and Sampson, J., 2007; S. K. Nair et al., 2015; Sampson et al., 2008; Sampson & Mitchell,

2015).

9.3 Replacing DCs with RNA-NP Vaccines

While ex vivo generation of TTRNA-loaded DCs holds considerable promise, the advancement of cellular vaccines is fraught with developmental challenges making it difficult to generate vaccines for the population at large (Farrell et al., 2011; D. A.

Mitchell et al., 2008; D. A. Mitchell & Nair, 2000a, 2000b; D. A. Mitchell & Sampson,

2009; S. K. Nair et al., 2015; Sampson & Mitchell, 2015). To circumvent the challenges of cellular therapeutics, we have utilized commercially available and clinically translatable nanoliposomes to deliver TTRNA to APCs in vivo. NPs such as DOTAP protect mRNA transcripts from degradation while simultaneously delivering them to APCs in lymphoid organs where peripheral T cells can be activated against intracranial malignancies (Templeton et al., 1997). Given the relative ease in which RNA-NPs can be manufactured, it is expected that these vaccines could supplant DCs in cellular immunotherapy trials. Since RNA loaded NPs can serve as an attractive “off-the-shelf”

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therapeutic platform with considerable scale-up capacity, we compared their immunogenicity and efficacy with DC vaccines.

9.4 RNA-NPs versus DC vaccines

If RNA-NPs are comparable in their immune effects to RNA-pulsed DC vaccines, then our formulations would supplant DC vaccines based on relative ease and capacity for enhancement. To compare antigen specific immunity generated by DC vaccines versus RNA-NPs, we vaccinated naïve C57Bl/6 mice that had been spiked with OT-I cell transfer and compared the effects of i.v. OVA RNA-NP complexes with i.d. DCs pulsed with OVA mRNA. There was no significant difference in expansion of OVA specific T cells from RNA-NPs versus DC vaccines (Fig. 58).

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Figure 58: Comparison of OVA specific T cell immunity from RNA-NP vaccines versus DC vaccines

C57Bl/6 mice, spiked with OT-Is, were vaccinated with a single OVA RNA-NP vaccine or DC vaccine pulsed with OVA mRNA before splenocytes were harvested one week later. OVA specific T cell immunity induced by RNA-NP vaccines was comparable to DC vaccines pulsed with OVA mRNA based on percentage of OVA specific CD8+ T cells in murine splenocytes.

To further demonstrate that immune responses generated by RNA-NPs were not exclusively a model antigen effect, we evaluated the expansion of tumor-specific T cells in naïve C57Bl/6 mice from TTRNA-NP vaccines compared with TTRNA-pulsed DCs

(Flores et al., 2015). We extracted TTRNA from an infiltrative pre-clinical high grade glioma cell line, KR158B-luc, and vaccinated C57Bl/6 mice with TTRNA-NPs or TTRNA pulsed DCs (Flores et al., 2015; Reilly et al., 2000). To track each animal’s response to vaccination, RNA-NP vaccines and DC vaccines were given after administration of ex vivo activated DsRed labeled T cells specific against KR158B-luc. Compared with mice

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receiving standard intradermal DC vaccines, mice vaccinated with RNA-NPs had a comparable expansion of tumor specific T cells (Fig. 59-60).

Figure 59: Representative flow plots comparing expansion of T cells specific against KR158B-luc from RNA-NP vaccines versus DC vaccines in murine splenocytes and lymph nodes

DS red+ tumor specific T cells were expanded in vitro from primed spleens of mice immunized against TTRNA extracted from KR158B-luc (Flores et al., 2015). These T cells were adoptively transferred after 5-7 days of in vitro activation into KR158B-luc intracranial tumor bearing C57Bl/6 mice followed by TTRNA-NP vaccines or TTRNA pulsed DCs (Flores et al., 2015). TTRNA-NPs expand TTRNA activated T cells comparably to DC based vaccines.

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Figure 60: Expansion of T cells specific against KR158B-luc from RNA-NP vaccines versus DC vaccines in murine splenocytes and lymph nodes.

DS red+ tumor specific T cells were expanded in vitro from primed spleens of mice immunized against TTRNA extracted from KR158B-luc (Flores et al., 2015). These T cells were adoptively transferred after 5-7 days of in vitro activation into KR158B-luc intracranial tumor bearing C57Bl/6 mice followed by TTRNA-NP vaccines or TTRNA pulsed DCs (Flores et al., 2015).

To demonstrate that TTRNA-NP could elicit anti-tumor efficacy against an infiltrative pre-clinical high grade glioma, naïve C57Bl/6 mice were implanted with a radiation resistant and temozolomide resistant infiltrative murine astrocytoma (KR158B- 116

luc) (Flores et al., 2015; Reilly et al., 2000). We have previously demonstrated that anti- tumor efficacy against this tumor is contingent on DC vaccines in a cellular immunotherapy model (Flores et al., 2015). In this model, there was no significant difference in the efficacy elicited by TTRNA pulsed DCs versus TTRNA-NPs (Fig. 61).

These results demonstrate that RNA-NP vaccines are comparable to DC vaccines for induction of anti-tumor efficacy in a cellular immunotherapy model for invasive high- grade glioma (Flores et al., 2015; Reilly et al., 2000).

Figure 61: Survival curve of mice vaccinated with TTRNA-NP vaccines versus TTRNA pulsed DC vaccines in a cellular immunotherapy model for high grade invasive glioma

C57Bl/6 mice were stereotactically implanted with KR158B-luc astrocytoma cells and received a single dose of 9Gy TBI on Day 4 followed by an intravenous injection of 5 x104 lin- bone marrow derived stem cells within 6 h of TBI and intravenous injection of 107 tumor specific T lymphocytes 1 day after TBI (Flores et al., 2015). This was immediately followed by vaccination of 2.5 x 105 total tumor RNA pulsed DCs or TTRNA-NP vaccines. The second and third DC/RNA-NP vaccines were administered at weekly intervals. Mice receiving RNA-NPs had comparable survival to mice receiving standard DC vaccines (*p = 0.0356, Gehan-Breslow-Wilcoxon test).

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9.5 Discussion

We have not witnessed toxicity using TTRNA-NPs in our pre-clinical experiments and have not observed autoimmunity using TTRNA-DC vaccines in our clinical trials for patients with medulloblastoma (Gururangan et al., 2013; Sampson &

Mitchell, 2015). Based on these data, we do not expect toxicity from TTRNA-NPs upon translation into human phase I trials for GBM; however, there may be unknown antigens within the TTRNA cohort from glial tumors with the potential to induce off- target effects (D. A. Mitchell et al., 2008; D. A. Mitchell & Nair, 2000a, 2000b). If this is true, subtractive hybridization may be utilized to selectively omit amplification of shared transcripts between normal cells and tumor cells (Badapanda, 2013; Buzdin,

Kovalskaya-Alexandrova, Gogvadze, & Sverdlov, 2006; Chen, Shen, & Tew, 2001; D. A.

Mitchell et al., 2008). Thus, it is expected that vaccination with TTRNA will provide a safe and personalized approach to re-direct host immunity against GBM antigens.

Since TTRNA pulsed DC vaccines remain limited by their cost, complexity and time to generation, alternative strategies are requisite (D. A. Mitchell et al., 2015; D. A.

Mitchell et al., 2008; D. A. Mitchell & Nair, 2000a, 2000b). After leukopheresis collection, autologous DC manufacturing requires several weeks. This production time provides a significant delay for patients with aggressive malignancies and may preclude candidates from trial with rapid disease progression. Alternatively, RNA-NPs can be generated in a few days after a patient’s biopsy and abrogate the need for a leukopheresis. In a pre-

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clinical cellular immunotherapy model for invasive malignant glioma, RNA NPs were comparable to standard DC vaccines in mediating anti-tumor activity. Since RNA-NP vaccines bypass the complexity of cellular therapeutics, are amenable to central distribution, have significant capacity for enhancement, and can be made within days of tumor resection, these formulations supplant DCs providing near immediate immune induction against inciting malignancies. RNA-NPs can thus be harnessed as a more feasible and effective therapy for patients with glioblastoma and other refractory malignancies.

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10. Conclusions and Future Directions

10.1 Summary

GBM is the most common malignant brain tumor; despite indiscriminate and toxic multimodal therapy including surgery, radiation, and chemotherapy, patients diagnosed usually succumb to their disease within 12-15 months (Finlay et al., 2008; D.

A. Mitchell et al., 2008; Sengupta, Marrinan, Frishman, & Sampath, 2012; Stupp et al.,

2009; Stupp et al., 2005). Since the non-specific and intensive treatment regimens utilized in these patients can lead to substantial neurologic impairment, novel therapies are needed that can target tumor cells without collateral damage to the surrounding normal brain tissue (Graham et al., 1997; D. A. Mitchell et al., 2008). The unique specificity of the immune system and the recent demonstrated activity of immune-based treatments in advanced cancers (in randomized phase III clinical trials), sustains immunotherapy as a promising modality to meet this essential need (Ansell et al., 2015;

Borghaei et al., 2015; Bot, 2010; Brahmer et al., 2012; Brower, 2010; Fecci et al., 2007; P. E.

Fecci et al., 2006; Hodi et al., 2010; Kantoff et al., 2010; D. A. Mitchell et al., 2011; D. A.

Mitchell et al., 2008; D. A. Mitchell & Sampson, 2009; S. K. Nair et al., 2014; Sampson et al., 2010; L. A. Sanchez-Perez et al., 2013; Sayour, Sanchez-Perez, et al., 2015; Sonabend et al., 2012; Yuan et al., 2011).

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To circumvent the challenges associated with cellular therapeutics, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable NPs that can be combined with tumor derived RNA to peripherally activate T cells against GBM antigens (Copland et al., 2003; Ferrari, 2005; D.

A. Mitchell et al., 2008; Nguyen et al., 2012; Peer et al., 2007; Perche et al., 2011). Since the advancement of novel NP platforms into clinical evaluation has been slow, we chose to evaluate nanoliposomal formulations that have been utilized in several clinical trials, are readily available in cGMP formulation, and can be engineered to up-regulate innate and adaptive host immunity (Ferrari, 2005; D. A. Mitchell et al., 2008; Peer et al., 2007).

(Fasol et al., 2012; Lu et al., 2012; Porteous et al., 1997; Strieth et al., 2013).

While nanoliposomes have suffered from poor in vivo targeting techniques and reticuloendothelial deposition, limiting their utility in the context of drug and gene delivery, we hypothesized that RNA encapsulated nanoliposomes would localize to RES sites, transfect peripheral APCs therein, and induce antigen specific T cell immunity against intracranial malignancies (Klein et al., 2007; Klugewitz et al., 2002; Lorenzer,

Dirin, Winkler, Baumann, & Winkler, 2015; Polakos et al., 2007; Simberg et al., 2004). We screened several translatable NP formulations for their ability to transfect DCs in vitro with GFP mRNA and demonstrated that the NP DOTAP was the most promising translatable formulation. RNA-NP vaccines formulated in DOTAP were shown to induce in vivo gene expression and preserve RNA stability over time. We determined

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that i.v. injection of RNA-NPs was requisite for inducing functional antigen specific immunity, which was superior to standard peptide vaccines formulated in CFA.

Intravenously administered RNA-NPs localized to the spleen and liver and WBCs from both organs expanded antigen specific T cells in naïve mice. After RNA-NP vaccination, splenic and hepatic CD11c+ cells had an increase in the percentage of B7 co-stimulatory molecules with compensatory PD-L1 expression. We enhanced the immunogenicity and anti-tumor efficacy of RNA-NP vaccines by combining RNA-NPs with immune checkpoint blockers (i.e. anti-PD-L1 mAb) and by co-encapsulating immunomodulatory

RNAs encoding for bioactive cytokines (i.e. GM-CSF mRNA). Finally, we demonstrated that RNA-NP vaccines mediate anti-tumor efficacy against intracranial and subcutaneous melanomas and engender therapeutic anti-tumor efficacy in a cellular immunotherapy model against a radiation/temozolomide resistant invasive murine high grade glioma.

10.2 Future Directions

Unlike many promising nanomaterials, mired by unknown reactivities in humans, we have demonstrated that translatable nanoliposomes localize to the spleen and liver transfecting CD11c+ cells therein without the need for engineering targeting moieties. Since DOTAP is available as a cGMP-grade formulation (Avanti) bypassing the rate-limiting steps of scale-up synthesis, these combinatorial RNA-NP vaccines can

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be translated with significant ease over other nanoscale formulations (Sayour, Sanchez-

Perez, et al., 2015). Another advantage of this platform is the unique ability to package

RNAs into NP formulations not only to re-direct baseline immune responses against cancer antigens but also to deliver combinatorial therapies using a single delivery platform (McNeil, 2009; Peer et al., 2007). Next generation RNA-NP vaccines can be comprised of tumor mRNA co-delivered with RNAs (i.e. RNAs encoding for mAbs or siRNAs) that interfere with host immunosuppressive mechanisms (i.e. PD-L1) identified on GBM (Bloch et al., 2013; Wintterle et al., 2003). These packaged and combinatorial

RNA-NP vaccines afford this technology malleability and a wide range of applicability.

10.3 Conclusion

Although cancer vaccines have suffered from weak immunogenicity and the milieu of a regulatory host microenvironment, we have advanced a NP vaccine formulation that can precipitate activated T cell frequencies necessary to engender long term anti-tumor efficacy against intracranial tumors (Rosenberg, 2012). Since RNA-NP vaccines can simultaneously bypass the complexity of cellular therapeutics (while cutting down the time to generation of personalized vaccines), the translation of RNA nanoliposomal vaccines promises to significantly improve treatment outcomes for patients with GBM. They also promise to offer a wide range of clinical application for all

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malignancies that can be targeted using RNA obtained from surgical resection of solid tumors.

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References

Adiseshaiah, P. P., Hall, J. B., & McNeil, S. E. (2010). Nanomaterial standards for efficacy and toxicity assessment. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2(1), 99- 112. doi: 10.1002/wnan.66

Ancliff, P. J., Gale, R. E., Liesner, R., Hann, I. M., & Linch, D. C. (2001). Mutations in the ELA2 gene encoding neutrophil elastase are present in most patients with sporadic severe congenital neutropenia but only in some patients with the familial form of the disease. Blood, 98(9), 2645-2650.

Ansell, S. M., Lesokhin, A. M., Borrello, I., Halwani, A., Scott, E. C., Gutierrez, M., . . . Armand, P. (2015). PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma. N Engl J Med, 372(4), 311-319. doi: 10.1056/NEJMoa1411087

Aviv, H., Voloch, Z., Bastos, R., & Levy, S. (1976). Biosynthesis and stability of globin mRNA in cultured erythroleukemic Friend cells. Cell, 8(4), 495-503.

Aytekin, C., Germeshausen, M., Tuygun, N., Tanir, G., Dogu, F., & Ikinciogullari, A. (2010). Kostmann disease with developmental delay in three patients. Eur J Pediatr, 169(6), 759-762. doi: 10.1007/s00431-010-1151-5

Azzoni, A. R., Ribeiro, S. C., Monteiro, G. A., & Prazeres, D. M. (2007). The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression. J Gene Med, 9(5), 392-402. doi: 10.1002/jgm.1031

Babu, R., & Adamson, D. C. (2012). Rindopepimut: an evidence-based review of its therapeutic potential in the treatment of EGFRvIII-positive glioblastoma. Core Evid, 7, 93-103. doi: 10.2147/CE.S29001

Badapanda, C. (2013). Suppression subtractive hybridization (SSH) combined with bioinformatics method: an integrated functional annotation approach for analysis of differentially expressed immune-genes in insects. Bioinformation, 9(4), 216-221. doi: 10.6026/97320630009216

Barbalat, R., Ewald, S. E., Mouchess, M. L., & Barton, G. M. (2011). Nucleic acid recognition by the innate immune system. Annu Rev Immunol, 29, 185-214. doi: 10.1146/annurev-immunol-031210-101340

125

Bawa, Raj (2008). Nanoparticle-based Therapeutics in Humans: A survey. Nanotechnology Law and Business, 5(2), 135-155.

Bennett, C. L., Christie, J., Ramsdell, F., Brunkow, M. E., Ferguson, P. J., Whitesell, L., . . . Ochs, H. D. (2001). The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet, 27(1), 20-21. doi: 10.1038/83713

Benteyn, D., Van Nuffel, A. M., Wilgenhof, S., Corthals, J., Heirman, C., Neyns, B., . . . Bonehill, A. (2013). Characterization of CD8+ T-cell responses in the peripheral blood and skin injection sites of melanoma patients treated with mRNA electroporated autologous dendritic cells (TriMixDC-MEL). Biomed Res Int, 2013, 976383. doi: 10.1155/2013/976383

Blasius, A. L., & Beutler, B. (2010). Intracellular toll-like receptors. Immunity, 32(3), 305- 315. doi: 10.1016/j.immuni.2010.03.012

Bloch, O., Crane, C. A., Kaur, R., Safaee, M., Rutkowski, M. J., & Parsa, A. T. (2013). Gliomas promote immunosuppression through induction of B7-H1 expression in tumor-associated macrophages. Clin Cancer Res, 19(12), 3165-3175. doi: 10.1158/1078-0432.CCR-12-3314

Boczkowski, D., Nair, S. K., Snyder, D., & Gilboa, E. (1996). Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med, 184(2), 465-472.

Borghaei, H., Paz-Ares, L., Horn, L., Spigel, D. R., Steins, M., Ready, N. E., . . . Brahmer, J. R. (2015). Nivolumab versus Docetaxel in Advanced Nonsquamous Non- Small-Cell Lung Cancer. N Engl J Med, 373(17), 1627-1639. doi: 10.1056/NEJMoa1507643

Bot, A. (2010). The landmark approval of Provenge, what it means to immunology and "in this issue": the complex relation between vaccines and autoimmunity. Int Rev Immunol, 29(3), 235-238. doi: 10.3109/08830185.2010.490777

Bradrick, S. S., Walters, R. W., & Gromeier, M. (2006). The hepatitis C virus 3'- untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Res, 34(4), 1293-1303. doi: 10.1093/nar/gkl019

Brahmer, J. R., Tykodi, S. S., Chow, L. Q., Hwu, W. J., Topalian, S. L., Hwu, P., . . . Wigginton, J. M. (2012). Safety and activity of anti-PD-L1 antibody in patients

126

with advanced cancer. N Engl J Med, 366(26), 2455-2465. doi: 10.1056/NEJMoa1200694

Brower, V. (2010). Approval of provenge seen as first step for cancer treatment vaccines. J Natl Cancer Inst, 102(15), 1108-1110. doi: djq295 [pii]

10.1093/jnci/djq295

Brunkow, M. E., Jeffery, E. W., Hjerrild, K. A., Paeper, B., Clark, L. B., Yasayko, S. A., . . . Ramsdell, F. (2001). Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet, 27(1), 68-73. doi: 10.1038/83784

Bung, C., Bochkaeva, Z., Terenin, I., Zinovkin, R., Shatsky, I. N., & Niepmann, M. (2010). Influence of the hepatitis C virus 3'-untranslated region on IRES-dependent and cap-dependent translation initiation. FEBS Lett, 584(4), 837-842. doi: 10.1016/j.febslet.2010.01.015

Bungener, L., Serre, K., Bijl, L., Leserman, L., Wilschut, J., Daemen, T., & Machy, P. (2002). Virosome-mediated delivery of protein antigens to dendritic cells. Vaccine, 20(17-18), 2287-2295.

Buzdin, A., Kovalskaya-Alexandrova, E., Gogvadze, E., & Sverdlov, E. (2006). GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. Nucleic Acids Res, 34(9), e67. doi: 10.1093/nar/gkl335

Calderwood, S. K., Murshid, A., & Gong, J. (2012). Heat shock proteins: conditional mediators of inflammation in tumor immunity. Front Immunol, 3, 75. doi: 10.3389/fimmu.2012.00075

Carralot, J. P., Probst, J., Hoerr, I., Scheel, B., Teufel, R., Jung, G., . . . Pascolo, S. (2004). Polarization of immunity induced by direct injection of naked sequence- stabilized mRNA vaccines. Cell Mol Life Sci, 61(18), 2418-2424. doi: 10.1007/s00018-004-4255-0

Carralot, J. P., Weide, B., Schoor, O., Probst, J., Scheel, B., Teufel, R., . . . Pascolo, S. (2005). Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas. Genet Vaccines Ther, 3, 6. doi: 10.1186/1479-0556-3-6

127

Chen, Z. J., Shen, H., & Tew, K. D. (2001). Gene expression profiling using a novel method: amplified differential gene expression (ADGE). Nucleic Acids Res, 29(10), E46.

Chiang, C. L., Kandalaft, L. E., & Coukos, G. (2011). Adjuvants for enhancing the immunogenicity of whole tumor cell vaccines. Int Rev Immunol, 30(2-3), 150-182. doi: 10.3109/08830185.2011.572210

Chikh, G., & Schutze-Redelmeier, M. P. (2002). Liposomal delivery of CTL epitopes to dendritic cells. Biosci Rep, 22(2), 339-353.

Choi J, Sun Wang, N. . (2011). Nanoparticles in Biomedical Applications and Their Safety Concerns. InTech 299-314.

Coban, C., Kobiyama, K., Aoshi, T., Takeshita, F., Horii, T., Akira, S., & Ishii, K. J. (2011). Novel strategies to improve DNA vaccine immunogenicity. Curr Gene Ther, 11(6), 479-484.

Conry, R. M., LoBuglio, A. F., Wright, M., Sumerel, L., Pike, M. J., Johanning, F., . . . Curiel, D. T. (1995). Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Res, 55(7), 1397-1400.

Copland, M. J., Baird, M. A., Rades, T., McKenzie, J. L., Becker, B., Reck, F., . . . Davies, N. M. (2003). Liposomal delivery of antigen to human dendritic cells. Vaccine, 21(9-10), 883-890.

Crispe, I. N. (2009). The liver as a lymphoid organ. Annu Rev Immunol, 27, 147-163. doi: 10.1146/annurev.immunol.021908.132629

Crispe, I. N. (2011). Liver antigen-presenting cells. J Hepatol, 54(2), 357-365. doi: 10.1016/j.jhep.2010.10.005

Daniels, G. A., Sanchez-Perez, L., Diaz, R. M., Kottke, T., Thompson, J., Lai, M., . . . Vile, R. G. (2004). A simple method to cure established tumors by inflammatory killing of normal cells. Nat Biotechnol, 22(9), 1125-1132. doi: 10.1038/nbt1007

Demeneix, B., & Behr, J. P. (2005). Polyethylenimine (PEI). Adv Genet, 53, 217-230.

Desmet, C. J., & Ishii, K. J. (2012). Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination. Nat Rev Immunol, 12(7), 479-491. doi: 10.1038/nri3247

128

Dudley, M. E., Wunderlich, J. R., Robbins, P. F., Yang, J. C., Hwu, P., Schwartzentruber, D. J., . . . Rosenberg, S. A. (2002). Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science, 298(5594), 850- 854. doi: 10.1126/science.1076514

Edlich, B., Hogdal, L. J., Rehermann, B., & Behrens, S. E. (2010). Dendritic cells transfected with Her2 antigen-encoding RNA replicons cross-prime CD8 T cells and protect mice against tumor challenge. Vaccine, 28(49), 7764-7773. doi: DOI 10.1016/j.vaccine.2010.09.054

El Andaloussi, A., Han, Y., & Lesniak, M. S. (2006). Prolongation of survival following depletion of CD4+CD25+ regulatory T cells in mice with experimental brain tumors. J Neurosurg, 105(3), 430-437. doi: 10.3171/jns.2006.105.3.430

Falcone, D., & Andrews, D. W. (1991). Both the 5' untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro. Mol Cell Biol, 11(5), 2656-2664.

Fang, J. W. (1999). In vitro stability of hepatitis C virus RNA. Methods Mol Med, 19, 381- 384. doi: 10.1385/0-89603-521-2:381

Farrell, D., Ptak, K., Panaro, N. J., & Grodzinski, P. (2011). Nanotechnology-based cancer therapeutics--promise and challenge--lessons learned through the NCI Alliance for Nanotechnology in Cancer. Pharm Res, 28(2), 273-278. doi: 10.1007/s11095-010- 0214-7

Fasol, U., Frost, A., Buchert, M., Arends, J., Fiedler, U., Scharr, D., . . . Mross, K. (2012). Vascular and pharmacokinetic effects of EndoTAG-1 in patients with advanced cancer and liver metastasis. Ann Oncol, 23(4), 1030-1036. doi: 10.1093/annonc/mdr300

Fecci, P. E., Ochiai, H., Mitchell, D. A., Grossi, P. M., Sweeney, A. E., Archer, G. E., . . . Sampson, J. H. (2007). Systemic CTLA-4 blockade ameliorates glioma-induced changes to the CD4+ T cell compartment without affecting regulatory T-cell function. Clin Cancer Res, 13(7), 2158-2167. doi: 10.1158/1078-0432.CCR-06-2070

Fecci, P. E., Sweeney, A. E., Grossi, P. M., Nair, S. K., Learn, C. A., Mitchell, D. A., . . . Sampson, J. H. (2006). Systemic anti-CD25 monoclonal antibody administration safely enhances immunity in murine glioma without eliminating regulatory T cells. Clin Cancer Res, 12(14 Pt 1), 4294-4305. doi: 10.1158/1078-0432.CCR-06-0053

129

Fecci, Peter E., Sweeney, Alison E., Grossi, Peter M., Nair, Smita K., Learn, Christopher A., Mitchell, Duane A., . . . Sampson, John H. (2006). Systemic Anti-CD25 Monoclonal Antibody Administration Safely Enhances Immunity in Murine Glioma without Eliminating Regulatory T Cells. Clin Cancer Res, 12(14), 4294- 4305.

Ferrari, M. (2005). Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer, 5(3), 161-171. doi: 10.1038/nrc1566

Finlay, J. L., Dhall, G., Boyett, J. M., Dunkel, I. J., Gardner, S. L., Goldman, S., . . . Children's Cancer, Group. (2008). Myeloablative chemotherapy with autologous bone marrow rescue in children and adolescents with recurrent malignant astrocytoma: outcome compared with conventional chemotherapy: a report from the Children's Oncology Group. Pediatr Blood Cancer, 51(6), 806-811. doi: 10.1002/pbc.21732

Fitzgerald, K. A., McWhirter, S. M., Faia, K. L., Rowe, D. C., Latz, E., Golenbock, D. T., . . . Maniatis, T. (2003). IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol, 4(5), 491-496. doi: 10.1038/ni921

Flores, C., Pham, C., Snyder, D., Yang, S., Sanchez-Perez, L., Sayour, E., . . . Mitchell, D. A. (2015). Novel role of hematopoietic stem cells in immunologic rejection of malignant gliomas. Oncoimmunology, 4(3), e994374. doi: 10.4161/2162402X.2014.994374

Fotin-Mleczek, M., Duchardt, K. M., Lorenz, C., Pfeiffer, R., Ojkic-Zrna, S., Probst, J., & Kallen, K. J. (2011). Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity. J Immunother, 34(1), 1-15. doi: 10.1097/CJI.0b013e3181f7dbe8

Fotin-Mleczek, M., Zanzinger, K., Heidenreich, R., Lorenz, C., Thess, A., Duchardt, K. M., & Kallen, K. J. (2012). Highly potent mRNA based cancer vaccines represent an attractive platform for combination therapies supporting an improved therapeutic effect. J Gene Med, 14(6), 428-439. doi: 10.1002/jgm.2605

Garfall, A. L., Maus, M. V., Hwang, W. T., Lacey, S. F., Mahnke, Y. D., Melenhorst, J. J., . . . Stadtmauer, E. A. (2015). Chimeric Antigen Receptor T Cells against CD19 for Multiple Myeloma. N Engl J Med, 373(11), 1040-1047. doi: 10.1056/NEJMoa1504542

Gattinoni, L., Finkelstein, S. E., Klebanoff, C. A., Antony, P. A., Palmer, D. C., Spiess, P. J., . . . Restifo, N. P. (2005). Removal of homeostatic cytokine sinks by 130

lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med, 202(7), 907-912. doi: 10.1084/jem.20050732

Geall, A. J., Verma, A., Otten, G. R., Shaw, C. A., Hekele, A., Banerjee, K., . . . Mandl, C. W. (2012). Nonviral delivery of self-amplifying RNA vaccines. Proc Natl Acad Sci U S A, 109(36), 14604-14609. doi: 10.1073/pnas.1209367109

Germeshausen, M., Deerberg, S., Peter, Y., Reimer, C., Kratz, C. P., & Ballmaier, M. (2013). The spectrum of ELANE mutations and their implications in severe congenital and cyclic neutropenia. Hum Mutat, 34(6), 905-914. doi: 10.1002/humu.22308

Graham, M. L., Herndon, J. E., 2nd, Casey, J. R., Chaffee, S., Ciocci, G. H., Krischer, J. P., . . . Friedman, H. S. (1997). High-dose chemotherapy with autologous stem-cell rescue in patients with recurrent and high-risk pediatric brain tumors. J Clin Oncol, 15(5), 1814-1823.

Gros, A., Robbins, P. F., Yao, X., Li, Y. F., Turcotte, S., Tran, E., . . . Rosenberg, S. A. (2014). PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors. J Clin Invest, 124(5), 2246-2259. doi: 10.1172/JCI73639

Grudzien-Nogalska, E., Jemielity, J., Kowalska, J., Darzynkiewicz, E., & Rhoads, R. E. (2007). Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells. RNA, 13(10), 1745-1755. doi: 10.1261/rna.701307

Grudzien-Nogalska, E., Stepinski, J., Jemielity, J., Zuberek, J., Stolarski, R., Rhoads, R. E., & Darzynkiewicz, E. (2007). Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability. Methods Enzymol, 431, 203- 227. doi: 10.1016/S0076-6879(07)31011-2

Grupp, S. A., Kalos, M., Barrett, D., Aplenc, R., Porter, D. L., Rheingold, S. R., . . . June, C. H. (2013). Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med, 368(16), 1509-1518. doi: 10.1056/NEJMoa1215134

Guo, C., Manjili, M. H., Subjeck, J. R., Sarkar, D., Fisher, P. B., & Wang, X. Y. (2013). Therapeutic cancer vaccines: past, present, and future. Adv Cancer Res, 119, 421- 475. doi: 10.1016/B978-0-12-407190-2.00007-1

Gururangan, S., Grant, G. A., Driscoll, T., Archer, G., Sayour, E. J., Herndon, J. E., . . . Mitchell, D. A. (2013). Adoptive Lymphocyte Therapy (Alt) Plus Dendritic Cell Vaccination (Dcv) after Myeloablative (Ma) or Non-Myeloablative (Nma)

131

Conditioning in Patients with Recurrent Central Pnet (C-Pnet). Pediatr Blood Cancer, 60, 13-13.

Hacker, H., Redecke, V., Blagoev, B., Kratchmarova, I., Hsu, L. C., Wang, G. G., . . . Karin, M. (2006). Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature, 439(7073), 204-207. doi: 10.1038/nature04369

Harries, M., Ellis, P., & Harper, P. (2005). Nanoparticle albumin-bound paclitaxel for metastatic breast cancer. J Clin Oncol, 23(31), 7768-7771. doi: 10.1200/JCO.2005.08.002

Heath, J. R., & Davis, M. E. (2008). Nanotechnology and cancer. Annu Rev Med, 59, 251- 265. doi: 10.1146/annurev.med.59.061506.185523

Heidenreich, R., Jasny, E., Kowalczyk, A., Lutz, J., Probst, J., Baumhof, P., . . . Fotin- Mleczek, M. (2014). A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile. Int J Cancer. doi: 10.1002/ijc.29402

Heimberger, A. B., Abou-Ghazal, M., Reina-Ortiz, C., Yang, D. S., Sun, W., Qiao, W., . . . Fuller, G. N. (2008). Incidence and prognostic impact of FoxP3+ regulatory T cells in human gliomas. Clin Cancer Res, 14(16), 5166-5172. doi: 10.1158/1078- 0432.CCR-08-0320

Heimberger, A.B., Sun, W., Hussain, S.F., Dey, M., Crutcher, L., Aldape, K., Gilbert, M., Hassenbusch, S.J., Sawaya, R., Schmittling, B., Archer, G.E., Mitchell, D.A., Bigner, D.D., Sampson, J.H (2007). Immunological responses in a patient with glioblastoma multiforme treated with sequential courses of temozolomide and immunotherapy: Case study. Neuro Oncology, In press.

Heiser, A., Maurice, M. A., Yancey, D. R., Coleman, D. M., Dahm, P., & Vieweg, J. (2001). Human dendritic cells transfected with renal tumor RNA stimulate polyclonal T- cell responses against antigens expressed by primary and metastatic tumors. Cancer Res, 61(8), 3388-3393.

Hess, P. R., Boczkowski, D., Nair, S. K., Snyder, D., & Gilboa, E. (2006). Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen. Cancer Immunol Immunother, 55(6), 672-683. doi: 10.1007/s00262-005-0064-z

132

Hodi, F. S., O'Day, S. J., McDermott, D. F., Weber, R. W., Sosman, J. A., Haanen, J. B., . . . Urba, W. J. (2010). Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med, 363(8), 711-723. doi: 10.1056/NEJMoa1003466

Hoekstra, D., Rejman, J., Wasungu, L., Shi, F., & Zuhorn, I. (2007). Gene delivery by cationic lipids: in and out of an endosome. Biochem Soc Trans, 35(Pt 1), 68-71. doi: 10.1042/BST0350068

Holcik, M., & Liebhaber, S. A. (1997). Four highly stable eukaryotic mRNAs assemble 3' untranslated region RNA-protein complexes sharing cis and trans components. Proc Natl Acad Sci U S A, 94(6), 2410-2414.

Honda, K., Ohba, Y., Yanai, H., Negishi, H., Mizutani, T., Takaoka, A., . . . Taniguchi, T. (2005). Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature, 434(7036), 1035-1040. doi: 10.1038/nature03547

Jackson, C. A., Messinger, J., Palmer, M. T., Peduzzi, J. D., & Morrow, C. D. (2003). Gene expression in the muscle and central nervous system following intramuscular inoculation of encapsidated or naked poliovirus replicons. Virology, 314(1), 45-61.

Jacobs, J. F., Idema, A. J., Bol, K. F., Grotenhuis, J. A., de Vries, I. J., Wesseling, P., & Adema, G. J. (2010). Prognostic significance and mechanism of Treg infiltration in human brain tumors. J Neuroimmunol, 225(1-2), 195-199. doi: 10.1016/j.jneuroim.2010.05.020

Joshi, M. D., Unger, W. J., Storm, G., van Kooyk, Y., & Mastrobattista, E. (2012). Targeting tumor antigens to dendritic cells using particulate carriers. J Control Release, 161(1), 25-37. doi: 10.1016/j.jconrel.2012.05.010

Kallen, K. J., Heidenreich, R., Schnee, M., Petsch, B., Schlake, T., Thess, A., . . . Fotin- Mleczek, M. (2013). A novel, disruptive vaccination technology: self-adjuvanted RNActive((R)) vaccines. Hum Vaccin Immunother, 9(10), 2263-2276. doi: 10.4161/hv.25181

Kalos, M., Levine, B. L., Porter, D. L., Katz, S., Grupp, S. A., Bagg, A., & June, C. H. (2011). T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med, 3(95), 95ra73. doi: 10.1126/scitranslmed.3002842

Kantoff, P. W., Higano, C. S., Shore, N. D., Berger, E. R., Small, E. J., Penson, D. F., . . . Investigators, Impact Study. (2010). Sipuleucel-T immunotherapy for castration-

133

resistant prostate cancer. N Engl J Med, 363(5), 411-422. doi: 10.1056/NEJMoa1001294

Karyampudi, L., Lamichhane, P., Scheid, A. D., Kalli, K. R., Shreeder, B., Krempski, J. W., . . . Knutson, K. L. (2014). Accumulation of memory precursor CD8 T cells in regressing tumors following combination therapy with vaccine and anti-PD-1 antibody. Cancer Res, 74(11), 2974-2985. doi: 10.1158/0008-5472.CAN-13-2564

Kasper, B., Tidow, N., Grothues, D., & Welte, K. (2000). Differential expression and regulation of GTPases (RhoA and Rac2) and GDIs (LyGDI and RhoGDI) in neutrophils from patients with severe congenital neutropenia. Blood, 95(9), 2947- 2953.

Kawai, T., & Akira, S. (2006). Innate immune recognition of viral infection. Nat Immunol, 7(2), 131-137. doi: 10.1038/ni1303

Kawai, T., & Akira, S. (2010). The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol, 11(5), 373-384. doi: 10.1038/ni.1863

Khattri, R., Cox, T., Yasayko, S. A., & Ramsdell, F. (2003). An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol, 4(4), 337-342. doi: 10.1038/ni909

Kim, J. J., Trivedi, N. N., Wilson, D. M., Mahalingam, S., Morrison, L., Tsai, A., . . . Weiner, D. B. (1998). Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine. Oncogene, 17(24), 3125-3135. doi: 10.1038/sj.onc.1201736

Kim, J. J., Trivedi, N. N., Wilson, D. M., Mahalingam, S., Morrison, L., Tsai, A., . . . Shoemaker, H. (1998). Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine. Oncogene, 17(24), 3125-3135.

Klein, I., Cornejo, J. C., Polakos, N. K., John, B., Wuensch, S. A., Topham, D. J., . . . Crispe, I. N. (2007). Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages. Blood, 110(12), 4077-4085. doi: 10.1182/blood-2007-02-073841

Klugewitz, K., Blumenthal-Barby, F., Schrage, A., Knolle, P. A., Hamann, A., & Crispe, I. N. (2002). Immunomodulatory effects of the liver: deletion of activated CD4+ effector cells and suppression of IFN-gamma-producing cells after intravenous protein immunization. J Immunol, 169(5), 2407-2413.

134

Kofler, R. M., Aberle, J. H., Aberle, S. W., Allison, S. L., Heinz, F. X., & Mandl, C. W. (2004). Mimicking live flavivirus immunization with a noninfectious RNA vaccine. Proc Natl Acad Sci U S A, 101(7), 1951-1956. doi: 10.1073/pnas.0307145101

Kore, A. R., & Shanmugasundaram, M. (2008). Synthesis and biological evaluation of trimethyl-substituted cap analogs. Bioorg Med Chem Lett, 18(3), 880-884. doi: 10.1016/j.bmcl.2007.12.049

Kozak, M. (1994). Features in the 5' non-coding sequences of rabbit alpha and beta- globin mRNAs that affect translational efficiency. J Mol Biol, 235(1), 95-110.

Kreiter, S., Diken, M., Selmi, A., Diekmann, J., Attig, S., Husemann, Y., . . . Sahin, U. (2011). FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines. Cancer Res, 71(19), 6132-6142. doi: 10.1158/0008-5472.CAN-11-0291

Kreiter, S., Diken, M., Selmi, A., Tureci, O., & Sahin, U. (2011). Tumor vaccination using messenger RNA: prospects of a future therapy. Curr Opin Immunol, 23(3), 399- 406. doi: 10.1016/j.coi.2011.03.007

Kreiter, S., Selmi, A., Diken, M., Koslowski, M., Britten, C. M., Huber, C., . . . Sahin, U. (2010). Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity. Cancer Res, 70(22), 9031- 9040. doi: 10.1158/0008-5472.CAN-10-0699

Kreiter, S., Vormehr, M., van de Roemer, N., Diken, M., Lower, M., Diekmann, J., . . . Sahin, U. (2015). Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature, 520(7549), 692-696. doi: 10.1038/nature14426

Krug, A., Towarowski, A., Britsch, S., Rothenfusser, S., Hornung, V., Bals, R., . . . Hartmann, G. (2001). Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur J Immunol, 31(10), 3026-3037. doi: 10.1002/1521-4141(2001010)31:10<3026::AID-IMMU3026>3.0.CO;2-H

Latchman, Y., Wood, C. R., Chernova, T., Chaudhary, D., Borde, M., Chernova, I., . . . Freeman, G. J. (2001). PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol, 2(3), 261-268. doi: 10.1038/85330

Learn, C. A., Fecci, P. E., Schmittling, R. J., Xie, W., Karikari, I., Mitchell, D. A., . . . Sampson, J. H. (2006). Profiling of CD4+, CD8+, and CD4+CD25+CD45RO+FoxP3+ T cells in patients with malignant glioma reveals differential expression of the immunologic transcriptome compared with T cells 135

from healthy volunteers. Clin Cancer Res, 12(24), 7306-7315. doi: 10.1158/1078- 0432.CCR-06-1727

Liu, M. A. (2010). Immunologic basis of vaccine vectors. Immunity, 33(4), 504-515. doi: 10.1016/j.immuni.2010.10.004

Lohr, J., Ratliff, T., Huppertz, A., Ge, Y., Dictus, C., Ahmadi, R., . . . Herold-Mende, C. (2011). Effector T-cell infiltration positively impacts survival of glioblastoma patients and is impaired by tumor-derived TGF-beta. Clin Cancer Res, 17(13), 4296-4308. doi: 10.1158/1078-0432.CCR-10-2557

Lorenzer, C., Dirin, M., Winkler, A. M., Baumann, V., & Winkler, J. (2015). Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics. J Control Release, 203, 1-15. doi: 10.1016/j.jconrel.2015.02.003

Lu, C., Stewart, D. J., Lee, J. J., Ji, L., Ramesh, R., Jayachandran, G., . . . Roth, J. A. (2012). Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans. PLoS One, 7(4), e34833. doi: 10.1371/journal.pone.0034833

Markov, O. O., Mironova, N. L., Maslov, M. A., Petukhov, I. A., Morozova, N. G., Vlassov, V. V., & Zenkova, M. A. (2012). Novel cationic liposomes provide highly efficient delivery of DNA and RNA into dendritic cell progenitors and their immature offsets. J Control Release, 160(2), 200-210. doi: 10.1016/j.jconrel.2011.11.034

Martinon, F., Krishnan, S., Lenzen, G., Magne, R., Gomard, E., Guillet, J. G., . . . Meulien, P. (1993). Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA. Eur J Immunol, 23(7), 1719-1722. doi: 10.1002/eji.1830230749

Maude, S. L., Frey, N., Shaw, P. A., Aplenc, R., Barrett, D. M., Bunin, N. J., . . . Grupp, S. A. (2014). Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med, 371(16), 1507-1517. doi: 10.1056/NEJMoa1407222

McNeil, S. E. (2009). Nanoparticle therapeutics: a personal perspective. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 1(3), 264-271. doi: 10.1002/wnan.6

Miao, H., Choi, B. D., Suryadevara, C. M., Sanchez-Perez, L., Yang, S., De Leon, G., . . . Sampson, J. H. (2014). EGFRvIII-Specific Chimeric Antigen Receptor T Cells Migrate to and Kill Tumor Deposits Infiltrating the Brain Parenchyma in an

136

Invasive Xenograft Model of Glioblastoma. PLoS One, 9(4), e94281. doi: 10.1371/journal.pone.0094281

Mitchell, D. A., Batich, K. A., Gunn, M. D., Huang, M. N., Sanchez-Perez, L., Nair, S. K., . . . Sampson, J. H. (2015). Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients. Nature, 519(7543), 366-369. doi: 10.1038/nature14320

Mitchell, D. A., Cui, X., Schmittling, R. J., Sanchez-Perez, L., Snyder, D. J., Congdon, K. L., . . . Sampson, J. H. (2011). Monoclonal antibody blockade of IL-2 receptor alpha during lymphopenia selectively depletes regulatory T cells in mice and humans. Blood, 118(11), 3003-3012. doi: 10.1182/blood-2011-02-334565 blood-2011-02-334565 [pii]

Mitchell, D. A., Fecci, P. E., & Sampson, J. H. (2008). Immunotherapy of malignant brain tumors. Immunol Rev, 222, 70-100. doi: 10.1111/j.1600-065X.2008.00603.x

Mitchell, D. A., & Nair, S. K. (2000a). RNA transfected dendritic cells as cancer vaccines. Curr Opin Mol Ther, 2(2), 176-181.

Mitchell, D. A., & Nair, S. K. (2000b). RNA-transfected dendritic cells in cancer immunotherapy. J Clin Invest, 106(9), 1065-1069. doi: 10.1172/JCI11405

Mitchell, D. A., & Sampson, J. H. (2009). Toward effective immunotherapy for the treatment of malignant brain tumors. Neurotherapeutics, 6(3), 527-538. doi: 10.1016/j.nurt.2009.04.003

Mitchell, D., Cui, X., Chewning, T., Friedman, H., and Sampson, J. (2007). Induction of enhanced immunologic responses during hematopoeitic recovery from temozolomide-induced lymphopenia. Neuro-Oncology, 9(4), 506 (Abstracts for the Twelfth Annual Meeting of the Society for Neuro-Oncology).

Mitchell, P., & Tollervey, D. (2000). mRNA stability in eukaryotes. Curr Opin Genet Dev, 10(2), 193-198.

Mockey, M., Bourseau, E., Chandrashekhar, V., Chaudhuri, A., Lafosse, S., Le Cam, E., . . . Midoux, P. (2007). mRNA-based cancer vaccine: prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes. Cancer Gene Ther, 14(9), 802-814. doi: 10.1038/sj.cgt.7701072

137

Morse, M. A., Hobeika, A. C., Osada, T., Berglund, P., Hubby, B., Negri, S., . . . Lyerly, H. K. (2010). An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer. J Clin Invest, 120(9), 3234-3241. doi: 10.1172/JCI42672

Murshid, A., Gong, J., & Calderwood, S. K. (2012). The role of heat shock proteins in antigen cross presentation. Front Immunol, 3, 63. doi: 10.3389/fimmu.2012.00063

Nair, S. K., De Leon, G., Boczkowski, D., Schmittling, R., Xie, W., Staats, J., . . . Mitchell, D. A. (2014). Recognition and killing of autologous, primary glioblastoma tumor cells by human cytomegalovirus pp65-specific cytotoxic T cells. Clin Cancer Res. doi: 10.1158/1078-0432.CCR-13-3268

Nair, S. K., Driscoll, T., Boczkowski, D., Schmittling, R., Reynolds, R., Johnson, L. A., . . . Mitchell, D. A. (2015). Ex vivo generation of dendritic cells from cryopreserved, post-induction chemotherapy, mobilized leukapheresis from pediatric patients with medulloblastoma. J Neurooncol, 125(1), 65-74. doi: 10.1007/s11060-015-1890-2

Nair, S. K., Heiser, A., Boczkowski, D., Majumdar, A., Naoe, M., Lebkowski, J. S., . . . Gilboa, E. (2000). Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nat Med, 6(9), 1011-1017. doi: 10.1038/79519

Nair, S., Zhou, F., Reddy, R., Huang, L., & Rouse, B. T. (1992). Soluble proteins delivered to dendritic cells via pH-sensitive liposomes induce primary cytotoxic T lymphocyte responses in vitro. J Exp Med, 175(2), 609-612.

Nakano, H., Yanagita, M., & Gunn, M. D. (2001). CD11c(+)B220(+)Gr-1(+) cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J Exp Med, 194(8), 1171-1178.

Napolitani, G., Rinaldi, A., Bertoni, F., Sallusto, F., & Lanzavecchia, A. (2005). Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1- polarizing program in dendritic cells. Nat Immunol, 6(8), 769-776. doi: 10.1038/ni1223

Nguyen, D. N., Mahon, K. P., Chikh, G., Kim, P., Chung, H., Vicari, A. P., . . . Anderson, D. G. (2012). Lipid-derived nanoparticles for immunostimulatory RNA adjuvant delivery. Proc Natl Acad Sci U S A, 109(14), E797-803. doi: 10.1073/pnas.1121423109

138

Oganesyan, G., Saha, S. K., Guo, B., He, J. Q., Shahangian, A., Zarnegar, B., . . . Cheng, G. (2006). Critical role of TRAF3 in the Toll-like receptor-dependent and - independent antiviral response. Nature, 439(7073), 208-211. doi: 10.1038/nature04374

Okazaki, T., Chikuma, S., Iwai, Y., Fagarasan, S., & Honjo, T. (2013). A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat Immunol, 14(12), 1212-1218. doi: 10.1038/ni.2762

Paller, C. J., & Antonarakis, E. S. (2012). Sipuleucel-T for the treatment of metastatic prostate cancer: promise and challenges. Hum Vaccin Immunother, 8(4), 509-519. doi: 10.4161/hv.18860

Peer, D., Karp, J. M., Hong, S., Farokhzad, O. C., Margalit, R., & Langer, R. (2007). Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol, 2(12), 751-760. doi: 10.1038/nnano.2007.387

Perche, F., Benvegnu, T., Berchel, M., Lebegue, L., Pichon, C., Jaffres, P. A., & Midoux, P. (2011). Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA. Nanomedicine, 7(4), 445-453. doi: 10.1016/j.nano.2010.12.010

Peres, E., Wood, G. W., Poulik, J., Baynes, R., Sood, S., Abidi, M. H., . . . Abella, E. (2008). High-dose chemotherapy and adoptive immunotherapy in the treatment of recurrent pediatric brain tumors. Neuropediatrics, 39(3), 151-156. doi: 10.1055/s- 0028-1093333

Polakos, N. K., Klein, I., Richter, M. V., Zaiss, D. M., Giannandrea, M., Crispe, I. N., & Topham, D. J. (2007). Early intrahepatic accumulation of CD8+ T cells provides a source of effectors for nonhepatic immune responses. J Immunol, 179(1), 201-210.

Ponsaerts, P., Van Tendeloo, V. F., & Berneman, Z. N. (2003). Cancer immunotherapy using RNA-loaded dendritic cells. Clin Exp Immunol, 134(3), 378-384.

Porteous, D. J., Dorin, J. R., McLachlan, G., Davidson-Smith, H., Davidson, H., Stevenson, B. J., . . . Innes, J. A. (1997). Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther, 4(3), 210-218. doi: 10.1038/sj.gt.3300390

Porter, D. L., Hwang, W. T., Frey, N. V., Lacey, S. F., Shaw, P. A., Loren, A. W., . . . June, C. H. (2015). Chimeric antigen receptor T cells persist and induce sustained 139

remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med, 7(303), 303ra139. doi: 10.1126/scitranslmed.aac5415

Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., & June, C. H. (2011). Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med, 365(8), 725-733. doi: 10.1056/NEJMoa1103849

Qiu, P., Ziegelhoffer, P., Sun, J., & Yang, N. S. (1996). Gene gun delivery of mRNA in situ results in efficient transgene expression and genetic immunization. Gene Ther, 3(3), 262-268.

Ransohoff, R. M., Kivisakk, P., & Kidd, G. (2003). Three or more routes for leukocyte migration into the central nervous system. Nat Rev Immunol, 3(7), 569-581. doi: 10.1038/nri1130

Rapoport, A. P., Stadtmauer, E. A., Binder-Scholl, G. K., Goloubeva, O., Vogl, D. T., Lacey, S. F., . . . June, C. H. (2015). NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med, 21(8), 914-921. doi: 10.1038/nm.3910

Rausch, S., Schwentner, C., Stenzl, A., & Bedke, J. (2014). mRNA vaccine CV9103 and CV9104 for the treatment of prostate cancer. Hum Vaccin Immunother, e29553. doi: 10.4161/hv.29553

Reilly, K. M., Loisel, D. A., Bronson, R. T., McLaughlin, M. E., & Jacks, T. (2000). Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects. Nat Genet, 26(1), 109-113. doi: 10.1038/79075

Restifo, N. P., Dudley, M. E., & Rosenberg, S. A. (2012). Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol, 12(4), 269-281. doi: 10.1038/nri3191

Ribas, A. (2007). Anti-CTLA4 Antibody Clinical Trials in Melanoma. Update Cancer Ther, 2(3), 133-139. doi: 10.1016/j.uct.2007.09.001

Rittig, S. M., Haentschel, M., Weimer, K. J., Heine, A., Muller, M. R., Brugger, W., . . . Brossart, P. (2011). Intradermal vaccinations with RNA coding for TAA generate CD8+ and CD4+ immune responses and induce clinical benefit in vaccinated patients. Mol Ther, 19(5), 990-999. doi: 10.1038/mt.2010.289

140

Rodgers, N. D., Wang, Z., & Kiledjian, M. (2002). Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping. RNA, 8(12), 1526-1537.

Rodriguez-Gascon, A., del Pozo-Rodriguez, A., & Solinis, M. A. (2014). Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles. Int J Nanomedicine, 9, 1833-1843. doi: 10.2147/IJN.S39810

Rosenberg, S. A. (2012). Raising the bar: the curative potential of human cancer immunotherapy. Sci Transl Med, 4(127), 127ps128. doi: 10.1126/scitranslmed.3003634

Rosenberg, S. A., & Dudley, M. E. (2004). Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. Proc Natl Acad Sci U S A, 101 Suppl 2, 14639-14645. doi: 10.1073/pnas.0405730101

Saito, T., Owen, D. M., Jiang, F., Marcotrigiano, J., & Gale, M., Jr. (2008). Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature, 454(7203), 523-527. doi: 10.1038/nature07106

Sampson, J. H., Aldape, K. D., Archer, G. E., Coan, A., Desjardins, A., Friedman, A. H., . . . Heimberger, A. B. (2011). Greater chemotherapy-induced lymphopenia enhances tumor-specific immune responses that eliminate EGFRvIII-expressing tumor cells in patients with glioblastoma. Neuro Oncol, 13(3), 324-333. doi: 10.1093/neuonc/noq157

Sampson, J. H., Archer, G. E., Mitchell, D. A., Heimberger, A. B., & Bigner, D. D. (2008). Tumor-specific immunotherapy targeting the EGFRvIII mutation in patients with malignant glioma. Semin Immunol, 20(5), 267-275.

Sampson, J. H., Heimberger, A. B., Archer, G. E., Aldape, K. D., Friedman, A. H., Friedman, H. S., . . . Bigner, D. D. (2010). Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma. J Clin Oncol, 28(31), 4722-4729. doi: 10.1200/JCO.2010.28.6963

Sampson, J. H., & Mitchell, D. A. (2015). Vaccination strategies for neuro-oncology. Neuro Oncol, 17 Suppl 7, vii15-vii25. doi: 10.1093/neuonc/nov159

Sanchez-Perez, L. A., Choi, B. D., Archer, G. E., Cui, X., Flores, C., Johnson, L. A., . . . Sampson, J. H. (2013). Myeloablative temozolomide enhances CD8(+) T-cell

141

responses to vaccine and is required for efficacy against brain tumors in mice. PLoS One, 8(3), e59082. doi: 10.1371/journal.pone.0059082

Sanchez-Perez, L., Choi, B. D., Reap, E. A., Sayour, E. J., Norberg, P., Schmittling, R. J., . . . Sampson, J. H. (2013). BLyS levels correlate with vaccine-induced antibody titers in patients with glioblastoma lymphodepleted by therapeutic temozolomide. Cancer Immunol Immunother, 62(6), 983-987. doi: 10.1007/s00262- 013-1405-y

Sanchez-Perez, L., Kottke, T., Daniels, G. A., Diaz, R. M., Thompson, J., Pulido, J., . . . Vile, R. G. (2006). Killing of normal melanocytes, combined with heat shock protein 70 and CD40L expression, cures large established melanomas. J Immunol, 177(6), 4168-4177.

Sanchez-Perez, L., Kottke, T., Diaz, R. M., Ahmed, A., Thompson, J., Chong, H., . . . Vile, R. G. (2005). Potent selection of antigen loss variants of B16 melanoma following inflammatory killing of melanocytes in vivo. Cancer Res, 65(5), 2009-2017. doi: 10.1158/0008-5472.CAN-04-3216

Sayour, E. J., McLendon, P., McLendon, R., De Leon, G., Reynolds, R., Kresak, J., . . . Mitchell, D. A. (2015). Increased proportion of FoxP3+ regulatory T cells in tumor infiltrating lymphocytes is associated with tumor recurrence and reduced survival in patients with glioblastoma. Cancer Immunol Immunother, 64(4), 419- 427. doi: 10.1007/s00262-014-1651-7

Sayour, E. J., Sanchez-Perez, L., Flores, C., & Mitchell, D. A. (2015). Bridging infectious disease vaccines with cancer immunotherapy: a role for targeted RNA based immunotherapeutics. J Immunother Cancer, 3, 13. doi: 10.1186/s40425-015-0058-0

Scheel, B., Aulwurm, S., Probst, J., Stitz, L., Hoerr, I., Rammensee, H. G., . . . Pascolo, S. (2006). Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA. Eur J Immunol, 36(10), 2807-2816. doi: 10.1002/eji.200635910

Scheel, B., Braedel, S., Probst, J., Carralot, J. P., Wagner, H., Schild, H., . . . Pascolo, S. (2004). Immunostimulating capacities of stabilized RNA molecules. Eur J Immunol, 34(2), 537-547. doi: 10.1002/eji.200324198

Scheel, B., Teufel, R., Probst, J., Carralot, J. P., Geginat, J., Radsak, M., . . . Pascolo, S. (2005). Toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mRNA. Eur J Immunol, 35(5), 1557-1566. doi: 10.1002/eji.200425656 142

Schlake, T., Thess, A., Fotin-Mleczek, M., & Kallen, K. J. (2012). Developing mRNA- vaccine technologies. RNA Biol, 9(11), 1319-1330. doi: 10.4161/rna.22269

Schuster, S. J., Neelapu, S. S., Gause, B. L., Janik, J. E., Muggia, F. M., Gockerman, J. P., . . . Kwak, L. W. (2011). Vaccination with patient-specific tumor-derived antigen in first remission improves disease-free survival in follicular lymphoma. J Clin Oncol, 29(20), 2787-2794. doi: 10.1200/JCO.2010.33.3005

Sebastian, M., Papachristofilou, A., Weiss, C., Fruh, M., Cathomas, R., Hilbe, W., . . . Zippelius, A. (2014). Phase Ib study evaluating a self-adjuvanted mRNA cancer vaccine (RNActive(R)) combined with local radiation as consolidation and maintenance treatment for patients with stage IV non-small cell lung cancer. BMC Cancer, 14, 748. doi: 10.1186/1471-2407-14-748

Sengupta, S., Marrinan, J., Frishman, C., & Sampath, P. (2012). Impact of temozolomide on immune response during malignant glioma chemotherapy. Clin Dev Immunol, 2012, 831090. doi: 10.1155/2012/831090

Serre, K., Machy, P., Grivel, J. C., Jolly, G., Brun, N., Barbet, J., & Leserman, L. (1998). Efficient presentation of multivalent antigens targeted to various cell surface molecules of dendritic cells and surface Ig of antigen-specific B cells. J Immunol, 161(11), 6059-6067.

Sharma, S., tenOever, B. R., Grandvaux, N., Zhou, G. P., Lin, R., & Hiscott, J. (2003). Triggering the interferon antiviral response through an IKK-related pathway. Science, 300(5622), 1148-1151. doi: 10.1126/science.1081315

Sheikh, N. A., Petrylak, D., Kantoff, P. W., Dela Rosa, C., Stewart, F. P., Kuan, L. Y., . . . Urdal, D. L. (2013). Sipuleucel-T immune parameters correlate with survival: an analysis of the randomized phase 3 clinical trials in men with castration-resistant prostate cancer. Cancer Immunol Immunother, 62(1), 137-147. doi: 10.1007/s00262- 012-1317-2

Simberg, D., Weisman, S., Talmon, Y., & Barenholz, Y. (2004). DOTAP (and other cationic lipids): chemistry, biophysics, and transfection. Crit Rev Ther Drug Carrier Syst, 21(4), 257-317.

Sonabend, A. M., Ogden, A. T., Maier, L. M., Anderson, D. E., Canoll, P., Bruce, J. N., & Anderson, R. C. (2012). Medulloblasoma: challenges for effective immunotherapy. J Neurooncol, 108(1), 1-10. doi: 10.1007/s11060-011-0776-1

143

Sonabend, A. M., Rolle, C. E., & Lesniak, M. S. (2008). The role of regulatory T cells in malignant glioma. Anticancer Res, 28(2B), 1143-1150.

Steinman, R. M. (2001a). Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation. Mount Sinai Journal of Medicine, 68(3), 106-166.

Steinman, R. M. (2001b). Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation. Mt Sinai J Med, 68(3), 160-166.

Steitz, J., Britten, C. M., Wolfel, T., & Tuting, T. (2006). Effective induction of anti- melanoma immunity following genetic vaccination with synthetic mRNA coding for the fusion protein EGFP.TRP2. Cancer Immunol Immunother, 55(3), 246-253. doi: 10.1007/s00262-005-0042-5

Strieth, S., Dunau, C., Michaelis, U., Jager, L., Gellrich, D., Wollenberg, B., & Dellian, M. (2013). Phase I/II clinical study on safety and antivascular effects of paclitaxel encapsulated in cationic liposomes for targeted therapy in advanced head and neck cancer. Head Neck. doi: 10.1002/hed.23397

Strobel, I., Berchtold, S., Gotze, A., Schulze, U., Schuler, G., & Steinkasserer, A. (2000). Human dendritic cells transfected with either RNA or DNA encoding influenza matrix protein M1 differ in their ability to stimulate cytotoxic T lymphocytes. Gene Ther, 7(23), 2028-2035. doi: 10.1038/sj.gt.3301326

Stupp, R., Hegi, M. E., Mason, W. P., van den Bent, M. J., Taphoorn, M. J., Janzer, R. C., . . . National Cancer Institute of Canada Clinical Trials, Group. (2009). Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol, 10(5), 459-466. doi: 10.1016/S1470-2045(09)70025-7

Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J., . . . National Cancer Institute of Canada Clinical Trials, Group. (2005). Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med, 352(10), 987-996. doi: 10.1056/NEJMoa043330

Sundaram, G. S., Harpstrite, S. E., Kao, J. L., Collins, S. D., & Sharma, V. (2012). A New Nucleoside Analogue with Potent Activity against Mutant sr39 Herpes Simplex Virus-1 (HSV-1) Thymidine Kinase (TK). Org Lett. doi: 10.1021/ol300728a

144

Templeton, N. S., Lasic, D. D., Frederik, P. M., Strey, H. H., Roberts, D. D., & Pavlakis, G. N. (1997). Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol, 15(7), 647-652. doi: 10.1038/nbt0797-647

Ulmer, J. B., Mason, P. W., Geall, A., & Mandl, C. W. (2012). RNA-based vaccines. Vaccine, 30(30), 4414-4418. doi: 10.1016/j.vaccine.2012.04.060 van Broekhoven, C. L., Parish, C. R., Demangel, C., Britton, W. J., & Altin, J. G. (2004). Targeting dendritic cells with antigen-containing liposomes: a highly effective procedure for induction of antitumor immunity and for tumor immunotherapy. Cancer Res, 64(12), 4357-4365. doi: 10.1158/0008-5472.CAN-04-0138

Vignuzzi, M., Gerbaud, S., van der Werf, S., & Escriou, N. (2001). Naked RNA immunization with replicons derived from poliovirus and Semliki Forest virus genomes for the generation of a cytotoxic T cell response against the influenza A virus nucleoprotein. J Gen Virol, 82(Pt 7), 1737-1747.

Wang, Z., & Kiledjian, M. (2000). The poly(A)-binding protein and an mRNA stability protein jointly regulate an endoribonuclease activity. Mol Cell Biol, 20(17), 6334- 6341.

Weide, B., Carralot, J. P., Reese, A., Scheel, B., Eigentler, T. K., Hoerr, I., . . . Pascolo, S. (2008). Results of the first phase I/II clinical vaccination trial with direct injection of mRNA. J Immunother, 31(2), 180-188. doi: 10.1097/CJI.0b013e31815ce501

Weide, B., Pascolo, S., Scheel, B., Derhovanessian, E., Pflugfelder, A., Eigentler, T. K., . . . Garbe, C. (2009). Direct injection of protamine-protected mRNA: results of a phase 1/2 vaccination trial in metastatic melanoma patients. J Immunother, 32(5), 498-507. doi: 10.1097/CJI.0b013e3181a00068

West, E. E., Jin, H. T., Rasheed, A. U., Penaloza-Macmaster, P., Ha, S. J., Tan, W. G., . . . Ahmed, R. (2013). PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells. J Clin Invest, 123(6), 2604-2615. doi: 10.1172/JCI67008

Wilgenhof, S., Van Nuffel, A. M., Benteyn, D., Corthals, J., Aerts, C., Heirman, C., . . . Neyns, B. (2013). A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients. Ann Oncol, 24(10), 2686-2693. doi: 10.1093/annonc/mdt245

Wintterle, S., Schreiner, B., Mitsdoerffer, M., Schneider, D., Chen, L., Meyermann, R., . . . Wiendl, H. (2003). Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis. Cancer Res, 63(21), 7462-7467. 145

Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A., & Felgner, P. L. (1990). Direct gene transfer into mouse muscle in vivo. Science, 247(4949 Pt 1), 1465-1468.

Wrzesinski, C., Paulos, C. M., Gattinoni, L., Palmer, D. C., Kaiser, A., Yu, Z., . . . Restifo, N. P. (2007). Hematopoietic stem cells promote the expansion and function of adoptively transferred antitumor CD8 T cells. J Clin Invest, 117(2), 492-501. doi: 10.1172/JCI30414

Yang, I., Tihan, T., Han, S. J., Wrensch, M. R., Wiencke, J., Sughrue, M. E., & Parsa, A. T. (2010). CD8+ T-cell infiltrate in newly diagnosed glioblastoma is associated with long-term survival. J Clin Neurosci, 17(11), 1381-1385. doi: 10.1016/j.jocn.2010.03.031

Ying, H., Zaks, T. Z., Wang, R. F., Irvine, K. R., Kammula, U. S., Marincola, F. M., . . . Restifo, N. P. (1999). Cancer therapy using a self-replicating RNA vaccine. Nat Med, 5(7), 823-827. doi: 10.1038/10548

Yu, J., & Russell, J. E. (2001). Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA. Mol Cell Biol, 21(17), 5879-5888.

Yuan, J., Adamow, M., Ginsberg, B. A., Rasalan, T. S., Ritter, E., Gallardo, H. F., . . . Gnjatic, S. (2011). Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab. Proc Natl Acad Sci U S A, 108(40), 16723-16728. doi: 10.1073/pnas.1110814108

Yue, Q., Zhang, X., Ye, H. X., Wang, Y., Du, Z. G., Yao, Y., & Mao, Y. (2014). The prognostic value of Foxp3+ tumor-infiltrating lymphocytes in patients with glioblastoma. J Neurooncol, 116(2), 251-259. doi: 10.1007/s11060-013-1314-0

Zhang, L., Gu, F. X., Chan, J. M., Wang, A. Z., Langer, R. S., & Farokhzad, O. C. (2008). Nanoparticles in medicine: therapeutic applications and developments. Clin Pharmacol Ther, 83(5), 761-769. doi: 10.1038/sj.clpt.6100400

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Biography

Elias Joseph Sayour was born on December 18, 1981, in Brooklyn, N.Y. to Antoine and Zakieh Sayour. He attended Xaverian High School in Brooklyn, graduating in 1999. He then matriculated at Fordham University and graduated in the spring of 2003 with a B.S. in Biology and Chemistry. He was accepted into medical school at the University of Buffalo School of Medicine and Biomedical Sciences and received his Doctor of Medicine in 2007. After completing residency at the Cohen Children’s Medical Center of New York (CCMCNY) in 2010, he was accepted into a pediatric hematology-oncology fellowship at Duke University Medical Center. Given his strong interest in pursuing a career as a physician-scientist, he enrolled in the Duke Graduate School in 2012 to pursue a Ph.D. in Experimental Pathology.

Publications:

1. Sanchez-Perez L, Choi BD, Reap E, Sayour EJ Norberg P, Schmittling RJ, Archer GE, Herndon JE 2nd, Mitchell DA, Heimberger AB, Bigner DD, Sampson JH. BLyS levels correlate with vaccine-induced antibody titers in patients with glioblastoma lymphodepleted by therapeutic temozolomide. Cancer Immunol Immunother. 2013 Jun;62(6):983-7

2. Miao H, Choi BD, Suryadevara CM, Sanchez-Perez L, Yang S, De Leon G, Sayour EJ McLendon R, Herndon JE 2nd, Healy P, Archer GE, Bigner DD, Johnson LA, Sampson JH. EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma. PLoS One. 2014 Apr 10;9(4):e94281

3. Mitchell DA, Sayour EJ, Reap E, Schmittling R, DeLeon G, Norberg P, Desjardins A, Friedman AH, Friedman HS, Archer G, Sampson JH. Severe adverse immunologic reaction in a patient with glioblastoma receiving autologous dendritic cell vaccines combined with GMCSF and dose-intensified temozolomide. Cancer Immunol Res.2015 Apr;3(4):320-5

4. Flores C, Pham C, Snyder D, Yang S, Sanchez-Perez L, Sayour E Cui X, Kemeny H, Friedman H, Bigner DD, Sampson JH, Mitchell DA. Novel role of hematopoietic stem cells in the immunologic rejection of malignant gliomas. OncoImmunology. 2015 Jan 22;4(3)

5. Sayour EJ, McLendon P, McLendon R et al. Increased proportion of FoxP3+ regulatory T cells in tumor infiltrating lymphocytes is associated with tumor

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recurrence and reduced survival in patients with glioblastoma. Cancer Immunol Immunother. 2015 Apr;64(4):419-27

6. Sayour EJ, Sanchez-Perez L, Flores C, and Mitchell DA. Bridging infectious disease vaccines with cancer immunotherapy: a role for targeted RNA based immunotherapeutics. J Immunother Cancer. 2015 Apr 21;3:13.

7. Sayour EJ, and Eldjerou L. A novel ELANE gene mutation in a patient with severe congenital neutropenia and intermittent thrombocytopenia. Pediatr Blood Cancer. 2015 Oct;62(10):1870-1.

8. Pham CD, Flores C, Yang C, Pinheiro EM, Yearley JH, Sayour E, Pei Y, Moore C, McLendon RE, Huang J, Sampson JH, Wechsler-Reya RJ, Mitchell DA. Differential immune microenvironments and response to immune checkpoint blockade amongst molecular subtypes of murine medulloblastoma. Clin Cancer Res. 2015 Sep 24. pii: clincanres.0713.2015. [Epub ahead of print]

ABSTRACTS 1) Sayour EJ, Pham C, Sanchez-Perez , Snyder D, Mitchell D. RNA Nanoparticle vaccines targeting medulloblastoma. Pediatric Blood and Cancer: Volume: 58 Issue: 7 Special Issue: SI Pages: 1078-1078 Published: JUL 2012

2) Sayour EJ, Sanchez-Perez L, Pham C, Snyder D, Xie WH, Cui XY, Bigner DD, Sampson JH, Mitchell DA, Developing RNA NP vaccines targeting pediatric and adult brain tumors. Neuro-Oncology. Volume: 14 Supplement: 6 Pages: 39- 39 Published: OCT 2012

3) Sayour EJ, McLendon P, Reynolds R, Bigner DD, Sampson JH, McLendon R, Mitchell DA. Assessment of intratumoral tregs in primary and recurrent GBM. Neuro-Oncology. Volume: 14 Supplement: 6 Pages: 39-39 Published: OCT 2012

4) Sayour EJ, Pham C, Sanchez-Perez L, Snyder D, Flores C, Kemeny H, Xie, WH, Cui XY, Bigner D, Sampson J, Mitchell D. RNA nanoparticle vaccines re-direct host-immunity against intracranial malignancies. Neuro-Oncology Volume: 15 Supplement: 1Pages: 36-36 Meeting Abstract: 0134 Published: APR 2013.

5) Sayour EJ, Pham C, Sanchez-Perez L Snyder D, Flores C, Kemeny H, Xie, WH, Cui XY, Bigner D, Sampson J, Mitchell D. Engineering RNA nanoparticle

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vaccines to target pediatric brain tumors. Pediatric Blood and Cancer. Volume: 60 Supplement: 2 Pages: S4-S4 Published: JUN 2013. Oral Paper Presentation at annual ASPHO meeting, Miami FL, May 2013.

6) Gururangan S, Gerald G, Driscoll TA, Archer GA, Sayour EJ at al. Adoptive lymphocyte therapy (ALT) plus dendritic cell vaccination (DCV) after myeloablative (MA) or non-myeloablative (NMA) conditioning in patients with recurrent central PNET (c-PNET). Pediatric Blood and Cancer: Volume: 60 Supplement: 3 Pages: 13-13 Published: SEP 2013

7) Sayour EJ, De Leon, Pham , Sanchez-Perez L, Snyder D, Kemeny H, Flores C, Mitchell D. Harnessing the immunologic capacity of the liver against glioblastoma multiforme. Neuro-Oncology. Volume: 16 Supplement: 5 Meeting Abstract: IT-31 Published: NOV 2014

8) Flores C, Pham C, Snyder D, Yang S, Sanchez-Perez L, Sayour E, Cui X, Kemeny H, Friedman H, Bigner DD, Sampson JH, Mitchell DA. Synergistic cellular interactions in adoptive immunotherapy leads to immunologic rejection of malignant glioma. Neuro-Oncology. Volume: 16 Supplement: 5 Meeting Abstract: IT-31 Published: NOV 2014

9) Chulanetra M, Sayour E, Eldjerou L, Lagmay J, Milner R, Slayton W, and Chang LJ. Novel GD2-specific chimeric antigen receptor-modified T cells targeting osteosarcoma. 2015 Proceedings of the American Association for Cancer Research.

10) Sayour EJ, Flores C, Pham C, De Leon, Kemeny H, Sanchez-Perez L. Mitchell D. RNA nanoparticle vaccines facilitate and sustain adoptive cellular therapy targeting pediatric intracranial malignancies Pediatr Blood Cancer Volume: 62 Supplement: 2 Pages: 24-24 Published: JUN 2015. Oral Paper Presentation at annual ASPHO meeting, Phoenix Az, May 2015.

11) Sayour EJ, De Leon, Pham C, Flores C, Sanchez-Perez L. Mitchell D. RNA nanoparticle vaccines re-program host immunity in favor of enhanced effector responses against intracranial malignancies. 2015 peds SNO meeting, San Diego CA.

12) Sayour EJ, De Leon, Pham C, Sanchez-Perez L Flores C, Mitchell D. RNA nanoparticle vaccines re-program host-immunity against glioblastoma inducing therapeutic anti-tumor activity. Selected for Oral Presentation at annual SNO meeting, San Antonio, Tx Nov. 2015.

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PROFESSIONAL AWARDS, DISTINCTIONS, AND SPECIAL RECOGNITIONS Thrasher Foundation Early Career Award, 2012 Young Investigator Travel Award, Annual ASPHO conference, 2013 St. Baldrick’s Post-doctoral Fellowship Award, 2013 American Brain Tumor Association Discovery Award, 2014 BeeZ Foundation Award, 2014 Hyundai Hope on Wheels Hope Award, 2014 ASPHO Young Investigator Award, 2015

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