Development of immunotherapy against prostate cancer using lentivirally-transduced dendritic cells expressing murine erbB2 as a model tumor-associated antigen

by

Miriam Esmat Mossoba

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Medical Biophysics University of Toronto

© Copyright by Miriam E. Mossoba (2008)

Miriam E. Mossoba

Doctor of Philosophy, 2008

Graduate Department of Medical Biophysics

University of Toronto

Thesis Title: Development of immunotherapy against prostate cancer using lentivirally-

transduced dendritic cells expressing murine erbB2 as a model tumor-associated antigen

Abstract:

Prostate cancer is a leading cause of cancer deaths in North American men. Current

treatments are often not curative, particularly in cases of advanced metastatic disease.

Immunotherapy is a promising approach to treating cancer as it harnesses the immune

system’s ability to mount potent responses against tumor-associated antigens (TAAs).

Dendritic cells (DCs) play a central role in mediating antigen-specific immunity and have

been recently used with some success in clinical trials. The difficulties associated with

obtaining sufficient quantities of DCs from cancer patients provided the rationale for

developing low-dose DC-based immunotherapy approaches in my thesis project. DCs

were genetically engineered using a lentiviral vector (LV) to express erbB2tr, a kinase-

deficient version of erbB2. The human form of erbB2, HER2/neu, is overexpressed in

20% of primary prostate tumors and 80% of their metastases, making this TAA an

attractive target. Using this LV system, efficient transgene delivery into DCs was

achieved without compromising DC function or phenotype. Administering low prime and

boost doses (2x105 or 2x103) of LV-transduced DCs to mice yielded potent and long-term

anti-tumor responses against murine prostate tumors engineered to overexpress erbB2tr.

The 2x105 DC dose yielded complete tumor protection and was associated with humoral

and cellular responses. The 2x103 dose also offered complete protection in some mice,

ii

suggesting that we had reached a lower threshold DC dose. This novel finding prompted

us to determine if co-transducing DCs with an additional LV carrying the cDNA for an

immunomodulatory factor could augment the efficacy of our low-dose strategy. We

chose to test both the DC survival-enhancing RANKL protein and DC function-

enhancing IL-12 in combination with erbB2tr. Although DCs co-transduced with the

LV/RANKL and LV/erbB2tr did not appear to offer enhanced anti-tumor benefits in a

prophylactic setting, co-transduction with LV/IL-12 and LV/erbB2tr did. The

incorporation of IL-12 into the low-dose immunization strategy led to robust long-term

tumor protection and relatively high levels of Th1 immunity. This is the first

demonstration of the efficacy of low-dose DC-mediated immunotherapy using lentiviral

vectors as gene transfer tools. These studies establish a platform for DC-mediated

therapies that can be realistically translated to the clinic.

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Acknowledgments

I first wish to thank all of my previous supervisors for leading me to the path to

graduate school. I also thank my 10th grade biology teacher, Mrs. Bernard, for

introducing me to the concept of gene therapy. I endured many years of waiting to

actually work on gene therapy. An enormous thank you goes to my graduate supervisor,

Dr. Jeffrey Medin. I am very fortunate to have been accepted into Dr. Medin’s lab and I

am so grateful for all he has done for me. His mentorship continues to direct my career.

The lab environment he created taught me many things about the ‘real world’ of science

without coddling me.

Thank you to Drs. Barber and Ohashi for their guidance during my PhD, especially

towards the end. Their help was truly invaluable. I would also like to acknowledge all my

friends and family, as well as all past and present members of the Medin lab for their

advice and help, particularly during the insanely late nights in the lab. Special thanks go

to Drs. Chris Siatskas, Makoto Yoshimitsu, and Jagdeep S Walia, who played a big part

in my education.

I appreciate all the support and help I received from our collaborators, especially

from Dr. Fowler as well as from his past and present lab members.

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TABLE OF CONTENTS Title Page i Abstract ii Acknowledgements iv Table of Contents v List of Figures ix List of Tables xi List of Abbreviations xii Chapter 1: Introduction 1

1.1 Prostate cancer 2

1.1.1 Development 2 1.1.2 Treatments 3 1.1.3 Experimental mouse models 4

1.2 Immunology of cancer 6 1.2.1 Natural self-tolerance against tumor-associated

antigens (TAAs) 6 1.2.2 Evasion of cancer from immunity 9 1.2.3 Immunotherapy against cancer 11

1.3 Dendritic cells (DCs) 12 1.3.1 Origin and development 12 1.3.2 The role of DCs in inducing antigen-specific

immunity 14 1.4 Non-viral DC manipulations for immunotherapy 16 1.5 Generation of recombinant viruses for transducing DCs 18

1.5.1 Overview 18 1.5.2 Adenoviruses 19 1.5.3 Poxviruses 20 1.5.4 Retroviruses 20

1.6 Mouse model studies using virally-transduced DCs 24 1.6.1 Studies using DCs transduced with recombinant

adenoviruses 24 1.6.2 Studies using DCs transduced with recombinant

onco-retroviruses 28 1.6.3 Studies using DCs transduced with recombinant

lentiviruses 29 1.7 Clinical trials using DCs transduced with recombinant viruses 31

1.7.1 Trials using DCs transduced with recombinant

v

adenoviruses 31 1.7.2 Trials using DCs transduced with recombinant

vaccinia viruses 33 1.7.3 Trials using DCs transduced with recombinant

fowlpox viruses 33 1.8 HER2/neu (erbB2) as a model TAA 34

1.8.1 Biology of HER2/neu 34 1.8.2 Association with cancer 36 1.8.3 HER2/neu-targeted therapeutics 36

1.9 Immunomodulatory proteins 37 1.9.1 Interleukin-12 (IL-12) 37 1.9.2 Receptor activator of nuclear factor κB ligand

(RANKL) 39 1.10 Hypothesis and goal of thesis project 41

Chapter 2: Tumor protection following vaccination with low doses of lentivirally transduced DCs expressing the self-antigen erbB2 42

2.1 Abstract 43 2.2 Introduction 44 2.3 Materials and Methods 46

2.3.1 Lentiviral vector (LV) construction and preparation of high titer stocks 46

2.3.2 Mice and cell lines 46 2.3.3 Murine DC generation and transduction 47 2.3.4 Flow cytometric analysis of DCs, tumor cells, and splenocytes 47 2.3.5 Allogeneic Mixed Lymphocyte Reaction 48 2.3.6 Immunizations and tumor inoculations 49 2.3.7 Measurement of anti-erbB2tr antibody from mouse plasma 49 2.3.8 Cytokine secretion assays 50 2.3.9 CD4+, CD8+, and NK cell depletion 51 2.3.10 Statistical analysis 51 2.4 Results 52 2.4.1 DCs are efficiently transduced with lentivirus 52

2.4.2 Lentiviral transduction does not alter DC phenotype or allostimulatory capacity 54

2.4.3 Vaccination with low doses of LV-modified DCs generates antigen-specific tumor protection 56

2.4.4 Mice vaccinated with DC-erbB2tr show strong antigen- specific humoral immunity 59

2.4.5 Analysis of cytokine secretion from splenocytes 60 2.4.6 Role of CD4+, CD8+, and NK cells in mediating tumor

protection 63 2.5 Discussion 68

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Chapter 3: Co-transduction with an IL-12, but not RANKL, lentivector enhances low-dose DC-erbB2tr immunotherapy against erbB2-expressing prostate tumors 73

3.1 Abstract 74 3.2 Introduction 76 3.3 Materials and Methods 80

3.3.1 Lentiviral vector (LV) construction and preparation of high titer lentivirus 80

3.3.2 Mice and tumor cell lines 80 3.3.3 Murine DC generation and transduction 81

3.3.4 Western blot analyses 81 3.3.5 Analysis of transgene and cell surface marker expression

on DCs 82 3.3.6 Immunizations and tumor inoculations 82 3.3.7 Measurement of anti-erbB2tr antibody from mouse plasma 83 3.3.8 Cytokine secretion assays 83

3.3.9 Analysis of tumor-infiltrating lymphocytes 84 3.3.10 Statistical analysis 84

3.4 Results 85 3.4.1 DC populations can be simultaneously transduced with

two independent LVs to efficiently co-express a TAA and an immunomodulatory gene 85

3.4.2 DCs expressing erbB2tr and IL-12 or RANKL exhibit varying levels of DC markers 88

3.4.3 Vaccination using DC-erbB2tr/IL-12, but not DC-erbB2tr/RANKL, elicits erbB2tr-specific anti-tumor protection 91

3.4.4 Mechanism of anti-tumor protection in groups vaccinated with DCs expressing IL-12 is not dependent on an anti-erbB2tr antibody response 93

3.4.5 DC-erbB2/IL-12 vaccinated mice show strong erbB2tr-specific Th1 immunity following tumor challenge 95

3.4.6 DC-erbB2tr/IL-12 immunizations fail to reduce established aggressive tumor growth 100

3.5 Discussion 102 Chapter 4: Discussion 107

4.1 Summary of findings 108 4.2 Future directions 111

4.2.1 Modifying the immunization strategy 111 4.2.2 Modifying the prostate model 113 4.2.3 Testing new applications 114

vii

4.3 Conclusions 115

References 118

viii

List of Figures Figure 1.1 Schematic diagram of a three-plasmid system

used to generate recombinant lentiviruses. 23 Figure 2.1 Schematic diagram of lentiviral vector (LV)

contructs used in these studies. 53 Figure 2.2 Transduction efficiency of LV/erb and LV/enGFP on DCs. 53 Figure 2.3 Transducing DCs with LV/erb and LV/enGFP does not

affect DC phenotype or allostimulatory capacity. 55 Figure 2.4 Immunization with low doses of DC-erbB2tr generates

potent anti-tumor responses. 58 Figure 2.5 DC-erbB2tr immunization dose of 2x105 cells causes a

sustained erbB2-specific humoral response. 61 Figure 2.6 Immunization using DCs transduced with LV-erb offers

antigen-specific Th1 immunity. 62 Figure 2.7 Cytokine production analysis of fractionated splenocytes

shows erbB2tr-specific Th1 immunity. 66 Figure 2.8 Involvement of CD4+, CD8+, and NK cells in mediating

RM1-erbB2tr tumor protection. 67 Figure 3.1 Efficiency of transducing DCs with LVs to express enGFP

or erbB2tr alone, or in combination with IL-12 or RANKL. 86 Figure 3.2 Effect of IL-12 and RANKL on DC signaling. 89 Figure 3.3 Effect of transducing DCs with LV/IL-12 or LV/RL on the

cell surface expression of DC markers. 90 Figure 3.4 Immunizing mice with low doses of DC-erbB2tr/IL-12

leads to robust tumor protection. 92 Figure 3.5 Assessment of humoral immune response against erbB2 in

vaccinated and non-vaccinated mice. 94 Figure 3.6 Vaccinations with DCs transduced with LV/erb and LV/IL-12

offers erbB2tr-specific Th1 immunity. 96 Figure 3.7 Neither Th1 nor Th2 immune responses against erbB2tr was

ix

upregulated in mice following immunizations with DC-erbB2tr/RANKL. 98

Figure 3.8 Mice vaccinated with DC-erbB2tr exhibited systemically

high tumor-infiltrating CD8+ lymphocytes. 99 Figure 3.9 Immunization of tumor-bearing mice with DCs transduced

with LV/erb and LV/IL-12 does not delay tumor growth. 101

x

List of Tables Table 1.1 Recent studies of virally-transduced DCs used in

mouse tumor models. 25 Table 1.2 Clinical trials using virally-transduced DCs. 32

xi

List of Abbreviations Ad adenovirus Ad5 adenovirus serotype 5 AIRE autoimmune regulator AR androgen receptor ADT androgen-deprivation therapy APC antigen-presenting cell BAFF B cell activating factor belonging to the TNF family BIM Bcl-2 Interacting Mediator of Cell Death BM bone marrow BMT bone marrow transplant CEA carcinoembryonic antigen FLIP caspase-8 homologue FLICE-inhibitory protein cppt central polypurine tract CLP common lymphoid progenitor CMP common myeloid progenitor CFA Complete Freund's Adjuvant CTEC cortical thymic epithelial cell CAR coxsackie-adenovirus receptor CTL cytotoxic T lymphocyte CTLA4 cytotoxic T-lymphocyte antigen 4 DC dendritic cell DP double positive ds double-stranded E1 early region 1 EF1α Elongation Factor 1α FcRs Fc receptor Th helper T HA hemagglutinin HSPC hematopoietic stem-progenitor cell HIFU high-intensity focused ultrasound HRPC hormone-refractory prostate cancer HER human epidermal growth factor receptor family HIV-1 human immunodeficiency virus type 1 IDO indolamine 2,3-dioxygenase ICAM-1 intercellular adhesion molecule 1 IFN-γ interferon-γ IL-12 interleukin-12 erbB2tr kinase-deficient form of erbB2 LV lentiviral vector LFA-3 Leukocyte Function Antigen-3 LPS lipopolysaccharide LTR long terminal repeat LHRH luteinizing hormone-releasing hormone

xii

MHC major histocompatibility complex MMP matrix metalloprotease MFI mean fluorescence intensity mTEC medullary thymic epithelial cell MART-1 melanoma antigen recognized by T cells 1 MIICs MHC class II-rich compartments MAPK mitogen-activated protein kinase MLR mixed lymphocyte reaction MVA modified vaccinia virus Ankara MPR mouse prostate reconstitution NSCLC non-small cell lung cancer NT non-transduced NF-κB nuclear factor-κB NIK nuclear factor-κB-inducing kinase OD optical density OCT optimal cutting temperature OPG osteoprotegerin OVA ovalbumin PAMP pathogen-associated molecular pattern PALS peri-arteriolar lymphatic sheath TSA peripheral tissue-specific antigen PI-3K phosphatidylinositol-3-kinase PEI polyethylenimine PIA proliferative inflammatory atrophy PSA prostate-specific antigen PSCA prostate stem cell antigen PSMA prostate-specific membrane antigen PIN prostatic intraepithelial neoplasia RIP rat insulin promoter RANKL receptor activator of nuclear factor κB ligand RBC red blood cell Treg regulatory T RRE rev response element SIN self-inactivating SHP1 SH2-domain-containing protein tyrosine phosphatase 1 SHIP SH2-domain-containing inositol phosphatase SP single positive ss single-stranded siRNA small interfering RNA SA splice acceptor SD splice donor SOCS suppressor of cytokine signaling TCR T-cell receptor TACE TNF-α-converting enzyme TLR Toll-like receptor TRAMP TRansgenic Adenocarcinoma Mouse Prostate

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TAP1 transporter associated with antigen processing 1 TRICOM TRIad of COstimulatory Molecules TIL tumor infiltrating lymphocyte TNF tumor necrosis factor TNF-α tumor necrosis factor-α TNM Tumor/Nodes/Metastases TAAs tumor-associated antigens TRAF6 tumor-necrosis factor-receptor-associated factor 6 TKI tyrosine kinase inhibitor TRP2 tyrosine-related protein 2 VSV-G vesicular stomatitis virus glycoprotein REL-B v-rel reticuloendotheliosis viral oncogene homolog B WT wild-type WPRE woodchuck hepatitis virus post-transcriptional regulatory element

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Chapter 1:

Introduction Some of the text presented in this chapter has been published in the following

review article: Mossoba ME., and Medin JA. (2006) Cancer immunotherapy using virally

transduced dendritic cells: animal studies and human clinical trials. Expert Rev Vaccines 5:717-32.

1

1.1 Prostate cancer

1.1.1 Development

Prostate cancer remains one of the leading cancers in men, with an estimated

186,320 new cases in the US and 24,700 in Canada this year [1, 2]. The rates of prostate

cancer vary worldwide, but African-American men have relatively high incidence rates

and associated death rates [1] possibly due in part to elevated levels of viral infections

[3]. Environmental exposures including dietary carcinogens [4] bacterial and viral

infections [3, 5] that could lead to inflammation within the prostate are associated with a

higher prostate cancer risk.

The progression from normal epithelial cells to invasive carcinoma begins with the

development of a state called proliferative inflammatory atrophy (PIA) [6, 7]. PIA can be

caused by several genetic or epigenetic changes. Examples include the inactivation of

tumor suppressor genes, such as NKX3.1 [8] and CDKN1B [9] as well as the activation

of oncogenes like MYC and hTERT [10]. Abnormal methylation patterns in CpG islands

[11] may also contribute to overall genetic instability resulting in the appearance of a PIA

state. PIA is associated with the infiltration of inflammatory cells, such as lymphocytes,

macrophages, and neutrophils, which can release reactive oxygen and nitrogen species.

Exposure to these reactive species can trigger DNA damage and cell injury, possibly

aiding the progression from PIA to low-grade prostatic intraepithelial neoplasia (PIN)

[12]. As its name suggests, PIN is characterized by the formation of neoplastic cells. The

genetic instability within these cells can then lead to an accumulation of genetic changes

and a lesion known as high-grade PIN [12]. Subsequent migration of these neoplastic

cells into areas beyond the prostate gland defines the final step towards the development

2

of invasive carcinoma and ultimately metastatic disease. The extent of metastases is

measured using the Tumor/Nodes/Metastases (TNM) system [13], where T1 and T2

clinically define organ-localized cancer and T3 and T4 indicate metastasis. Histological

assessment of the tumor cells is reported as a Gleason score of 1-10, with a value of 10

indicating the most abnormal looking cells [14].

1.1.2 Treatments

The TNM and Gleason grading systems help determine what treatment option will

be used for prostate cancer. Conventional treatments include surgery, radiotherapy, high-

intensity focused ultrasound (HIFU), chemotherapy, cryosurgery, and hormone ablative

therapy. Each of these options carries the risk of significant side effects [15-20],

prompting the need for novel treatment development.

Surgical removal of the prostate gland (radical prostatectomy) for clinically-

localized prostate tumors is associated with low rates of disease recurrence [21]. Due to

the risk of incomplete tumor resection, other treatment modalities may be chosen over

surgery for patients with more advanced disease including locally-advanced T3 cancer, in

which the tumor has grown beyond the prostate organ but not to distant sites [22].

Furthermore, administering neo-adjuvant treatment such as hormonal therapy prior to

radical prostatectomy has not been proven to offer a clear clinical benefit to patients with

locally-advanced prostate cancer [23].

External beam radiotherapy is also often used for locally-advanced cases, but

follow-up studies have demonstrated that up to 90% of treated patients experience tumor

persistence [24]. However, the combination of hormonal therapy, such as luteinizing

hormone-releasing hormone (LHRH) analog treatment, with external beam radiotherapy

3

has been shown to offer patients improved survival relative to radiotherapy alone [25].

Brachytherapy is an alternative radiotherapy treatment, involving the implantation of

small radioactive rods (or seeds) into the prostate gland and this form of therapy has

recently been shown to yield promising results with respect to 5-year survival rates [26].

When patients present with metastatic prostate cancer, androgen-deprivation

therapy (ADT) can help control the spread of the disease, but eventually hormone-

refractory disease often ensues [27]. The mechanisms of this transition from a hormone-

sensitive to a hormone-refractory prostate cancer (HRPC) are poorly understood.

Currently, it is unclear whether administration of chemotherapy to HRPC patients is

beneficial [28].

1.1.3 Experimental mouse models

Experimental mouse models of prostate cancer can be categorized into orthotopic

and ectopic models. To generate orthotopic transgenic models that develop prostate

cancer, viral oncogenes may be expressed in prostate tissue or the pathways involved in

prostate cancer may be genetically manipulated. One notable example is the TRansgenic

Adenocarcinoma Mouse Prostate (TRAMP) mouse, which develops epithelial tumors in

the dorsolateral prostate via the SV40 large T antigen expression driven by the prostate-

specific rat probasin promoter [29]. Generating transgenic models through the genetic

manipulation of prostate cancer signaling pathways is generally more common, as several

target genes have been identified. For example, mice that express the protein kinase Akt

specifically in the prostate under the probasin promoter develop PIN [30]. This model

offers the advantage of being able to test therapeutics specifically against the Akt

signaling pathway, although this model does not develop invasive or metastatic disease.

4

A more aggressive transgenic model was generated by the expressing a mutant androgen

receptor (AR) gene (AR-E231G) in the mouse prostate [31]. These mice develop

metastatic prostate cancer and can therefore be useful for studying the efficacy of

metastasis-targeted therapeutics.

An ectopic transgenic mouse model of prostate cancer was created in which only

the prostate is transgenic for MYC and RAS oncogenes [32]. This model is known as the

mouse prostate reconstitution (MPR) model and is generated in three steps. First, the fetal

urogenital sinus, from which the murine prostatic lobes develop, are isolated. Then, it is

infected with retroviruses to express MYC and RAS. Finally, it is implanted under a host

animal’s renal capsule. There it becomes a fully mature prostate that forms poorly-

differentiated prostatic adenocarcinomas [32]. Furthermore, if the host animals are p53-

deficient, then metastases form in a pattern that is similar to that in prostate cancer

patients [33].

Several cell lines have been derived from the above-described transgenic models.

For example, TRAMP-C1 [34] and RM-1 [35] are murine prostate tumor cell lines

derived from TRAMP and MPR (on a C57BL/6 background) mouse models,

respectively. Injecting these lines into the mouse prostate or the dorsolateral flank

generates orthotopic or ectopic syngeneic mouse models, respectively. Injecting human

prostate cell lines into mice, on the other hand, creates xenogeneic models. Examples of

commonly used human cell lines are DU145 [36], PC3 [37], and LNCaP [38], which

were derived from metastatic prostate tumors. The use of xenogeneic mouse models may

be of limited value for immunotherapy studies if the host animals are immunodeficient.

5

1.2 Immunology of Cancer

1.2.1 Natural self-tolerance against tumor-associated antigens (TAAs)

Although some antigens expressed by tumor cells are mutant proteins, the

majority of TAAs identified thus far are wild-type antigens that are also expressed by

normal cells and are therefore self-antigens. Examples of TAAs are prostate stem cell

antigen (PSCA), prostate-specific antigen (PSA), prostate-specific membrane antigen

(PSMA) and carcinoembryonic antigen (CEA). The immune system is equipped with

many mechanisms to ensure that cells, which express self-antigens including TAAs, are

spared from immune attack. These mechanisms fall under the categories central and

peripheral tolerance.

During central tolerance processes, positive selection of T cells takes place in the

thymic cortex following interaction between thymocytes and cortical thymic epithelial

cells (cTECs) [39]. As immature T cells move within the cortex, they have the

opportunity to encounter a self-peptide-MHC complex on cTECs and to become

positively selected. Studies have shown that the thymocytes at this stage are CD4+CD8+

double positive (DP) T cells and that their interaction with cTECs leads to the formation

of CD4+ or CD8+ single positive (SP) T cells. This transition is also associated with their

migration into the thymic medulla, and is in part mediated by upregulation of the

chemokine receptor CCR7 [40]. Here, medullary thymic epithelial cells (mTECs) and

DCs interact with the developing T cells during negative selection. The expression of co-

stimulatory molecules and peripheral tissue-specific antigens (TSAs) by mTECs allow

the developing T cells that enter the medulla to interact with antigens that are expressed

in the periphery. If the affinity of the interaction is high, then these T cells may undergo

6

clonal deletion or instead develop into a regulatory T cell. Thus, the expression of TSAs

serves as a critical step in guiding the immune system to define self-antigens and to

ensure that tolerance is established against them. The transcriptional regulator AIRE

(autoimmune regulator) helps drive the expression of TSAs [41]. Its importance is

underscored by the fact that mutations in AIRE in mice cause the autoimmune disorder

polyendocrinopathy-candidiasis-ectodermal-dystrophy syndrome, in which clonal

deletion is defective and multi-organ autoimmunity occurs [42, 43].

Proper differentiation and function of mTECs is dependent on factors such as

tumor-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) [44] and nuclear

factor-κB (NF-κB)-inducing kinase (NIK) [45, 46], which help regulate members of the

NF-κB family [47, 48]. The essential role of mTECs in mediating central tolerance is

highlighted by the autoimmune disorders that develop in mice that have deficiencies

relating to mTECs. For example, mice that are deficient in v-rel reticuloendotheliosis

viral oncogene homolog B (REL-B) [49, 50] or TRAF6 [44], or that have a mutation in

the NIK gene [45, 46] have a small medulla lacking mTECs. These mice display normal

positive selection, but have impaired negative selection, leading to autoimmunity and/or

severe inflammation [44-46, 51]. The DCs that reside in the medulla play a role in cross-

presenting TSAs produced by mTECs to developing T cells. This process of cross-

presentation is critical because it allows DCs to load self-peptides derived from

exogenous self-antigens onto major histocompatibility (MHC) class I molecules without

expressing TSAs themselves. Evidence for this process came from experiments using the

transgenic RIP-OVA mouse model, which expresses ovalbumin (OVA) from the rat

insulin promoter (RIP) [52]. Gallegos and Bevan (2004) demonstrated that BM-derived

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antigen-presenting cells are capable of capturing OVA from mTECs and cross-presenting

it to developing T cells in the thymus to mediate clonal deletion [52]. DCs also express

co-stimulatory molecules, including CD80 and CD86, which have been shown to aid in

the process of clonal deletion. For example, studies where CD80 or CD86 were

eliminated or neutralized have demonstrated that self-reactive T cells were not properly

purged [53, 54].

The depletion of lymphocytes that display high-affinity self-antigen recognition

during central tolerance processes does not preclude the escape of some potentially self-

reactive lymphocytes. Peripheral tolerance mechanisms provide an additional layer of

protection against autoimmunity. Thymus-produced CD4+CD25+ regulatory T (Treg)

cells help maintain peripheral self-tolerance, as they can recognize self-antigens

including TAAs [55, 56]. Depletion of Treg cells can result in autoimmune disease [57]

and when cancer patients are Treg cell-depleted, TAA-specific circulating effector T cells

are more readily detected in the peripheral blood [58]. Conversely, Treg cells can be

expanded for therapeutic purposes in treating autoimmune diseases [59, 60].

In addition to suppression by Treg cells, other mechanisms like anergy are in

place to help maintain peripheral tolerance. One intrinsic mechanism of anergy is

antigen-receptor down-regulation. For example, in B cells, defects in the transport of

newly formed IgM from the ER to the cell surface leads to tolerant B cells [61, 62].

Alternatively, negative feedback processes can elevate the threshold of proteins needed to

achieve lymphocyte activation, such as via the recruitment of SH2-domain-containing

protein tyrosine phosphatase 1 (SHP1), a negative regulator of B cell antigen receptor

signaling [63, 64]. Expression of inhibitory receptor CD5 in B and T cells can help

8

recruit SHP1 and inhibit further antigen-receptor signaling and activation [65-67]. SH2-

domain-containing-inositol phosphatase (SHIP) has also been implicated in the inhibition

of B cell activation. This mechanism involves the binding of SHIP to inhibitory receptor

FcγRIIB [68], whose deficiency in mice leads to autoimmunity [69]. In T cells, cytotoxic

T-lymphocyte antigen 4 (CTLA4) is another inhibitory receptor whose expression is

induced when the TCR is highly self-reactive [64]. CTLA4 competes with CD28 for

binding to co-stimulatory B7 molecules on APCs and inhibits T cell activation.

Potentially self-reactive lymphocytes can also be regulated by extrinsic

mechanisms. For example, B cells can be eliminated from the periphery following

competition with less reactive B cells. Prolonged ligation of a self-reactive BCR can

trigger an increase in pro-apoptotic BIM (for Bcl-2 Interacting Mediator of Cell Death)

expression, which would require elevated pro-survival BAFF (for B cell activating factor

belonging to the TNF family) expression levels for the B cell to be rescued from cell

death [70]. Failure to produce enough BAFF would result in the self-reactive B cell in

being out-competed by less reactive B cells, whose dependence on BAFF production for

survival is less [71].

1.2.2 Evasion of cancer from immunity

In addition to overcoming self-tolerance mechanisms, the immune system must

also circumvent immune-evasion strategies used by tumors. Tumor immunity is a

complex process in which tumors and the immune system regulate each other through

several different mechanisms. As a result of the selective pressures exerted by the

immune system, the tumors that successfully persist and develop into more advanced

cancers are derived from cells that were able to evade the immune system. This

9

phenotypic shaping of tumor cell populations by the immune system is called

‘immunoeditting’ [72].

Tumor cells have been found to evade elimination by the immune system through a

variety of mechanisms. Many tumors avoid recognition by cytotoxic T lymphocytes

(CTLs) through loss of MHC class I molecules and can develop into advanced cancer, as

is often the case with lung cancer [73]. Other cancers, such as colorectal carcinoma [74],

exhibit a down-modulation of proteins involved in antigen-processing, including

transporter associated with antigen processing 1 (TAP1) [75]. Due to the subsequent lack

of antigen presentation, a T-cell mediated immune response is evaded and tumors may

become more advanced. It is intriguing that even if an effective T cell response is

initiated against tumor cells, tumor lysis may be avoided. The mechanism for this process

involves the secretion of serine-protease inhibitor PI9 [76, 77] by tumor cells, preventing

effector T cells from releasing perforin and other cytolytic granules that permeablize

tumor cell membranes and initiate tumor cell death. In addition, tumor cells can resist

apoptosis by down-regulating the death receptor Fas [78] or even by up-regulating the

caspase-8 homologue FLICE-inhibitory protein (FLIP) for enhanced tumor survival [79].

Other than defensive mechanisms that tumors use to avoid elimination, several

‘immunosubversion’ (active immune-suppressive) mechanisms against the immune

system have also been reported. Tumors may inhibit CD8+ T cell proliferation [80] and

promote CD4+ T cell apoptosis by expressing indolamine 2,3-dioxygenase (IDO) [81].

Tumors may also secrete immunosuppressive cytokines and other factors, including IL-

10 and prostaglandin E2, which have been reported to inhibit APC function [82]. Finally,

solid tumor masses can also physically exclude immune cells [83]. The immune system

10

exerts a selective pressure on tumor cells such that the tumor cells that evade immunity

are selected.

1.2.3 Immunotherapy against cancer

Improving patients’ immune responses against TAAs is a major goal of cancer

immunotherapy, which can be classified into passive and active forms. Passive

immunotherapy entails administering patients with pre-formed immune complexes (such

as cytokines or antibodies) or with cells that possess anti-tumor reactivity. This approach

has been effectively tested in animal models and clinical trials. For example, the anti-

HER2/neu monoclonal antibody, Herceptin (trastuzumab), has been successfully tested as

a first-line therapy in breast cancer patients that over-express the cell surface TAA

HER2/neu, a member of the human epidermal growth factor receptor family [84]. It is

thought that Herceptin binds to the extracellular domain of HER2/neu, which induces its

downregulation via endocytosis and inhibits important signaling pathways (ras-Raf-

MAPK and PI3K/Akt), thereby blocking tumor cell-cycle progression [85]. Another

example of passive immunotherapy is the use of anti-CTLA-4 antibody, which functions

by antagonizing the T cell inhibitory molecule CTLA-4 in order to help prolong anti-

tumor T cell responses. Anti-CTLA-4 therapy has shown some efficacy against cancers

such as melanoma and prostate cancer in clinical trials [86, 87].

Adoptive transfer of cells that mediate direct anti-tumor reactivity began with the

demonstration that in vitro propagation of lymphoid cells using IL-2 could yield a

population of cells termed lymphokine-activated killing (LAK) cells [88, 89]. In murine

models and clinical trials, LAK cells were shown to have the ability to lyse tumors in a

non-MHC restricted manner [90, 91]. In recognition of the antigen-specific recognition of

11

tumors by T cells, efforts were subsequently made to derive tumor-specific T cell lines

and T cell clones from tumor-infiltrating lymphocytes (TILs) [92, 93]. The evaluation of

adoptive therapy using TILs was shown to be 100-fold more effective than the adoptive

transfer of LAK cells in murine models [94]. Since the first report demonstrating the

regression of cancer using TILs in melanoma patients [95], this form of passive

immunotherapy been used to treat a variety of other cancers with some success [96].

In active immunotherapy, the goal is to endow patients with the ability to create or

strengthen an existing immune response towards antigen(s). This approach can be

achieved in many ways including immunization with autologous tumor cells [97], tumor

lysates [98], specific TAA proteins [99], TAA-derived peptides [100], or even DNA

[101] or viruses [102] encoding TAAs. Another way to achieve active immunotherapy is

by injecting patients with DCs presenting the TAA(s) of interest. DCs are considered to

be the most potent antigen-presenting cells in the immune system [103]. They are critical

for initiating and sustaining strong immune responses. They also allow the formation of

memory T cells and B cells for later recall responses.

1.3 Dendritic cells (DCs)

1.3.1 Origin and development

The origin of murine DCs is the bone marrow, where at least two distinct DC

precursors reside, myeloid and lymphoid. These DC subsets have different phenotypes,

locations, and other characteristics. Although both express surface markers such as

CD11c, CD80, CD40, and MHC class II molecules, lymphoid DCs exclusively express

CD8α [104]. Both DC subsets are found in the spleen and lymph nodes, but lymphoid

DCs are mainly found in their peri-arteriolar lymphatic sheath (PALS) areas that are T

12

cell-rich, while myeloid DCs are usually located in the marginal zone. Although

lymphoid DCs are less phagocytic than myeloid DCs, both have the ability to prime T

cells efficiently in vivo [105, 106].

Evidence for a myeloid origin of DCs came from early studies showing that bone

marrow (BM) myeloid precursors could give rise to macrophages, granulocytes, and DCs

[107]. Other studies indicated that DCs residing in lymphoid tissues had a lymphoid

origin, since they expressed markers associated with lymphoid cells including CD8α,

CD4, and CD25 [108]. More recent in vitro and in vivo studies have demonstrated that

common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) can

each give rise to lymphoid (CD8α+) and myeloid (CD8α-) DCs [106]. This finding

suggests that although the origin of all DCs is the hematopoietic stem cell, DCs appear to

possess the unique property of being able to develop from both myeloid and lymphoid

differentiation pathways and that the subtype of a DC may not necessarily reveal its

lineage origin.

Whereas lymphoid DC precursors that form in the BM migrate to the thymus to

continue their development, myeloid DC precursors migrate into peripheral tissues and

take up antigens. Lymphoid DCs remain in the thymus to mediate central tolerance, as

described in section 1.2.1. Myeloid DCs remain in the periphery until they receive a

maturation signal, which leads to a down-regulation of receptors that take part in antigen

uptake, such as Fc receptors (FcRs) [109], and an up-regulation of proteins needed for T-

cell priming, such as CD80 and CD86 molecules of the immunoglobulin superfamily

[110, 111]. CD80 and CD86 may influence the differentiation pathway, expansion, or

cytokine production of helper T (Th) cells, depending on its antigenic experience; these

13

molecules may help naïve T cells secrete IL-4 and differentiate into Th2 cells, while Th1

cytokine production is mediated by CD80 and CD86 in the presence of IL-2 [112]. DC

maturation signals are inflammatory cytokines, such as TNF-α, or microbial-derived

antigens having pathogen-associated molecular pattern (PAMP) motifs. PAMPs interact

with pattern recognition receptors, such as Toll-like receptors (TLRs), expressed by DCs.

Lipopolysaccharide (LPS), for example, is a cell membrane component of Gram-negative

bacteria and interacts with TLR4 on DCs [113]. During the subsequent maturation

process, DCs present their captured antigens in the form of peptides bound to MHC

molecules on their cell surface [114]. Concurrently, the maturing myeloid DCs migrate

to secondary lymphoid organs [115, 116]. There they interact with lymphocytes using

their MHC-peptide complexes and co-stimulatory molecules to induce a primary immune

response before later undergoing apoptosis [117].

1.3.2 The role of DCs in inducing antigen-specific immunity

The ability of DCs to induce potent immune responses is due in part to the high

expression of MHC and costimulatory molecules on their surface relative to other

antigen-presenting cells (APCs) such as B cells [118]. To prime an antigen-specific

immune response, DCs process antigens intracellularly and present them in the context of

MHC molecules to T cells bearing the cognate antigen-specific T-cell receptor (TCR),

providing T cells with a ‘signal 1’. In vitro assays have indicated that the level of peptide-

MHC molecules presented on the DC surface can help influence the differentiation

pathway of Th cells; low peptide doses support Th1 cell development, while higher doses

promote Th2 cell development [119, 120]. Animal studies of infectious diseases have

demonstrated that the bacterial immunization dose can determine whether Th1 or Th2

14

immunity develops; low antigen doses are associated with Th1 immunity, while high

doses yield a both Th1 and Th2 responses [121, 122]. Furthermore, presentation of low

densities of pigeon cytochrome C (PCC) antigen on APCs has been found to efficiently

activate Th1 memory cell responses [123].

The presentation of antigenic peptides in the context of MHC class I and II

molecules is dependent on the nature of the antigen and how it is acquired by the DC. In

the classical endogenous pathway, peptides from self-antigens and intracellular pathogens

are loaded onto MHC class I molecules for subsequent presentation to CD8+ T cells.

Endogenous antigens are ubiquitinated and then cleaved in the proteasome into peptides

[124]. With the aid of TAP transporters, TAP1/2, peptides are translocated to the

endoplasmic reticulum for further processing and binding to MHC class I molecules

[125]. In addition to MHC class I presentation of endogenous antigens, DCs have the

ability to ‘cross-present’ [126] exogenous antigens, such as phagocytosed antigens, in the

context of MHC class I molecules, in a TAP-independent or -dependent manner [127-

129].

For DCs to prime CD4+ T cells, peptides are presented in the context of MHC class

II molecules. DCs collect soluble antigens via macropinocytosis or endocytosis and

particulate antigens via phagocytosis [109, 130]. Captured antigens are broken down

within endosomes and the resulting polypeptides are transported to MHC class II-rich

compartments (MIICs) for subsequent binding with MHC class II molecules [131, 132].

With the aid of cathepsin S in MIICs, the invariant chain that is associated with MHC

class II molecules is cleaved off to finalize peptide loading before the MHC-peptide

complex is transported to the surface of maturing DCs [133].

15

While mature DCs deliver ‘signal 1’ to T cells to activate them, they also deliver a

‘signal 2’ in order to sustain T cell activation. ‘Signal 2’ is provided by the interactions

between costimulatory molecules like CD80 and CD86 on DCs with CD28 on T cells and

helps to sustain T cell activation and survival [134, 135].

More recently, a ‘signal 3’ has been described as a signal that is delivered from the

APC to the T cell to help instruct its differentiation pathway. Interleukin-12 (IL-12), for

example, is produced by DCs to promote the development of CD4+ Th 1 cells and CD8+

CTLs [136], while Notch1 and Notch2, for example, signal Th2 cell development [137,

138]. Cytokine secretion by activated Th cells leads to the activation of many other cell

types including CTLs [139-141] and B cells [142-144]. The release of Th1 cytokines

[145] such as IFN-γ, IL-2, and TNF-α participates in the development of effector CTLs

within secondary lymphoid organs. Subsequent migration of activated CTLs into the

periphery allows them to migrate towards target cells and to lyse them. In contrast to the

Th1 arm of cellular immunity, the release of Th2 cytokines [145] such as IL-5 and IL-13

contributes to B cell activation. Activated B cells migrate out of secondary lymphoid

organs into the periphery to become mature plasma cells, capable of secreting antibodies

that target the pathogens or cells carrying the appropriate antigens. Thus, DCs are

important cells for inducing and regulating the specificity, strength, and type of immune

reactions.

1.4 Non-viral DC manipulations for immunotherapy

Several types of DC modifications have been tested to initiate or enhance active

immunotherapy against cancer. These schemas can be broadly classified into three

groups: peptide- or lysate-pulsing; RNA or DNA transfections; and viral transductions.

16

DC-pulsing involves co-incubating DCs with whole tumor lysates or with synthetic or

natural peptides against TAAs [146, 147]. This form of DC modification has shown

some efficacy in animal models [148]. Since the early use of peptide-pulsed DCs in

cancer patients [149], many clinical trials have assessed the use of DCs pulsed with a

single peptide or a cocktail of peptides derived from TAAs. Two recent Phase I clinical

trials used HLA-restricted carcinoembryonic antigen (CEA)-derived peptides to pulse

autologous DCs from colorectal cancer patients [150, 151]. Both of those trials showed

an increase in the frequency of CEA-specific T cells, but did not show reduction in

tumors. In another Phase I trial, melanoma patients were given DCs loaded with peptides

derived from four TAAs (MART-1, tyrosinase, MAGE-3, and gp100). These patients

experienced no measurable increase in antigen-specific T-cell responses and no objective

clinical benefit [152]. Tumor lysate-pulsed DCs have also shown modest

immunotherapeutic results in clinical trials. For example, in a recent Phase I trial for

hepatocellular carcinoma, 4 of 31 patients experienced a partial anti-tumor response due

to DC vaccination and 17 patients were found to have stabilized disease after treatment

[153], as defined by set guidelines [154]. Potential reasons for less-than-complete

responses include the limited repertoire offered by peptide-pulsing and the general

instability of the MHC/peptide complex following peptide- or lysate-loading.

To help overcome such potential limitations associated with pulsed DCs, other

methods to modify DCs have been developed. Transfections of DNA or RNA [155, 156]

into DCs have been directly compared to DC-pulsing methods using animal models.

Comparative studies have shown unequivocally the advantages of using nucleic acid

modification methods [157]. Reasons for this include greater MHC-peptide stability

17

(because of their de novo formation), a greater repertoire of peptides derived from each

antigen presented, and the additional possibility of co-expressing adjuvant proteins [158].

That said, it is not entirely clear whether plasmid or genomic DNA-loaded DCs are

clinically more effective against tumors than DCs transfected with in vitro-generated or

whole cell RNA [159]. There is a paucity of clinical trials on DNA-transfected DCs, but

in vivo mouse studies and in vitro human cell experiments indicate potential efficacy in

generating TAA-specific CTL responses [160, 161]. One of the first reported clinical

trials using DCs transfected with tumor RNA did not show dramatic clinical responses

[162]. However, trials using DCs transfected with in vitro-transcribed RNA encoding

TAAs, such as PSA, have demonstrated enhanced T-cell responses against this antigen.

In one study, a complete but transient clearance of circulating tumor cells was achieved

in 3 of 3 analyzed patients [163, 164].

1.5 Generation of recombinant viruses used for transducing DCs

1.5.1 Overview

Viruses are efficient vehicles for introducing foreign DNA into host cells. Several

types of virus have been exploited for delivering genes to DCs. Viral genomes have been

modified in a number of ways to render them useful for gene transfer applications. Genes

required for viral replication are removed and structural/packaging sequences are

separated from the viral backbone into helper plasmids. Removal of viral sequences also

allows for the introduction of a transgene expression cassette. Together, these viral

modifications reduce the chances of homologous recombination re-creating a wild-type

virus. Of note, helper plasmids lack a packaging domain, so that they themselves cannot

be packaged into a viral particle. Transfection of expression and helper plasmids into

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packaging cell lines produces recombinant virions that can be used to transduce target

cells, such as DCs. Here, I focus on three commonly used recombinant viruses in this

context: adenoviruses, poxviruses, and retroviruses.

1.5.2 Adenoviruses

Adenoviruses (Ad) are linear double-stranded (ds) DNA viruses. Following entry

into a cell after binding to the coxsackie-adenovirus receptor (CAR), they transcribe their

early region 1 (E1) genes, which are needed to initiate viral gene expression and genome

replication. Genome replication is also dependent on the expression of E2 and E4 genes.

The E3 region is dispensable for viral replication. Structural proteins necessary for

encapsulating newly-formed viruses are made late in the life-cycle. First generation Ad

vectors were E1-deleted to prevent viral replication. In second and third generation Ad

vectors, E2, E3, and/or E4 genes were also deleted to help reduce Ad immunogenicity.

Further deletions led to the development of ‘gutless’ Ad vectors, which depend on a

helper virus to provide essential genes in trans. The cloning capacity of these vectors is

up to 35 kb. Ad vectors have been widely used in the clinic; between 1989 and 2007, 342

gene therapy trials have used this strategy [165]. Transgene expression in DCs is

transient due to the episomal nature of Ad infection [166]. Interestingly, it has been

reported that binding of the fiber knob domain of Ad to CAR on DCs can actually induce

DC maturation [167]. Because human DCs express very low levels of CAR, Ad vectors

have been developed to permit efficient DC transduction [168]. Ad5f35, for example, is

an Ad serotype 5 (Ad5) vector containing the knob domain derived from Ad35 and has

been shown to efficiently transduce human DCs [169, 170].

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1.5.3 Poxviruses

Vaccinia, Fowlpox, and Canarypox belong to the poxvirus family. Their linear

dsDNA genomes are 100- to 300-kb long and have a hairpin loop at both ends.

Attenuated strains of these viruses have been created. The modified vaccinia virus

Ankara (MVA), for example, was isolated after over 500 passages in chicken embryonic

fibroblasts. By the end of these passages, approximately 31 kb of its genome was lost,

leading to impaired viral replication [171]. Attenuated strains of fowlpox virus and

canarypox virus, such as TROVAC and ALVAC, respectively, were derived in a similar

manner [171]. Between 1989 and 2007, 93 vaccinia and 88 other poxvirus gene therapy

clinical trials were carried out [165]. One of the major advantages to choosing

recombinant vaccinia is that it can carry over 25 kb of foreign DNA. There are reports,

however, that vaccinia virus-transduced DCs exhibit reduced antigen-presenting function

[172, 173].

1.5.4 Retroviruses

Retroviruses are 7- to 11-kb linear single-stranded (ss) RNA viruses. Following

host cell entry, the RNA genome is reverse-transcribed into dsDNA and stably integrated

into the host genome. This property of integrating into host genomes is advantageous for

gene therapy since long-term expression can be achieved in transduced cells and their

progeny. Onco-retroviruses and lentiviruses both belong to the Retroviridae family, but

the latter are more complex. Their genomes consist of long terminal repeat (LTR)

sequences at both ends, as well as structural proteins, polymerases, integrases, and

surface glycoproteins that are encoded by the Gag, Pol, and Env genes, respectively.

Onco-retroviruses and lentiviruses can carry up to 8 kb and 9 kb of exogenous DNA,

20

respectively [174, 175]. Whereas onco-retroviruses require actively dividing host cells for

integration and efficient gene expression, lentiviruses have the ability to integrate into the

genome of slowly-dividing cells since they have a more stable pre-integration complex. It

is reported that 307 gene therapy trials used onco-retroviruses between 1989 and 2007

[165]. Extensive work with lentiviruses in mouse models is now beginning to be

extended to gene therapy clinical trials, 11 of which have been completed and published

to date [165]. Lentiviral vectors have been used in the studies described in this thesis and

will be described in more detail.

The most widely used lentiviral vector (LV) for gene transfer into DCs is based on

the human immunodeficiency virus type 1 (HIV-1). In addition to carrying the Gag, Pol,

and Env genes of simple retroviruses, lentiviruses also contain a number of additional

regulatory and accessory proteins and sequences such as Rev, Tat, central polypurine

tract (cppt) and others. To reduce the risk of forming replication competent retroviruses,

lentiviral vectors have been designed such that many accessory genes have been deleted

or separated onto different plasmids [176] (Figure 1.1). To further increase vector

biosafety, a 400-bp deletion in the 3’ long terminal repeat (LTR) U3 region has been

introduced that renders the virus self-inactivating (SIN). Following transduction of target

cells and provirus integration, this inactive U3 region is present in the 5’ LTR as well

[177]. This modification also helps eliminate residual LTR promoter activity and

prevents the formation of full-length viral transcripts [178, 179]. It also allows for the use

of internal promoters that can achieve tissue-specific gene expression.

Recombinant lentiviruses can be produced by triple transfection of 293T cells using

the following three plasmids (Figure 1.1): (1) The transfer vector, which encodes the

21

transgene of interest (reporter gene or therapeutic gene, for example) driven by an

internal promoter, as well as cis-acting sequences required for packaging (ψ), reverse

transcription, and proviral integration; (2) The packaging vector, which carries Gag and

Pol sequences under the control of a constitutive promoter (eg. CMV); (3) The envelope

vector, which contains a sequence encoding an envelope glycoprotein such as the

vesicular stomatitis virus glycoprotein (VSV-G). Transfected 293T cells release viral

particles into the cell culture medium. Pseudotyping viral particles with VSV-G allows

for a broad tropism of infectivity and enables concentration by ultracentrifugation to

produce high titers. Functional viral titer can be measured by transducing 293T cells with

serially-diluted viral preparations followed by quantifying the percentage of transgene-

positive cells by flow cytometry. Alternatively, particle titer can be determined by

ELISA to quantify the levels of p24 antigen, which is present in the capsid. Other

titration methods using real-time quantitative PCR of RNA isolated from the viral

particles or from the transduced target cells have also been developed [180, 181].

22

Figure 1.1 Schematic diagram of a three-plasmid system used to generate recombinant lentiviruses. The transfer vector contains an expression cassette for the transgene(s) driven by the internal promoter Elongation Factor 1α (EF1α), the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), the packaging signal (ψ), splice donor (SD) and acceptor (SA) sites, and the rev response element (RRE). The packaging vector (pCMV ∆R8.91) encodes Gag, Pol, and other viral genes. The envelope vector (pMD.G) contains the VSV-G envelope sequence needed to pseudotype the recombinant lentivirus.

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1.6 Mouse model studies using virally-transduced DCs

In this section, recent (published in the last 5 years) mouse model studies using

DCs that have been transduced with adenoviruses, onco-retroviruses, or lentiviruses are

discussed. A summary of these studies is shown in Table 1.1.

1.6.1 Studies using DCs transduced with recombinant adenoviruses

Results from several studies employing recombinant Ad vectors are generating

varied immune responses but outcomes are encouraging overall. A paper by Steitz et al.

(2006) [182] has shown that the use of DCs transduced with Ad carrying the melanoma

enzyme tyrosine-related protein 2 (TRP2) was far more efficient in generating a TRP2-

specific immune response compared to a single peptide derived from TRP2 (TRP2aa180-

188). In that study, mice were first immunized with 2.5x105 transduced or peptide-pulsed

DCs. One week later, they were challenged with 4x105 B16 melanoma cells to generate a

model for lung metastases. Induction of CD4+ T cells was found to be critical for the

observed expansion of peptide-specific CD8+ T cells. Complete protection from tumors

was reported for mice immunized with the transduced DCs. This study highlights the

potential of using Ad-transduced DCs for overcoming tolerance to a defined TRP2

peptide.

In another metastatic model of melanoma, Broder et al. (2003) [183] used

recombinant Ad to transduce DCs with Melanoma Antigen Recognized by T cells-1

(MART-1) as the TAA. 5x105 DCs were injected 1 week before 500 B16 melanoma

cells were implanted in the brain. MART-1-specific CTL immunity was achieved along

with improved survival, although only 2 of 23 mice experienced complete protection. It

is difficult to reconcile this report with the previous one and this underscores the

24

25

importance of tumor location, since some locations are immune-privileged [184].

Broder et al. used a greater number of DCs to protect mice against a considerably lower

tumor burden. Although a CTL response was measured, the lack of tumor protection in

an immune-privileged organ, the brain, may illustrate the need for combining

immunotherapy with a brain-targeting strategy. It is unclear whether the induced levels

of immunity against MART-1 would have been sufficient to protect against tumor

challenge at another site.

As mentioned, in order to improve the efficiency of Ad entry into immune cells,

mutations have been made to Ad vectors. The low expression of the Ad-receptor CAR

on DCs prompted the development of a fiber mutant Ad vector (AdRGD). This mutant

uses av–integrin on DCs to gain efficient cell entry. Employing AdRGD to transduce

DCs to express the melanoma-associated antigen gp100, Okada et al. (2003) [185] found

better results compared to conventional Ad. In their experimental model, mice were

immunized with 1x106 DCs and then challenged with 2x105 C57BL/6-derived B16

melanoma cells one week later. The mutant fiber form of this Ad virus allowed for a

higher gene transduction efficiency compared to the conventional Ad vector used for

most gene therapy studies. Greater protection from tumor challenge was also achieved

using the fiber-mutant; 4 of 6 mice remained tumor-free for 60 days post-immunization

compared to 1 of 6 mice given the conventional Ad-gp100-transduced DCs. There was

also a correlation between enhanced protection and enhanced CTL and NK cell activities

against the gp100-expressing melanoma tumors [185].

Ad-transduced DCs have also been tested in transgenic tumor models, including a

breast cancer model. Using Ad encoding the extracellular and transmembrane domains

26

of neu, the rat homologue of the human TAA HER2/neu, transduced DCs were found to

be effective in delaying the onset of mammary tumors in neu-transgenic mice [186].

Whereas all mice that received non-transduced DCs had palpable tumors at 28 weeks, 14

of 21 mice that received 1x106 DCs were tumor-free. It is interesting to note that a

humoral mechanism was primarily involved in mediating this anti-tumor effect [186].

Another study tested Ad-p53 transduced DCs injected in three consecutive daily

injections of 1x106 cells directly into palpable MethA sarcoma tumors or MCA-207

fibrosarcoma tumors [187]. Although 5 of 8 mice bearing MethA tumors underwent

complete tumor regression following DC-p53 treatment, 4 of 8 mice that received non-

transduced DCs also showed complete regression. Only 2 of 5 mice harboring MCA-207

cells and immunized with DC-p53 cells survived for 80 days post-tumor challenge. Only

1 of 4 tumor-bearing mice that received uninfected DCs survived. It is unclear why in

this study the control groups such a high level of tumor regression.

To enhance the effectiveness of DCs, the use of cytokine gene-modified DCs has

been explored. For example, Tatsumi et al. (2003) [188] used two intratumoral injections

of 1x106 DCs transduced with Ad carrying both IL-12 and IL-18 to effectively treat local

and distant CMS4 or MethA sarcoma tumors. IL-12 and IL-18 are pro-inflammatory

cytokines that help elicit Th1/Tc1-type immunity. Using this combination of cytokines,

the viability of injected DCs was extended and splenocyte production of IFN-γ was

enhanced [188]. Although the DCs were not engineered to express a TAA, the authors

hypothesized that the extended DC viability allowed for a greater opportunity to cross-

present tumor antigens.

27

Another promising approach uses intratumorally-injected DCs that are transduced

with Ad vector encoding IFN-α. This cytokine can improve overall DC function and

even help promote cross-priming of T cells [189]. Kuwashima et al. (2005) [190] tested

the therapeutic efficacy of DCs transduced with Ad and thereby engineered to express

IFN-α. Mice with intracranial GL261 glioma tumors were injected intratumorally with

1x105 transduced DCs. Potent anti-tumor responses were measured through IFN-γ

production and cytolytic activities of draining LN cells.

1.6.2 Studies using DCs transduced with recombinant onco-retroviruses

Although many earlier studies have investigated the use of onco-retroviruses in

transducing DCs for inducing anti-tumor immunity, relatively few have been done in the

last few years. This trend may be due to the increasing popularity of lentiviruses, which

share many properties of onco-retroviruses and have the added benefit of dramatically

more efficient transduction and the ability to transduce slowly-dividing cells. On the

other hand, onco-retroviruses have been well studied and production can be standardized

relatively easily. A previous study was published from our laboratory showing the use of

onco-retrovirally transduced DCs in protecting mice from tumor development as well as

inducing regression of palpable tumors [191]. DCs were genetically modified to express

one of two prostate TAAs, PSA or PSMA. In tumor protection experiments, 4x105 PSA-

or PMSA-transduced DCs were injected into mice, followed by tumor challenge using

specifically engineered cells (1x106 or 3x106 cells) at either 1 or 18 weeks later. Mice

exhibited potent humoral and cellular responses against PSA or PSMA and yielded robust

protection against tumors. Immunological memory was also observed. In a more

therapeutic setting examining effects on pre-existing tumors, 5x106 PSA-expressing

28

tumor cells were administered s.c. before immunizing mice at 7 and 37 days later with

PSA-transduced DCs. Near complete elimination of PSA-tumors was observed, whereas

control tumors continued to increase in size over time [191].

1.6.3 Studies using DCs transduced with recombinant lentiviruses

A few papers describing outcomes after LV transduction of DCs in murine models

have been published in recent years. LVs are efficient in transducing DCs and unlike Ad,

they are reported neither to affect the maturational state of DCs nor to elicit virus-specific

immunity, unless unusually high MOI values (around 500) are used [192, 193].

A recent article showed some therapeutic efficacy of LV-transduced DCs

expressing a model antigen (ovalbumin (OVA)) against B16 melanoma engineered to

express the soluble form of OVA. Mice were inoculated with 2x105 tumor cells and then

immunized with 5x105 vector-transduced or OVA peptide-pulsed DCs three days later.

While the pulsed DCs did not confer a survival advantage over control groups, OVA-

transduced DCs allowed mice to survive longer and strongly inhibited tumor growth,

although these mice did not show actual tumor regression in that study [194].

In another investigation targeting therapy for melanoma, DCs were transduced with

a LV encoding murine TRP-2 to assess the protective effects of 1x105 genetically

modified DCs given to animals [195]. Ten days later, mice were injected with 1x105

B16-F10 melanoma cells. Strong protection against tumor challenge was achieved and 4

of 7 mice were free of tumors up to 80 days post-immunization; it was then further shown

that effect was dependent on both CD8+ and CD4+ T cells [195].

Although the classical approach of testing transduced DCs in mouse models has

been to inject BM-derived DCs before or after tumor inoculation, an alternative strategy

29

has been recently undertaken by Cui et al. and published in 2003 [196]. In that study,

hematopoietic stem-progenitor cells (HSPCs) were LV-transduced to engineer expression

of the model antigen hemagglutinin (HA) and transplanted into irradiated recipient mice

bearing B-cell lymphoma A20-HA tumors. This step produced transduced DCs of donor-

origin in the lymphoid organs of recipient mice. Subsequent adoptive transfer of 2.5x107

HA-specific splenocytes from transgenic mice carrying HA-specific CD4+ or CD8+ T

cells was used to clear A20-HA tumor cells. At 20 weeks post-bone marrow transplant

(BMT), mice receiving transduced DCs had a better survival rate than those receiving

non-transduced DCs (50% vs. 10%, P=0.026). Using a similar approach, Xhang et al.

(2008) treated pulmonary metastases in a mouse model using bone marrow cells that

were LV-transduced to express HA [197]. One week following inoculation of mice with

1x106 HA-expressing Renca cells, 4x106 LV-modified bone marrow cells and 1x107

splenocytes were transplanted into lethally-irradiated recipients. Another week later, mice

were treated with DC stimulators GM-CSF and/or CpG-containing oligonucleotides. This

approach efficiently activated antigen-specific T cells and improved the survival of

tumor-bearing mice. Compared to control mice, mice that received HA-expressing LV-

transduced bone marrow cells as well as both adjuvants yielded up to 50% survival by

day 53 compared to just 10% survival by day 33, demonstrating the utility of this

approach in a therapeutic setting [197].

To expand the applications of LV-transduced DCs in immunotherapy, methods

have been developed using LVs to help DCs overcome tolerance to self-TAAs. Shen et

al. (2004) [198] generated an LV encoding a small interfering RNA (siRNA) that down-

regulated the expression of the suppressor of cytokine signaling (SOCS) 1. SOCS1

30

functions as a negative regulator of DC antigen presentation. Silencing SOCS1 by

transducing DCs with LV-SOCS1-siRNA and then pulsing these DCs with TRP2 peptide

led to a significant enhancement of antigen-specific and potent anti-tumor immunity

[198]. In their experimental model, TRP+ B16 tumor-bearing mice were treated with 1

injection of 1x106 LV-transduced, TRP2 peptide-pulsed DCs. Tumor growth was nearly

completely blocked and this result correlated with TRP2-specific CTL activity, as

measured by IFN-γ ELISPOT and CTL assays. Thus, regulating the extent of tumor

antigen presentation on DCs by LV transduction of SOCS1 siRNA has been shown to be

a useful finding in enhancing DC-mediated immunotherapy.

1.7 Clinical trials using DCs transduced with recombinant viruses

In this section, recent clinical studies using DC that have been transduced with

recombinant viruses are discussed below. Table 1.2 shows a summary of these trials.

1.7.1 Trials using DCs transduced with recombinant adenoviruses

In 2006, Antonia et al. reported the results of a clinical trial testing the effects of

immunizing 29 patients having advanced small cell lung cancer with DCs that had been

transduced with a recombinant Ad vector and engineered to express wild-type p53 [199].

Most patients received 3 DC injections consisting of 2.4x105 to 5x106 p53+ DCs. Of the

29 patients in the study, 15 of them produced p53-specific T-cell immune responses.

Antibodies against Ad were also measurable in responsive patients, indicating the

presence of an anti-viral immune reaction. The only clinical benefit observed was a

partial response in 1 patient. Interestingly however, chemotherapy treatment following

the immunotherapy regimen resulted in 18 patients responding with an objective clinical

response. The authors postulate that this is due to chemotherapy inducing: (1) the down-

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32

regulation of tumor-derived immunosuppressive factors; (2) the up-regulation of p53 in

tumor cells for enhanced recognition by CTLs; and/or (3) the up-regulation of perforin or

granzyme production by CTLs for improved tumor killing. The combination of these two

treatment modalities may reveal the potential for achieving better clinical responses.

1.7.2 Trials using DCs transduced with recombinant vaccinia viruses

A recent Phase I study used MVA to transduce DCs in order to engineer expression

of the melanoma TAA, human tyrosinase [200]. Six Stage IV melanoma patients were

immunized with 1x108 gene-modified DCs by i.v. injection three times given 14 days

apart. Tyrosinase (368-376) and VV H3L (184-192) antigen-specific IFN-γ secreting

effector cells were detected by ELISPOT in 5 patients. Memory T cells specific for

tyrosinase were also produced. The patient demonstrating the most response experienced

reduction in one tumor nodule and remained tumor-free for over 850 days following

surgical resection of this nodule. Side effects of the treatment included a mild fever in 3

patients, mild erythema at the site of injection in 5 patients, and vitiligo in 2 patients.

1.7.3 Trials using DCs transduced with recombinant fowlpox viruses

In 2005, Morse et al. reported the results from a Phase I trial for treating 11

colorectal cancer patients and 3 non-small cell lung cancer (NSCLC) patients using DCs

transduced with fowlpox virus encoding CEA combined with a triad of costimulatory

molecules (collectively referred to as TRICOM) [201]. TRICOM consists of B7.1

(CD80), intercellular adhesion molecule 1 (ICAM-1 or CD54), and Leukocyte Function

Antigen-3 (LFA-3 or CD58). Of the 14 patients, 12 received at least 1 immunization

cycle. Ten patients experienced an increase in the frequency of CEA-specific CD4+ and

CD8+ T cells, although administration of a second round of immunization did not lead to

33

any further increase. One patient had minor regression of adenopathy and 5 patients had

disease stabilized for at least 3 months following the first immunization. Grade 3/4

toxicities that were observed could not be directly attributed to immunizations.

Overall, clinical trials using virally-transduced DCs appear to be safe and able to

confer heightened immunity specifically towards tumor-associated antigens to patients.

Although there are several advantages of this form of immunotherapy, the rates of

objective clinical tumor responses seem to be low and emphasize the need for further

developing this approach. Clinical trials testing other forms of immunotherapy, such as

peptide alone or peptide-pulsed DCs have also shown limited success, altogether having

an average objective clinical response of about 2.6% [202].

1.8 HER2/neu (erbB2) as a model TAA

1.8.1 Biology of HER2/neu

HER2/neu belongs to the human epidermal growth factor receptor (HER) family,

which also includes HER1 (EGFR), HER3, and HER4 [203]. The murine homolog of

HER2/neu is often referred to as erbB2 and the rat homolog is often referred to as neu,

since the HER2/neu gene was first cloned from a rat neuroglioblastoma [204]. All HER

members are Type I transmembrane proteins consisting of extracellular, transmembrane,

and intracellular domains. They homodimerize or heterodimerize allowing ligands to

bind and stabilize their extracellular domains. Ligand binding results in activation of the

intracellular tyrosine kinase domains and initiation of numerous possible downstream

signaling events. Interestingly, HER2/neu is considered an orphan receptor, as it lacks

the ability to bind ligands, while HER3 lacks a functional kinase domain. However, the

heterodimer formed between these two receptors generates a highly active receptor [205]

34

and HER2/neu seems to be the preferred binding partner for the other HER proteins

[203]. Each receptor dimer possesses a different collection of tyrosine phosphorylation

sites and accordingly sequesters different signaling molecules. The best-studied

HER2/neu-influenced signal transduction pathways involve mitogen-activated protein

kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3K) [206, 207].

Attributed functions of the HER family of proteins so far are broad, as they include

cell differentiation, migration, proliferation, and survival [208, 209]. The embryonic

lethality associated with erbB2-knockout mice emphasizes the essential role of this

protein during normal development [210]. Furthermore, there are three main areas of the

body whose development is highly dependent on HER2/neu: the cardiovascular system,

the nervous system, and the mammary glands [203]. Evidence for a role for erbB2 in the

cardiovascular system includes the finding that mice having a conditional erbB2 mutation

using the established Cre-Lox system, exhibit severe dilated cardiomyopathy [211]. In

the nervous system, the ablation of erbB2 using Cre-Lox technology results in the failure

of peripheral motorneuron axons to form mature neuromuscular junctions as they enter

the muscle [212]. Among the many stages of mammary gland development, erbB2

function is thought to be crucial during puberty; expression levels decrease thereafter and

remain low even during pregnancy, lactation, and involution. The role of erbB2 at these

later stages is still important, since adult animals that are transgenic for a dominant-

negative intracellular-deficient rat homolog of erbB2 mutant have problems lactating,

owing to failure of alveolar expansion [209].

35

1.8.2 Association with cancer

Overexpression of HER2/neu occurs in about 20% of primary prostate cancer cases

and 80% of their metastases [213]. HER2/neu is also overexpressed in several other

epithelial tumors, including breast, lung, and ovarian cancers [206, 214-216]. The

oncogenic effects of HER2 arise from activation of the MAPK and PI-3K signal

transduction pathways, leading to increased mitosis and the inhibition of apoptosis [206,

207]. In vitro it has been shown that HER2/neu overexpression in cells can lead to

chemo-resistance and even enhanced invasiveness [217-219]. Several studies have

reported that cancer patients with elevated HER2/neu levels have relatively poor

prognoses [220, 221]. Although the majority of HER2/neu-overexpressing cancers have

amplified copies of the HER2/neu gene, mutational activation of HER2/neu has also been

reported in some lung cancers [222].

1.8.3 HER2/neu-targeted therapeutics

Many of the therapies targeting HER2/neu have originally been designed for

treating breast cancer. Two main approaches have been developed: (1) the monoclonal

anti-HER2/neu antibody Herceptin (trastuzumab) and (2) small molecule general

inhibitors of tyrosine kinase. When Herceptin is used as a first-line treatment, it is

effective in reducing metastatic tumors in up to 26% of HER2/neu+ breast cancer patients

[223, 224]. Its mechanism of action is not entirely clear, but it is thought to involve

binding to the extracellular domain of HER2/neu in order to trigger internalization and

prevent signaling from the cell surface [225, 226], which in turn may lead to cell cycle

arrest [227]. The efficacy of Herceptin has been evaluated for advanced prostate cancer

patients in clinical trials, but thus far it has not shown to offer benefit [228]. Efforts are

36

underway to understand the resistance of cancers to Herceptin. Current hypotheses for

this resistance include the activation of alternative signaling pathways (such as via

insulin-like growth factor-I receptor) that promote proliferation and metastasis, and the

idea that Herceptin is unable to bind HER2/neu due to receptor masking by proteins like

MUC4, a membrane-associated mucin [229].

Small molecule tyrosine kinase inhibitors (TKIs) of HER2/neu such as lapatinib

has been used in the clinic for treating HER2/neu+ breast cancer, but offered limited

efficacy even though it was confirmed to at least effectively suppress MAPK activity

[230, 231]. It appears that most TKIs are generally unreliable single agent treatments,

perhaps due to the incomplete suppression of HER2-signalling [206].

1.9 Immunomodulatory proteins

1.9.1 Interleukin-12 (IL-12)

Interleukin-12 (IL-12) is a pleiotropic cytokine that functions as a key regulator of

innate and adaptive immunity. It is a 70-kDa heterodimer composed of two covalently

linked subunits, p35 and p40, and is mainly expressed by DCs, macrophages, and other

hematopoietic phagocytic cells. During T cell development, IL-12 induces the

differentiation of CD4+ T cells into Th1 cells. Activated Th1 cells secrete IFN-γ, which in

turn inhibits Th2 proliferation. Conversely, IL-10 and TGF-β produced by Th2 cells

block Th1 activation by mechanisms that include inhibiting IL-12 synthesis [232, 233].

During host defense against pathogens, IL-12 production by APCs can initially be

triggered by ligation of Toll-like receptors by bacterial products [234]. Subsequent

amplification of IL-12 production occurs following APC contact with antigen-specific T

cells via CD40L-CD40 and MHC-peptide-TCR interactions [234]. Ligation of the IL-12

37

receptor leads to signaling through the phosphorylation and activation of JAK2 and

TYK2 [235]. Activation of these Janus kinases is followed by the phosphorylation of

STAT3 and STAT4 [236, 237]. These transcription factors are then translocated to the

nucleus, resulting in activation of T cells and secretion of IFN-γ, which in turn activates

APCs to clear the pathogen.

IL-12 has also been shown to have an impact when used to treat primary and

metastatic tumors in mouse models [238-240] and clinical trials [241-243]. Its anti-tumor

effects are directly linked to its ability to induce IFN-γ secretion from lymphocytes. IFN-

γ may act directly on tumor cells to increase tumor antigen processing and presentation

on MHC class I molecules, and thus make tumors more susceptible to T cell recognition

[244]. IFN-γ may also affect stromal and endothelial cells in the tumor microenvironment

to inhibit angiogenesis and tumor invasion. This process involves the upregulation of

chemokines such as IP-10 and MIG [245, 246], which can recruit effector cells that in

turn remodel the extracellular matrix and inhibit matrix metalloprotease (MMP)

expression [247]. IFN-γ may also inhibit angiogenesis by down-regulating the expression

of adhesion molecules, such as the integrin αVβ3, by endothelial cells [248].

The multiple anti-tumor effects that IL-12 offers has provided the rationale for

using IL-12 to treat cancer. The success of its use has varied, but has provided some

patients objective tumor responses. For example, in a recent Phase II clinical trial, when

non-Hodgkin’s lymphoma patients were given IL-12 alone, 21% of patients exhibited

objective responses and had elevated levels of circulating CD8+ levels [249]. IL-12

therapy has also been used in combination with other anti-tumor agents, including

Herceptin. When this treatment combination was used in a Phase I trial in patients with

38

HER2+ tumors, 6% of patients had objective responses that were also associated with an

increase in IFN-γ secretion by NK cells [250].

Despite the modest success achieved by IL-12 therapy, there have been reports of

toxicity associated with it due to the high levels of IFN-γ secreted by lymphocytes. To

overcome the toxic effects of systemic IL-12 in patients, gene therapy studies have been

designed to achieve local IL-12 production at the tumor site and only low levels of

systemic IL-12 [251]. In one clinical trial [252], patients with metastatic gastrointestinal

carcinomas received intra-tumoral injections of DCs that were engineered to express IL-

12 by recombinant Ad. Among the 17 patients, 1 patient experienced a partial response.

In another study [253], plasmid DNA encoding IL-12 was injected directly into lesions of

9 malignant melanoma patients. The outcome was that the treatments were well tolerated

and 2 patients had stable disease and 1 patient achieved complete remission. Carefully

selecting combinatorial approaches using IL-12 may help extend its efficacy in treating

cancer.

1.9.2 Receptor activator of nuclear factor κB ligand (RANKL)

RANKL (also called tumor necrosis factor (TNF)-related activation-induced

cytokine [TRANCE]) is a member of the TNF ligand family and its receptor is receptor

activator of nuclear factor κB (RANK). The RANKL-RANK system is known to play

important roles in the immune system, bone metabolism, and the vascular system. In the

immune system, activated T cells express both membrane-bound and soluble forms of

RANKL. Soluble RANKL is generated through cleavage of the membrane-bound form

by the protease TNF-α-converting enzyme (TACE) [254]. Both forms of RANKL bind

to RANK expressed by DCs, leading to signal transduction via the NF-κB and c-Jun N-

39

terminal kinase pathways [255]. These signaling events result in the upregulation of the

anti-apoptotic factor Bcl-xL in DCs, leading to the enhancement of DC survival [255,

256] and even protection from FasL-mediated DC apoptosis [257]. RANKL also has the

ability to increase DC abundance in draining lymph nodes and enhance antigen-specific

effector and memory CTL responses [256, 258]. Engagement of RANK on T cells also

triggers signaling through the NF-κB pathway, again leading to enhanced cell survival.

Secretion of osteoprotegerin (OPG) by DCs helps down-modulate these signaling events,

as OPG acts as a decoy receptor.

The effects of T-cell-derived RANKL are not restricted to regulating DCs and T

cells. RANKL expressed by T cells can also activate RANK on osteoclast precursors to

promote their development into mature osteoclasts to mediate bone loss [259-262]. Bone

metabolism is further regulated through the ligation of RANKL by osteoclast-expressing

RANK and OPG produced by stromal cells and osteoblasts [263, 264]. The upregulation

of RANKL or RANK expression has been associated with the onset of several bone

diseases including post-menopausal osteoporosis [265] and myeloma bone disease [266].

In the vascular system, RANKL is expressed by endothelial cells, which also express

RANK. In these cells, RANK engagement by RANKL leads to signaling via the

PKB/Akt pathway to promote endothelial cell survival [267]. Blocking of this process

due to OPG production is associated with cardiovascular disease [268]. Thus for all three

systems, the relative levels of RANKL, OPG, and RANK must be well-balanced to

achieve proper regulation of immunity as well as bone and vascular development.

40

1.10 Hypothesis and goal of thesis project

DCs have a natural ability to elicit potent antigen-specific immunity. The work in

my thesis investigates how DCs can be manipulated and used to prevent against the

development of cancer. Given the difficulties associated with generating or obtaining

DCs from cancer patients to perform immunotherapy [269-271], we sought to develop

low-dose DC-based approaches that may translate to feasible doses on a human scale.

We hypothesized that if DCs could be made to efficiently express the self-antigen erbB2

through lentiviral transduction, then we could use relatively low doses of the engineered

DCs as an immunotherapy vaccine to induce erbB2-specific immunity and offer anti-

tumor effects in a mouse model of prostate cancer.

As described in Chapter 2, an experimental murine model was first developed to

test the ability of genetically engineered DCs to protect mice from forming ectopic

prostate tumors. In Chapter 3, I describe expanded research that investigated whether

immunomodulatory molecules could be incorporated into the vaccination strategy to help

strengthen anti-tumor immunity.

41

Chapter 2:

Tumor protection following vaccination with low doses of lentivirally transduced DCs expressing the self-antigen erbB2

A version of text presented in this chapter has been published in the following

original article: Mossoba ME, Walia JS, Rasaiah VI, Buxhoeveden N, Head R, Ying C, Foley JE,

Bramson JL, Fowler DH, and Medin JA (2008). Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2. Mol Ther, 16:607-617.

I performed all experiments for the work presented in this chapter, except for N

Buxhoeveden and JE Foley, who ran the multiplex immunoassays, and C Ying, who prepared the lineage-specific antibodies. JS Walia assisted with the thymidine incorporation assay and R Head assisted with handling of the mice.

42

2.1 Abstract

Gene therapy strategies may accelerate the development of prophylactic

immunotherapy against cancer. We synthesized a lentiviral vector encoding a kinase-

deficient form of erbB2 (erbB2tr) to efficiently transduce murine DCs. Murine erbB2

models a clinically-relevant tumor-associated self-antigen; its human homolog (HER-

2/neu) is overexpressed in breast cancer and 80% of metastatic prostate cancers.

Following one infection, ~47% of DCs overexpressed erbB2tr. To determine whether low

doses of transduced DCs could protect mice from tumors, we performed prime/boost

vaccinations with 2x103 or 2x105 erbB2tr-transduced DCs. Six weeks post-vaccination,

mice were simultaneously challenged with the aggressive RM-1 prostate cancer cell line

and an erbB2tr-expressing variant (RM-1-erbB2tr). Whereas control mice developed both

tumors, all recipients of 2x105 erbB2tr-transduced DCs developed only wild-type RM-1

tumors. One-third of mice vaccinated with just 2x103 erbB2tr-transduced DCs also

demonstrated erbB2tr-specific tumor protection. Protection against RM-1-erbB2tr tumors

was associated with sustained levels of anti-erbB2tr antibody production and also

correlated with erbB2tr-specific Th1 cytokine secretion. Depletion of CD4+, CD8+, or

NK cells prior to tumor challenge underscored their role in mediating tumor protection.

We conclude that administration of DCs expressing a self-antigen through efficient

lentivirus-based gene transfer activates cellular and humoral immunity, protecting host

animals against specific tumor challenge.

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2.2 Introduction

Cancer immunotherapy aims to overcome the inability of the immune system to

efficiently protect against the establishment of tumors or reject established tumors.

Dendritic cells (DCs) are potent antigen-presenting cells that have been widely used to

initiate or enhance tumor-associated antigen (TAA)-specific immune responses in animal

models and clinical settings. Numerous reports show that modifying DCs via TAA

peptide- or tumor lysate-pulsing can induce anti-tumor immunity [148, 150-153].

Transfecting DCs with nucleic acid sequences encoding TAAs carries the advantage of

inducing immunity towards a larger repertoire of naturally-derived MHC class I and II

compatible peptides. Comparative studies have shown that by transfecting DCs with

RNA, stronger anti-tumor effects can be achieved than by pulsing DCs with peptides

[155, 157, 272, 273].

Viral transduction of DCs offers similar advantages as RNA transfection, with the

added potential benefits of more efficient transgene delivery and stable transgene

expression, depending on the choice of virus. Retroviruses, including onco-retroviruses

and lentiviruses, can also be used to transduce DCs with one or more genes. Lentiviruses

are well-suited for transducing DCs because they are capable of efficiently transducing

slowly-dividing cells. Their integration into the host genome provides a way to generate

long-term stable transgene expression. In a few recent reports, lentiviruses have been

used to transduce murine and human DCs with TAAs [194, 195, 274-276]. Overall,

murine studies that have used virally-transduced DCs for immunotherapy have

demonstrated encouraging anti-tumor efficacy [277]. However, the majority of murine

44

studies published to date use human or rat homologs of TAAs instead of species-matched

TAAs.

In the current study, we chose to use a naturally occurring splice variant of the

murine erbB2 antigen (erbB2tr) as our target TAA in a mouse model of prostate cancer.

In this setting, murine erbB2 represents a true self-antigen, which better mirrors the

clinical setting. The human homolog of erbB2 (HER-2/neu) is overexpressed in 20% of

primary prostate tumors and 80% of patients with metastatic prostate cancer, making this

TAA a clinically relevant target for immunotherapy [213]. HER-2/neu is also

overexpressed in other malignancies including breast, ovarian, and lung tumors [278-

280]. A naturally occurring kinase-truncated variant of HER-2/neu has also been

described in human tumor cells [281].

Our aim was to generate immunity towards the self-antigen erbB2 in mice using

DCs that were genetically engineered to express erbB2tr. We hypothesized that

vaccinating mice with lentivirally-transduced DCs could impart long-term erbB2-specific

immunity and protection against subsequent challenge with erbB2-expressing tumors. In

our model we used an aggressive RM-1 prostate tumor cell line that we have modified to

express erbB2tr. We chose to focus on low-dose vaccination strategies, as limited

availability of syngeneic DCs may be a factor in human vaccination protocols. This study

provides a platform for the development of a low-dose DC immunotherapy strategy using

LVs as gene transfer tools engineering expression of target TAAs.

45

2.3 Materials and Methods

2.3.1 Lentiviral vector (LV) construction and preparation of high titer stocks

The enhanced green fluorescent protein (enGFP)-containing LV pHR'EF-GW-SIN

(LV/enGFP) was described previously [282]. To construct an erbB2tr-containing

recombinant LV (LV/erb), the enGFP cDNA sequence was excised from pHR'EF-GW-

SIN by EcoRI (New England Biolabs, Beverly, MA) digestion and replaced with the

cDNA sequence for erbB2tr (GI:28386210). This erbB2tr sequence was amplified by

PCR from the Invitrogen pYX-Asc plasmid (IMAGE 5702040) with Taq polymerase

(both Invitrogen, Burlington, ON), ligated into PCR-Script Amp(+) SK(+) (Stratagene,

La Jolla, CA), and excised by EcoRI digestion.

LV particles were generated by calcium-phosphate transfection of 293T cells

(kindly provided by Dr. Michele Calos, Stanford University, CA) with the plasmids

pCMVDR8.91, pMD.G [283], and either LV/enGFP or LV/erb. Viral supernatants were

collected at 24 and 48 hrs post-transfection, filtered using a 0.45 um filter, and

concentrated at 19,000xg for 2 hrs using an Optima L-100 XP Ultracentrifuge (Beckman

Coulter Canada Inc., Mississauga, ON). Concentrated virus preparations were serially

diluted and titered on 293T cells by FACS analysis as previously described [284].

2.3.2 Mice and cell lines

C57BL/6 (Jackson Laboratories, Bar Harbor, ME) and BALB/c (Charles River,

Wilmington, MA) mice were bred and housed under specific pathogen-free conditions at

the UHN Animal Resource Centre. RM-1 cells, a murine prostate cancer cell line

syngeneic to the C57BL/6 strain, were kindly provided by Dr. Timothy Thomson

46

(Baylor). The clonal RM-1-erbB2tr cell line was generated by transducing RM-1 cells to

overexpress a kinase-truncated form of erbB2 (erbB2tr) and then isolating single cell

clones. For these transductions, an onco-retroviral pUMFG-erbB2tr vector was

constructed (Mossoba and Medin, unpublished) and transfected into the E86 packaging

cell line to generate virus-producing E86 cells, as previously described [285]. In vitro

growth characteristics of RM-1-erbB2tr vs. WT RM-1 cells were nearly identical. All

animal experiments were performed under a protocol approved by the Animal Care

Committee at the UHN.

2.3.3 Murine DC generation and transduction

DCs were generated according to Lutz et al. (1999) [286] with slight modifications.

Briefly, bone marrow was flushed from femurs and tibiae of C57BL/6 mice using a 25G

needle. Red blood cells (RBCs) were lysed using RBC Lysing Buffer (Sigma, St. Louis,

MO). Remaining cells were plated in 10-cm petri dishes at a concentration of 2x105

cells/ml in a total volume of 10 ml/dish. DC media consisted of RPMI with 10% FBS

(PAA Laboratories, Etobicoke, ON), 1% penicillin/streptomycin, 5x10-5 M 2-

mercaptoethanol (both from Sigma), 40 ng/ml rmGM-CSF and 5 ng/ml rmIL-4 (both

from Peprotech, Rocky Hill, NJ). Cells were infected on day 3 of culture with either

LV/erb or LV/enGFP, or left uninfected. Half-volume media changes were done every

other day starting on day 4. On day 7, 50 ng/ml of rmTNF-α (Peprotech) was added for

24-48 hrs of DC maturation.

2.3.4 Flow cytometric analysis of DCs, tumor cells, and splenocytes

47

DCs and tumor cells were stained with an anti-erbB2 primary antibody (Ab4,

Oncogene Science, Tarzana, CA) and a PE-conjugated poly-adsorption goat anti-mouse

Ig secondary antibody (BD Biosciences Canada, Mississauga, ON) and cell surface

expression of erbB2tr was measured using a FACS Calibur (BD). For phenotypic

analysis of DCs, the following BD antibodies were used with appropriate isotype

controls: PE- or FITC- conjugated anti-CD11c (clone HL3), purified anti-CD80 (clone

1G10), and FITC-conjugated anti-CD86 (clone GL1), FITC-conjugated anti-I-Ab (clone

AF6-120.1). For analysis of splenocytes, the following antibodies were used with

appropriate isotype controls: anti-CD8-FITC, anti-TNF-α-PE, anti-CD4-PerCP, anti-IFN-

γ-APC, anti-IL-4-PE, anti-IL-2-APC. Levels of regulatory T cells were measured by cell

surface staining with anti-CD4-FITC, anti-CD25-APC, and intracellularly with anti-

FoxP3-PE according to manufacturer’s instructions (BD Biosciences).

2.3.5 Allogeneic Mixed Lymphocyte Reaction

Transduced and control DCs were harvested on day 9 of culture and dosed with 30

cGy in a Gammacell 3000 Elan 137Co irradiator (Nordion International Inc., Ottawa,

Canada). Freshly isolated splenocytes from C57BL/6 and BALB/c mice were B cell-

depleted using goat anti-mouse Ig magnetic beads (Dynal, Brown Deer, WI). The

remaining T cell-enriched population was plated in triplicate in a 96-well U-bottom plate

(BD) at 2x105 cells per well in T cell media. Next, serially-diluted irradiated DCs (range

of 0 to 0.6x105 cells/well) were added. Following 4 days of co-incubation, 1 uCi of

[3H]methyl-thymidine was added to each well for 20 hrs. Thymidine incorporation was

measured using a Beckman LS 1801 Liquid Scintillation Counter (Beckman).

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2.3.6 Immunizations and tumor inoculations

C57BL/6 mice were injected i.p with 2x105 or 2x103 DCs transduced with

LV/erbB2tr, LV/enGFP, or non-transduced controls in 200 ml of PBS. As a positive

control, one group of 5 mice was injected with 2x105 erbB2tr-transduced DCs along with

CFA (Sigma). These immunizations were repeated 2 weeks later. Six weeks after the

second immunization, 6 of 10 mice in each cohort were challenged with bilateral tumors

and the remaining mice were sacrificed for splenocyte cytokine secretion analyses (see

below). For the tumor challenge, each mouse was injected s.c. with 2x105 RM-1-NT and

RM-1-erbB2tr cells (in 200 ml of PBS) in the dorsal left and right flanks, respectively.

Starting six days later, the length (l), width (w), and height (h) of each tumor was

measured by caliper on a daily basis. Tumor volume was calculated by multiplying

lxwxh.

2.3.7 Measurement of anti-erbB2tr antibody from mouse plasma

Approximately 200 ul of blood was collected weekly from the tail vein of each

mouse into EDTA-coated tubes (Sarstedt, Montreal, Canada). Plasma was isolated by

centrifugation at 18,000xg at 4°C for 20 min. Plasma anti-erbB2 measurements were

performed using a flow cytometry-based ELISA method we developed that was based on

that described by Piechocki et al. (2002) [287]. Briefly, RM-1-erbB2tr and wild-type

(WT) RM-1 cells were first stained with diluted plasma samples or primary Ab4 antibody

(above) for 1 hr on ice followed by 2 washes with PBS. Secondary staining with PE-

conjugated poly-adsorption goat anti-mouse antibody was done for 1 hr on ice, again

followed by 2 PBS washes. 7-AAD was added to each sample to exclude dead cells from

49

flow cytometric analysis. The mean fluorescence intensity (MFI) value in the FL2

channel was measured on a FACS Calibur for each sample. A standard curve was

generated by plotting Ab4 antibody concentration versus the MFI values of the Ab4-

stained RM-1-erbB2tr cells. This curve was used to convert MFI values of plasma anti-

erbB2 levels from each mouse cohort into antibody concentration values. Each

experiment was performed three times and the SD of the means was calculated.

2.3.8 Cytokine secretion assays

Spleens from immunized and naïve control C57BL/6 mice were dissociated into

single-cell suspensions and treated with RBC lysis buffer. RBC-depleted splenocytes

were cryopreserved in freezing medium (90% FCS, 10% DMSO), then thawed when

needed using a method described by Maecker et al. (2005) [288]. Briefly, cryovials were

warmed to 37°C in a waterbath and the contents diluted dropwise with an equal volume

of warm media. Diluted cells were transferred to a 50 ml tube containing 8 ml of warm

media per cryovial of added cells and centrifuged at 290xg for 7 min. Cell pellets were

resuspended and brought to a final concentration of 5x106 cells/ml in RPMI medium

containing 10% FCS, 1% penicillin/streptomycin, 1% minimal essential amino acids

(Invitrogen), and 5x10-5 M 2-mercaptoethanol. Next, 200 ml of cell suspensions were

transferred to each well of 96-well round-bottom plates (BD) and incubated at 37°C for

18 hrs. Splenocytes were then collected from each well, counted, and plated in 24-well

plates at 3x106 cells per well in 1 ml. Approximately 2x105 freshly prepared DCs that

were left non-transduced or that were transduced with LV/erb, LV/enGFP were added to

each well. Co-cultures were incubated at 37°C for 24 hrs and supernatants were collected

and stored at -20°C. IFN-γ, IL-2, TNF-α, IL-4, and IL-10 levels were measured from

50

thawed supernatant samples by Bio-Plex multiplex sandwich immunoassays according to

the manufacturer’s protocol (Bio-Rad Laboratories, Hercules, CA).

2.3.9 CD4+, CD8+, and NK cell depletion

Five mice per group were immunized two times (two weeks apart) with either

erbB2tr- or enGFP-transduced DCs at the dose of 2x105 or 2x103 cells per immunization.

Approximately 6 weeks post-immunization, mice were depleted of CD4+, CD8+, or NK

cells by antibody administrations and bilaterally challenged with ~0.1x105 RM-1-NT and

RM-1-erbB2tr tumors (s.c.). For CD8+ T cell depletion, mice were injected i.p. with

200ug of anti-CD8 (clone 2.43) 3 days and 1 day before bilateral tumor inoculations.

After tumor injections, mice were given 200ug of antibody once per week. For CD4

depletion, mice were injected with 400ug of anti-CD4 (clone GKI.5) 3 days and 1 day

before bilateral tumor challenge. After tumor challenge, the dose of anti-CD4 was

reduced to 200ug per mouse and anti-CD4 was administered every 3-4 days. For NK cell

depletion, mice were given 200ug of anti-NK1.1 (clone PK136) 2 days and 1 day before

bilateral tumor inoculations. After tumor injections, mice were injected with 200ug of

anti-NK1.1 per mouse every 3-4 days. All i.p. injections were prepared in PBS in a final

volume of 400ul per mouse. Depletion of all three cell lineages from peripheral blood of

recipients was verified by flow cytometry in 7 animals per depletion group.

2.3.10 Statistical analysis

Student’s t tests were used to perform pairwise comparisons. Differences in means

were considered statistical significance at P< 0.05.

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2.4 Results

2.4.1 DCs are efficiently transduced with lentivirus

A lentiviral transfer vector encoding erbB2tr, a truncated (kinase-deficient) form of

the murine self-antigen erbB2 (LV/erb) was constructed (Fig. 2.1); an enGFP LV was

previously described [284]. Titers of produced LVs usually approximated between 5x106

and 3.6x108 functional infectious viral particles per ml. To determine the transduction

efficiency of LV/erb, we infected BM-derived murine DCs on day 3 of in vitro culture. In

an initial pilot experiment, we determined that between 20% and 70% of a DC population

was productively infected after one overnight incubation with LV/erb. Using these

erbB2tr-transduced DCs, we performed an in vivo pilot study designed to test the efficacy

of prime/boost vaccinations with LV-transduced DCs in mediating erbB2tr-specific anti-

tumor immunity. After observing the potent antigen-specific effects of erbB2tr-

expressing DCs in vivo, we initiated a second set of larger-scale experiments to

corroborate our pilot study. Freshly-derived DCs were transduced and their expression

levels of erbB2tr and enGFP were monitored over time. On culture day 7 for DCs used in

the first immunization, we observed that 32.6% of transduced DCs were erbB2tr+ and

47.9% were enGFP+, respectively (Fig. 2.2a). By day 9, when DCs were injected,

erbB2tr+ and enGFP+ cells had decreased to 16.7% and 22.3%, respectively. For the

second immunization, we also checked expression levels at day 5 and found that 47.4%

of DCs were erbB2tr+ and 70.2% were enGFP+ (Fig. 2.2b) at that time. The percentage of

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Figure 2.1. Schematic diagram of lentiviral vector (LV) constructs used in these studies. LTR, long-terminal repeat; SD, splice donor site; SA, splice acceptor sites; ψ, RNA packaging signal; EF1α, elongation factor 1 α promoter; SIN LTR, Self-inactivating LTR; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; Gag, viral structural proteins; RRE, Rev response element.

Figure 2.2. Transduction efficiency of LV/erb and LV/enGFP on DCs. BM cells were cultured in the presence of GM-CSF and IL-4. DC maturation was induced with TNF-α on culture day 8. Transductions were performed on culture day 3 using either erbB2tr- or enGFP-encoding lentiviruses. Transgene expression on DCs used for the first (A) and the second (B) scheduled immunizations was monitored by flow cytometry. Numbers indicate the percentage of transgene-positive cells for each filled histogram compared against appropriate isotype or non-stained controls (outlined histograms).

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erbB2tr+ DCs decreased steadily to 33.7% on day 7 and 2.7% on day 9. The percentage

of enGFP+ DCs was 79.7% and 73.7% on days 7 and 9, respectively.

2.4.2 Lentiviral transduction does not alter DC phenotype or allostimulatory

capacity

To determine whether transducing DCs with our recombinant LVs at reasonable

MOIs led to changes in phenotype, we first performed flow cytometry to compare the

expression of typical surface molecules on mature transduced and control DCs. We

assessed the percentage of cells expressing the myeloid marker CD11c, MHC II molecule

I-Ab, along with co-stimulatory molecules CD80 and CD86. DCs used for the first

scheduled vaccinations expressed similar levels of CD11c; 68.1% of the non-transduced

DC cultures were CD11c+ compared to 75.7% and 70.2% for erbB2tr- and enGFP-

transduced DC cultures, respectively (Fig. 2.3a). Further comparisons revealed that the

percentage of CD11c+ I-Ab+ DCs was nearly identical between non-transduced and

LV/erb-transduced DCs (39.3% vs. 38.1%, respectively). A minor difference in the

percentage of CD11c+CD80+ DCs was measured from non-transduced compared to

erbB2tr-transduced cultures (39.5% vs. 45.5%, respectively). Similarly, 37.5% of non-

transduced DCs and 43.8% of erbB2tr-transduced DCs were CD11c+CD86+ (Fig. 2.3a).

The DCs generated for the second vaccination exhibited similar trends (Fig. 2.3b).

The percentage of CD11c+ cells in the control cultures was 91.1%, compared to 90.3%

for erbB2tr-transduced DCs, and 79.8% for enGFP-transduced DCs. The percentages of

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Figure 2.3. Transducing DCs with LV/erb and LV/enGFP does not affect DC

phenotype or allostimulatory capacity. BM-derived DCs were transduced on culture day 3 and matured on culture day 8. Transduced and non-transduced (NT) immature (day 5 and/or 7) and mature (day 9) DCs prepared for the first (A) and the second (B) immunizations were stained with antibodies recognizing CD11c, I-Ab, CD80, and CD86 and analyzed by flow cytometry. Numbers indicate the percentage of positive cells in the indicated density plot quadrants. Mature day 9 DCs from C57BL/6 mice were also co-cultured with [3H]thymidine-pulsed syngeneic (C57BL/6) or allogeneic (BALB/c) splenocytes to compare the allostimulatory capacity of non-transduced DCs to either (C) erbB2tr- or (D) enGFP-transduced DCs. Co-cultures were plated in triplicate and the mean ± SD values are shown.

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CD11c+I-Ab+ were similar for control and erbB2tr-transduced DCs (60.0% vs. 67.0%,

respectively). Comparing the percentages of DCs expressing costimulatory molecules, we

found that 52.3% of non-transduced DCs and 59.0% of erbB2tr-transduced DCs were

CD11c+CD80+. A slight difference in the CD11c+CD86+ percentage was also detected

between the control and transduced DCs (62.3% vs. 65.0%, respectively).

To determine whether transduction with LV/enGFP or LV/erb affected DC

function, we compared the ability of non-transduced and transduced DCs to induce a

response in an allogeneic mixed lymphocyte reaction (MLR). We cultured H-2b-

expressing DCs (transduced and control) with either H-2d splenocytes from BALB/c mice

or H-2b splenocytes from C57BL/6 mice and measured splenocyte proliferation by

thymidine incorporation (Figs. 2.3c,d). No significant differences were found between

the allostimulatory capacities of non-transduced DCs and either LV/enGFP- or LV/erb-

transduced DCs.

2.4.3 Vaccination with low doses of LV-modified DCs generates antigen-

specific tumor protection

Although many studies employing DC-vaccination strategies [277] evaluate tumor

protection around 1-2 weeks post-vaccination, we chose to investigate the long-term

benefits of a prime/boost vaccination strategy by challenging mice ectopically with RM-1

prostate tumors 6 weeks after the second vaccination. RM-1 cells grow aggressively in

vivo, providing a stringent model for assessing tumor growth after vaccination;

subcutaneously implanting just 104 RM-1 cells will yield palpable tumors within 1 week

and can compromise mouse survival by 10 days post-implantation [289]. The RM-1

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tumor cell line lacks endogenous erbB2 expression according to our FACS analysis (Fig.

2.4a). Therefore, we generated a clonal cell population of erbB2tr-expressing RM-1 cells

(RM-1-erbB2tr) by onco-retroviral transduction followed by clonal isolation (Fig. 2.4a).

In our aforementioned pilot study, we first tested the efficacy of three

immunizations using doses of 2x105 and 2x103 of erbB2tr-transduced DCs to protect

against subsequent challenge with erbB2-expressing tumors. We vaccinated mice three

times with either erbB2tr-transduced or non-transduced DCs. Two weeks after the third

vaccination, we injected mice with non-transduced RM-1 cells (RM-1-NT) on one dorsal

flank and RM-1-erbB2tr cells on the opposite dorsal flank in order to generate a bilateral

tumor model in the same individual. Whereas many tumor protection studies utilizing

virally-transduced DCs typically inject between 0.5x106 and 1x106 DCs per

immunization [277], the focus of this pilot study was to investigate the possible benefits

of using markedly lower doses of transduced DCs. In that initial pilot study, we observed

that both erbB2 immunization regimens offered considerable protection against erbB2tr-

expressing RM-1 tumors specifically, compared to that obtained using non-transduced

DCs.

To examine these low-dose outcomes in more detail, we next performed a larger

study. We again used the dose of 2x105 DCs for immunizing one cohort of mice, and the

100-fold lower dose of 2x103 DCs for another. Importantly, however, we also reduced the

number of vaccinations per animal to just two. We injected mice twice with either

control, erbB2-transduced, or enGFP-transduced DCs, two weeks apart. We next

inoculated animals with the same tumor challenge described in our pilot study above. As

a positive control, one group of mice was immunized twice with 2x105 erbB2tr-

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Figure 2.4. Immunization with low doses of DC-erbB2tr generates potent anti-

tumor responses. Cell lines RM-1-erbB2tr and non-transduced RM-1 (RM-1-NT) were stained with anti-erbB2 antibody and analyzed by flow cytometry (A). Mice were immunized twice with either 2x105 (B) or 2x103 (C) DCs and then inoculated with RM-1-erbB2tr and RM-1-NT cells on opposite flanks. Plotted values are the mean ± SEM of 5 or 6 mice per group, except for the positive control group (n=3). *P<0.05, vs DC-erbB2tr vaccination; **P<0.005, vs DC-erbB2tr vaccination.

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transduced DCs mixed with the Complete Freund's Adjuvant (CFA) emulsion. Using this

prime/boost strategy, none of the 6 mice that were immunized with 2x105 erbB2tr-

transduced DCs showed RM-1-erbB2tr tumor growth, whereas RM-1-NT tumors grew

rapidly in each animal (Fig. 2.4b). In contrast, the control naïve mice and mice

immunized with 2x105 non-transduced or enGFP-transduced DCs developed both RM-1-

NT and RM-1-erbB2tr tumors with an aggressive growth profile that necessitated

euthanasia within 2 weeks. Strikingly, significant tumor protection from RM-1-erbB2tr

tumors was also observed in mice that were immunized with the 100-fold lower dose of

2x103 erbB2-transduced DCs (Fig. 2.4c). In this group, 2 of 6 mice displayed complete

tumor protection until the point of sacrifice at 2 weeks post-tumor challenge, and 2 of 6

mice showed reduced RM-1-erbB2tr growth compared to control cohorts.

2.4.4 Mice vaccinated with DC-erbB2tr show strong antigen-specific humoral

immunity

To begin investigating potential mechanisms by which DC-erbB2tr immunizations

could break tolerance against erbB2tr, we collected blood from each mouse on a weekly

basis and measured the plasma levels of anti-erbB2 antibodies. As shown in Figure 2.5a,

mice immunized with CFA + erbB2tr-transduced DCs (positive control) began producing

modest levels of anti-erbB2tr antibodies. Following the second vaccination, these mice

showed steadily increasing titers that peaked at about 45 days after the first DC injection.

Relatively high antibody levels were detected up to 70 days post-prime vaccination when

the mice were sacrificed. In our experimental groups, mice injected twice with 2x105

erbB2tr-transduced DCs showed a rapid increase in antibody titer after the boost

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vaccination. Indeed, within 10 days, the average anti-erbB2tr titer rose to over 5 times the

average level in control mice. After this peak, a steady decline was measured, but specific

anti-erbB2tr antibodies were still detectable at day 50, when mice were inoculated with

tumors. In the non-vaccinated group of mice, challenge with RM-1-erbB2tr tumors

caused a slight increase in antibody titers, revealing weak background immunogenicity of

these tumors. Injecting the lower dose of 2x103 transduced DCs did not lead to detectable

anti-erbB2tr antibodies at least within the sensitivity limits of this assay, despite the anti-

tumor effects observed above (Fig. 2.5b).

2.4.5 Analysis of cytokine secretion from splenocytes

To further evaluate mechanisms, we harvested the spleens from naïve and

immunized mice 6 weeks after the second vaccination. We re-stimulated splenocytes in

vitro for 24 hrs with freshly prepared transduced or control DCs and analyzed culture

supernatants for production of Th1 (IL-2, IFN-γ, and TNF-α) and Th2 (IL-4 and IL-10)

cytokines. We found that recipients of 2x105 erbB2tr-transduced DCs produced greater

levels of IL-2, IFN-γ, and TNF-α following in vitro re-stimulation with erbB2tr-

transduced DCs relative to controls (Fig. 2.6). In contrast, this erbB2tr-specific cellular

response was absent from the supernatants of all other mouse cohorts. To quantify the

levels of antigen-specificity of the Th1 response, we calculated a ‘specificity index’ by

normalizing cytokine concentration results from each group to the values obtained from

re-stimulation with non-transduced DCs (Fig. 2.6). We found that splenocytes from mice

vaccinated with 2x105 erbB2tr-transduced DCs produced approximately 370-fold more

IL-2 after re-stimulation with erbB2tr-transduced DCs than with non-transduced DCs. An

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Figure 2.5. DC-erbB2tr immunization dose of 2x105 cells causes a sustained

erbB2-specific humoral response. Plasma samples from naïve and immunized mice were pooled according to cohort and quantitatively tested for the presence of anti-erbB2 antibodies by a flow cytometry-based ELISA. Graphs show a comparison between anti-erbB2tr levels from control groups and either (A) 2x105 or (B) 2x103 DC dose groups. Plotted values are the mean ± SEM of three independent assays. *P<0.01, vs vaccination with DC-NT, DC-enGFP, or no vaccination.

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Figure 2.6. Immunization using DCs transduced with LV-erb offers antigen-

specific Th1 immunity. Splenocytes from naïve and immunized mice were co-cultured in triplicate with LV-transduced or non-transduced DCs for 24 hours and supernatants were analyzed for IL-2, IFN- γ, and TNF-α by Bio-Plex multiplex sandwich immunoassays. Specificity index values were calculated by normalizing cytokine concentration values from each 24-hr re-stimulation condition to the values obtained from re-stimulation with non-transduced DCs.

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even greater specificity index value was calculated for the relative increase in IFN-γ; over

1100-fold more IFN-γ was produced after erbB2tr-specific re-stimulation. We also found

a large (635-fold) increase in TNF-α production following in vitro re-stimulation with

erbB2tr-transduced DCs relative to controls. Levels of IL-4 and IL-10 in the co-culture

supernatants were generally very low and specificity towards erbB2tr was not observed.

We confirmed the specificity of our cytokine responses towards erbB2tr antigen

using fractionated splenocytes. Six weeks after immunizing mice with erbB2tr- or

control-DCs, percentages of CD4+ or CD8+ T cells producing IL-2, IFN-γ, TNF-α, or IL-

4 were measured by intracellular flow cytometry (Fig. 2.7). Splenocytes from mice that

received 2x105 erbB2tr-transduced DCs produced high levels of IL-2 and IFN-γ. In this

immunization cohort, approximately 57% of CD8+ cells and 48% of CD4+ cells were IL-

2+, compared to < 9% of cells from all negative control groups. In addition, 13.3% and

5.3% of splenocytes from this cohort were CD8+IFN-γ+ and CD4+IFN-γ+, respectively,

compared to ≤ 2% of splenocytes from all negative control groups. With the exception of

the positive control group, production of TNF-α and IL-4 were quite low in CD8+ and

CD4+ splenocytes among all cohorts, with levels ranging from about 1% to 4% double

positive cells. Despite not observing a clear pattern of TNF-α production specifically

towards erbB2tr, our overall findings of Th1 cytokine upregulation from fractionated

splenocytes supports our erbB2-specificity index data in Figure 2.6.

2.4.6 Role of CD4+, CD8+, and NK cells in mediating tumor protection

To evaluate the importance of CD4+, CD8+, and NK cells in determining tumor

protection, we repeated our study using mice depleted of these cell lineages. First, mice

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were immunized two times (two weeks apart) with either erbB2tr- or enGFP-transduced

DCs at the dose of 2x105 or 2x103 cells per immunization. Approximately 6 weeks post-

immunization, mice were depleted of CD4+, CD8+, or NK cells by antibody

administrations and bilaterally challenged with RM-1-NT and RM-1-erbB2tr tumors. As

expected, RM-1-NT tumor growth was not significantly affected by cell depletion (Fig.

2.8a). Whereas RM-1-erbB2tr tumor growth was inhibited in non-depleted recipients of

2x105 erbB2tr-transduced DCs, there was some enhanced growth following all three

depletion conditions (Fig. 2.8b). Interestingly, mice that were immunized with 2x103

erbB2tr-transduced DCs relied on a protection mechanism that appeared to be highly

dependent on CD4+ T cells. In this cell depletion study, the use of enGFP-transduced

DCs as control vaccines did not yield clear differences in tumor growth between depleted

and non-depleted conditions, but overall rates of tumor growth in this iteration of the

experiment were slower than expected.

To further understand the mechanism of tumor protection, we used ELISAs to

analyze the production of IFN-γ and IL-4 in the supernatants of cultured splenocytes

harvested from depleted and non-depleted immunized mice (Figs. 2.8c,d). Although

cytokine levels were generally difficult to detect in all mice that were immunized with

2x103 erbB2tr-transduced DCs, we were able to measure cytokines produced by

splenocytes from mice immunized with the higher dose of DCs. Production of IFN-γ was

reduced by 44.5-fold in NK cell-depleted mice and 3.3-fold in CD4+ T cell-depleted mice

compared to non-depleted mice (Fig. 2.8c). CD8+ T cell depletion also yielded a

relatively small reduction in IFN-γ splenocyte secretion on average (1.2-fold), but was

associated with a high level of variability (Fig. 2.8c). The depletion of each of the three

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lineages also correlated with an increase in IL-4 production in mice that received 2x105

erbB2tr-transduced DCs (Fig. 2.8d). This finding reflects how Th1/Th2 cross-regulation

can upregulate Th2 immunity when Th1 cytokine production is low.

To evaluate the role of a fourth cell type in mediating anti-tumor immunity, we

studied the impact of gene-modified DC immunization on the behavior of regulatory T

cells (Treg cells), defined as CD4+CD25+FoxP3+cells. We used flow cytometry to

investigate whether there were changes in Treg levels in spleens of all immunized and

naïve mice. However, significant differences in Treg levels between experimental groups

were not detected.

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Figure 2.7. Cytokine production analysis of fractionated splenocytes shows erbB2tr-specific Th1 immunity. Mice were immunized twice with non-transduced, enGFP-transduced, or erbB2tr-transduced DCs (two weeks apart) and then 6 weeks later, 4 of 10 mice were sacrificed from each group to check the cytokine levels from their splenocytes. Percentages of (A) CD8+ or (B) CD4+ T cells producing IFN-γ, IL-2, TNF-α, and IL-4 were measured by intracellular flow cytometry.

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Figure 2.8. Involvement of CD4+, CD8+, and NK cells in mediating RM-1-erbB2tr tumor protection. Five mice per group were first vaccinated with 2x103 or 2x105 erbB2tr- or enGFP-transduced DCs two times, two weeks apart. Approximately 6 weeks after the last immunization, mice were depleted of CD4+, CD8+, or NK cells, or left non-depleted, and injected bilaterally with RM-1-NT and RM-1-erbB2tr tumors. (A) RM-1-NT or (B) RM-1-erbB2tr tumor volumes were calculated based on caliper measurements of length x width x height. Graphed values are the mean ± SEM of 4 or 5 mice per group. Splenocytes were prepared from each mouse at two weeks post-tumor challenge and cultured at a concentration of 2x106 cells/ml for 48 hrs. Analysis of (C) IFN-γ and (D) IL-4 production was done in triplicate by ELISA.

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2.5 Discussion

This study is the first to demonstrate the use of lentivirally transduced DCs in an

immuno-gene therapy cancer model targeting the self-antigen erbB2 in mice. Despite the

growing number of DC-mediated immuno-gene therapy studies, complete protection

against cancer has been mostly elusive [277]. One limitation likely relates to the level of

antigen presentation by DCs. Thus, the method of engineering DCs to present TAAs may

play an important role in the potency of immunotherapy schemas. Our use of

recombinant retroviral vectors encoding full-length or large portions of TAAs may permit

transduced DCs to present a broad repertoire of natural immunogenic tumor antigen

peptides in stable MHC complexes. Indeed, we have previously demonstrated the utility

of DCs transduced with onco-retroviruses to express xenogeneic human prostate TAAs in

mediating anti-tumor immunity in mice [191]. In our current study, we employed an LV-

based system and found that DCs could be even more efficiently transduced to express

antigens, without compromising their phenotype or function. This finding is especially

important given that the functional effects of lentiviral transduction of murine DCs with

erbB2 have not been previously investigated and our LV/GFP transduction efficiency is

higher than those reported by other groups [194, 290].

To address the possibility of oncogenic effects due to the use of integrating vectors,

many groups have already extensively assessed the safety of lentiviral transduction [291-

294]. In addition, although genetically modifying DCs to express murine erbB2 is

original, data from other DC studies employing human or rat homologs have shown that

this type of manipulation does not lead to oncogenic effects in DCs [186, 295-297]. To

further decrease the possibility of affecting the target myeloid DC population by

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overexpressing a heterologous signaling molecule, we employed a kinase-deficient

version of erbB2 for our lentiviral construct. Although recent evidence suggests that

some cases of acute myeloid leukemia may have a myeloid DC origin [298, 299],

myeloid DCs may be less amenable to oncogenesis than plasmacytoid DCs, whose

malignant counterparts are well documented [300-303]. In the future, gene therapy

protocols may also incorporate additional safety mechanisms, such as the tmpk/AZT

suicide gene therapy strategy that our laboratory developed [304].

While monitoring enGFP and erbB2tr levels in transduced DC cultures, we

observed a decrease in transgene-positive DCs between culture day 5 and 9. The

decrease was more pronounced in DC populations transduced with erbB2tr compared

with enGFP. Some researchers have attributed this phenomenon to the elimination of

genetically-modified DCs by T cells that contaminate DC cultures [305]. The possibility

that this elimination occurred in our cultures cannot be ruled out, since our DC generation

protocol allows for the persistence of low levels of lymphocyte contaminants [286].

However, the finding that enGFP levels can remain stable over time does not fully

support this notion. Instead, it may simply point to the possibility that peptide formation

from protein degradation within DCs occurs more readily for some antigens than for

others. Our flow cytometry data, which shows decreased whole antigen expression, may

be capturing the transition from whole protein expression to peptide formation as DCs

develop into mature APCs.

It is important to consider the number of DCs being employed for immunization

strategies, as an effort to minimize the DC dose could improve the feasibility of

employing such strategies in clinical settings. Availability of effective patient DCs may

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be a limiting factor [269-271]. We used only 2 immunizations, in order to decrease the

number of DCs required, and were still able to induce specific immune-mediated anti-

tumor responses. As stated previously, typical in vivo immunotherapy approaches

involving virally transduced DCs administer between 0.5x106 to 1x106 DCs per

immunization [277]. We found that a dose as low as 2x103 DCs offered partial protection

against erbB2tr-expressing tumors, suggesting that the minimum effective DC dose falls

in the range between our tested doses. In addition, these results were obtained in a

relatively long-term setting, as mice were tumor challenged 6 weeks after the last

immunization. As a direct result of testing these low-dose DC vaccinations, we also gain

the ability to establish a workable threshold that can be used as a sensitive read-out in

future studies to determine if other co-manipulations further enhance the immunotherapy

effect.

In agreement with the recent results of Sakai et al. (2004) [186], we also observed a

humoral response in vaccinated mice that correlated with tumor protection in the 2x105

DC-dose group. Mice vaccinated with 2x103 erbB2tr-transduced DCs had comparatively

low antibody titers. In addition, long-term Th1 immunity was observed in the 2x105 DC-

dose mice. It was not detected in the 2x103 DC dose cohort despite the finding that 2 of 6

of these mice were protected from developing RM-1-erbB2tr tumors and an additional 2

mice had markedly reduced tumor volumes compared to controls. This may reflect the

detection limit of the assays, and may also point to the very sensitive nature of this

system. It may be that specific anti-tumor immune responses can be induced with very

subtle changes to the immune marker profiles. In our Th1 assays, the erbB2tr specificity

of cytokine production in the 2x105 DC-erbB2tr cohort is clear. Note that we did detect

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low levels of Th1 reactivity towards enGFP, likely because enGFP is a xeno-antigen and

therefore inherently foreign to the mouse species. That we did not observe significant

levels of Th2 cytokines at 6 weeks post-vaccination is consistent with the waning of anti-

erbB2tr antibody levels over time.

Our DC vaccination strategy using erbB2-transduced cells was well tolerated in

animals. Although the self-antigen erbB2 is naturally expressed at varying levels

throughout the mouse body including the lungs, intestines, and brain [306],

manifestations of auto-immune toxicity were not observed in the 10 weeks after initiation

of the vaccination schedule. Other groups have also reported that anti-tumor immunity in

mouse models can occur without damaging normal tissues, even when self-antigens are

targeted [307-309]. Although the exact reasons for the lack of observed auto-immune

disease in these studies are not clear, it is thought that it may relate to the higher than

normal level of antigen-presentation by over-expressing tumor cells [308, 310, 311] or to

differences in antigen recognition between tumor cells and normal cells [312].

In conclusion, our results show that vaccination using relatively low doses of DCs

transduced with a recombinant LV to express a truncated form of HER-2/neu can safely

and effectively protect mice against tumor development in an antigen-specific manner.

This is the first implementation of this stable gene transfer method for this broadly

important antigen in tumor biology. Tumor protection was associated with antigen-

specific cellular and humoral immunity. The recombinant LV system we utilized served

as an efficient gene transfer vehicle, which did not adversely affect DCs. Efforts to

develop low dose DC-immunotherapy strategies will be of value in clinical situations

where patient DCs may be scarce. Such cancer immunotherapy vaccines may be

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particularly applicable before tumors are established and in early stage disease, and could

reduce the need for more intensive treatments with systemic toxicity such as

chemotherapy or radiation therapy.

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Chapter 3: Co-transduction with an IL-12, but not RANKL, lentivector enhances low-dose

DC-erbB2tr immunotherapy against erbB2-expressing prostate tumors The text presented in this chapter has been submitted for publication to Cancer

Immunology, Immunotherapy.

ME Mossoba, J Symes, JE Foley, J Mariotti, VI Rasaiah, JS Walia, S Loisel-Meyer, DH Fowler, JA Medin (2008). Co-transduction with an IL-12, but not RANKL, lentivector enhances low-dose DC-erbB2tr immunotherapy against erbB2-expressing prostate tumors.

I performed all experiments for the work presented in this chapter, except for J Symes and VI Rasaiah, who helped run some of the Western blots, JE Foley and J Mariotti, who ran the multiplex immunoassays, and JS Walia, who constructed one of the vectors. S Loisel-Meyer assisted with measuring tumors.

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3.1 Abstract Dendritic cell (DC)-based immunotherapy has the potential to protect against tumor

formation and even reduce existing tumors at primary and metastatic sites. To aid in the

translation of DC-based immunotherapies to the clinic, we are developing strategies in

mouse models that may be achievable on the human scale. We have previously shown

that vaccination with doses as low as 2x103 lentiviral vector (LV)-transduced DCs could

offer some animals complete antigen-specific protection against aggressive prostate

tumors. In this prior model, DCs were engineered to express a kinase-truncated version of

erbB2 (erbB2tr), a self-antigen that is the murine orthologue of the human tumor-

associated antigen (TAA) HER2/neu. Here, we sought to test whether combining erbB2tr

expression with that of immunomodulatory proteins IL-12 or RANKL, could further

augment anti-tumor immunity to achieve stronger tumor protection. We hypothesized

that IL-12 expression on DCs would promote erbB2tr-specific Th1 immunity and that

RANKL would also enhance T cell stimulation through its known effect on DC life-span.

First, we demonstrated the feasibility of co-transducing functional DCs with two LVs

simultaneously, one LV encoding either enGFP or erbB2tr cDNA sequences and the

other encoding IL-12 or RANKL cDNA sequences. In our mouse model of prostate

cancer, we show that immunizing mice twice (two weeks apart) with low doses of DCs

expressing erbB2tr and IL-12 offered long-term tumor protection. This anti-tumor effect

correlated with an upregulation in Th1 immunity, as defined by IL-2, IFN-γ, and TNF-α

secretion from stimulated splenocytes. Vaccination with DCs expressing higher levels of

IL-12 alone also yielded significant tumor protection, but was not associated with

erbB2tr-specific immunity. By comparison, co-expressing RANKL with erbB2tr in DCs

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offered neither tumor protection nor anti-erbB2 immunity. This study reveals that IL-12

is a better modulator than RANKL in the context of low-dose DC-based immunotherapy

strategies that combine elements of antigen specificity with factors generating

enhancement of DC-mediated immunity.

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3.2 Introduction

In recent years, dendritic cell (DC)-based immunotherapy has been shown to

possess potential for the treatment of cancer [277]. DCs naturally play a critical role in

controlling the balance between immunity and tolerance [103]. They can also be

modified to enhance their ability to stimulate selective immune responses against specific

tumor-associated antigens (TAAs). This often requires that they be manipulated in some

fashion to effectively present TAA-derived peptides. While many methods exist to

accomplish this task, lentiviral vector (LV) transduction allows for efficient, stable, and

strong transgene expression [313].

No matter which method is employed to manipulate the antigen-presenting cells,

the use of TAA-expressing DCs for prevention or regression of tumors in mouse models

have generally followed a protocol of administration of high DC doses - on the order of

106 cells per immunization [277]. Considering the average weight of a human and mouse

to be 75 kg and 30 g, respectively, a DC dose of 106 manipulated cells in a mouse on a

per weight basis would be equivalent to ~2.5x109 human DCs. Given that it is more

difficult to collect DCs or generate them from DC precursors (monocytes and CD34+

cells) from cancer patients than from healthy donors [314, 315] and that most clinical

protocols use ~107 DCs per immunization [316], then mouse studies using high DC doses

simply do not model treatments that can be readily applied to a clinical setting. In fact,

this marked dose discrepancy may possibly explain why differential outcomes are seen

between mouse studies and human clinical protocols. Indeed, the appropriate DC dose for

inducing TAA-specific immunity remains unknown - especially as regards the clinical

context. The optimal dose is likely variable and dependent on many factors including the

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health of the donor, the inherent immunogenicity of the target TAA(s), DC purity and

stage of maturity, and the ability of ex vivo-generated DCs modified by various methods

to home to the appropriate locations within the lymphatic system.

Our laboratory has been working towards the development of a DC-based

immunotherapy strategy for the prevention and treatment of prostate cancer [191, 317].

Recently we have chosen to focus on self-antigens and have targeted the TAA, HER2/neu

(erbB2), as it is overexpressed in both primary and metastatic prostate tumors along with

breast, lung, and ovarian cancers [206, 213, 318]. We have previously shown that a

potent and specific vaccine can be made following LV-transduction of bone marrow

(BM)-derived DCs engineered to express a truncated (kinase-deficient) version of erbB2

(erbB2tr) [317]. In that study, we used a bilateral murine tumor model wherein mice were

injected in one flank with erbB2tr-expressing murine prostate tumor cells (RM-1-

erbB2tr) and the non-erbB2tr expressing parental line (RM-1-NT) in the other. We

showed that immunizing mice two times, two weeks apart, with 2x105 DCs that were LV-

transduced to express erbB2tr completely protected mice from tumor challenge at six

weeks post-immunization. This protection was associated with both humoral and cellular

immunity. Furthermore, reducing the vaccination dose to only 2x103 cells per vaccination

yielded erbB2tr-specific tumor protection in some animals. Based on these findings and

this relative threshold that we uncovered, we sought here to further develop a low-dose

DC-based immunotherapy strategy against prostate cancer.

To enhance the role of DCs in immunotherapy, strategies employing function-

enhancing cytokines [319, 320] or other proteins that help prevent apoptosis [321],

exposure to heat-shock proteins [322], or knock-down of SOCS1 expression [198] have

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all been attempted. In particular, the use of Interleukin-12 (IL-12) has been well

documented [251, 323]. As a pleiotropic cytokine that is expressed by many cells

including DCs, IL-12 has been shown to augment several immune response pathways,

including CTL- and NK-based immunity [324]. IL-12 signals through the JAK-STAT

pathway to stimulate the release of Interferon-γ (IFN-γ) and tumor necrosis factor-α

(TNF-α) from T lymphocytes and NK cells, thus enhancing their functional responses

[325]. In addition, IL-12 has offered some benefit to cancer patients when used alone

[249] and as an adjuvant [326]. Receptor Activator of NF-kB Ligand (RANKL) is

another protein that has been implicated in DC stimulation, but its effects on BM-derived

DCs towards the goal of augmenting anti-tumor immunity have not been extensively

studied. It is known, however, that RANKL can upregulate expression of the anti-

apoptotic molecule Bcl-xL in DCs to extend their life-span in vitro and their persistence

within draining lymph nodes in vivo [327]. In addition, treatment of DCs with RANKL

has been shown to improve DC function by increasing expression of inflammatory

cytokines such as IL-1, IL-6, IL-12, and IL-15 [327-329]. RANKL is normally expressed

on T lymphocytes and binds its cognate receptor, RANK, on DCs [330]. However,

adding RANKL to DCs may allow DCs within a population to activate each other and

also allows shed RANKL to activate transduced DCs in an autocrine fashion.

For the current study, we chose to co-express erbB2tr with either IL-12 or RANKL

in DCs in an effort to develop a more effective low-dose DC vaccine. We hypothesized

that we could combine our previously-demonstrated specificity towards erbB2tr with the

immune-enhancing effects of IL-12 by co-transducing DCs with LVs carrying these two

cDNAs. We successfully designed a co-transduction protocol to achieve functional

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expression of erbB2tr along with either IL-12 or RANKL in DCs, and then assayed the

independent effects of LV co-transduction on DC purity, the state of DC maturation, and

APC function. We next employed the co-transduced DCs in a murine prostate cancer

immunotherapy model using low doses of DCs for immunizations. It was found that the

DCs co-transduced with LV/erb and LV/IL-12 but not with LV/RANKL provides

additional tumor protection effects compared to LV/erb-transduced DCs alone. Further,

our results show this effect is associated primarily with Th1 immunity towards erbB2.

This study is an important step in refining the methods used to modulate DCs towards

achieving tumor protection, and furthers our aim to facilitate translation of dose-effective

DC immunotherapy into the clinic.

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3.3 Materials and Methods

3.3.1 Lentiviral vector (LV) construction and preparation of high titer

lentivirus

Construction of the following transfer vectors was described previously: pHR'EF-

erbB2tr-W-SIN (LV/erb) [317], pHR'EF-IL-12-W-SIN (LV/IL-12) [331], and pHR'EF-

GW-SIN (LV/enGFP) [282]. To construct pHR'EF-RANKL-W-SIN (LV/RL), we

excised the enGFP cDNA sequence from LV/enGFP using restriction enzyme EcoRI

(New England Biolabs, Beverly, MA) and inserted the cDNA encoding RANKL (kindly

provided by Dr. A.K. Stewart, Mayo Clinic Arizona).

We generated LV particles by polyethylenimine (PEI) transfection of 293T cells

(kindly provided by Dr. Michele Calos, Stanford University, CA) as described previously

[304] using the plasmids pCMVDR8.91, pMD.G [282], and our transfer vectors. Viral

supernatants were harvested using a 0.45 µm filter at 48 hrs and 72 hrs post-transfection.

Vector was then concentrated at 19,000xg for 2 hrs using an Optima L-100 XP

Ultracentrifuge (Beckman Coulter Canada Inc., Mississauga, ON) and titered on 293T

cells as previously described [282].

3.3.2 Mice and tumor cell lines

Male C57BL/6 mice (age 7-10 weeks) (Jackson Laboratories, Bar Harbor, ME)

were housed under specific pathogen-free conditions at the UHN Animal Resource

Centre. The RM-1 cell line is a murine prostate cancer cell line that is derived from the

C57BL/6 strain. We generated the clonal RM-1-erbB2tr cell line, which expresses a

kinase-truncated form of erbB2 (erbB2tr) previously described [317]. All mouse

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experiments were performed following a protocol approved by the UHN Animal Care

Committee.

3.3.3 Murine DC generation and transduction

DCs were generated according to Lutz et al. (1999) [286] with some modifications

as already described [317]. Cells were either left uninfected or were infected on culture

day 3 with one LV or with a combination of 2 LVs. Media was changed after 4 hours and

then half-volume media changes were done every other day. To induce DC maturation on

culture day 7, 50 ng/ml of rmTNF-α (Peprotech) was added for 24-48 hrs.

3.3.4 Western blot analyses

Transduced and non-transduced DCs were lysed 24 hours after addition of TNF-α

in NP40 buffer (50mM Tris pH 7.4, 1mM EDTA, 150mM NaCl, 1% NP-40) containing

protease inhibitors (Sigma, Oakville). Protein quantification was done using the DC

Protein Assay (Bio-Rad, Hercules, CA). For Stat4 (89 kDa) and Stat4-P (89 kDa)

analyses, 20 ug of protein were run on a 7% polyacrylamide gel alongside the All Blue

(broad range) ladder (Bio-Rad). Following semi-dry transfer, membranes were blocked in

5% milk and stained with either purified rabbit anti-mouse Stat4 (C-20; 1:1000) (Santa-

Cruz Biotechnology, Inc, Santa Cruz, CA), purified rabbit anti-mouse Stat4-P (1:500;

Zymed Laboratories, South San Francisco, CA), or anti-β-actin (1:2000; Chemicon

International, Temecula, CA). For RANKL (35 kDa) and Bcl-xL (27 kDa) detection, 15

ug of protein were run on a 10% gel. Following semi-dry transfer, membranes were

blocked in 5% milk and probed with either biotin-conjugated rat anti-mouse TRANCE

(RANKL) (1:1000; eBioscience), purified mouse anti-mouse Bcl-xL (2H12) (1:200;

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eBioscience), or anti-β-actin. Membranes were probed with the appropriate HRP-linked

secondary antibody and bands visualized using Western Lightning Chemiluminescence

Kit (Perkin Elmer).

3.3.5 Analysis of transgene and cell surface marker expression on DCs

To assess erbB2tr levels on DCs using a FACS Calibur (BD Biosciences Canada,

Mississauga, ON), cells were stained on culture day 5 as already described [317].

RANKL expression was assessed on day 5 using an anti-TRANCE primary antibody

(IKK2/5, eBioscience, San Diego, CA) and an APC-conjugated anti-rat secondary

antibody (BD). To measure IL-12 production from DCs, day 5 cell culture supernatants

were harvested and analyzed using a murine IL-12 ELISA kit (BD). To analyze the cell

surface profile of DCs, we used the following antibodies along with proper isotype

controls: PE- or FITC- conjugated anti-CD11c (clone HL3), purified anti-CD80 (clone

1G10), and FITC-conjugated anti-CD86 (clone GL1), FITC-conjugated anti-I-Ab (clone

AF6-120.1) (all from BD). For all FACS analyses, dead cells were gated out using 7-

aminoactinomycin D (Sigma).

3.3.6 Immunizations and tumor inoculations

For tumor protection experiments, C57BL/6 mice were injected i.p with 2x103

transduced or control DCs (in 200 ul of PBS). One group of mice was injected with 2x103

erbB2tr-transduced DCs along with CFA (Sigma). Two weeks later, mice were

immunized a second time. Five of ten mice per group were sacrificed for subsequent

splenocyte cytokine secretion analyses (see below). The remaining mice were inoculated

s.c. in one flank with 2x105 RM-1-NT and RM-1-erbB2tr cells (in 200 ml of PBS) in

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opposite flanks. Tumors were measured using a caliper and volumes were calculated by

multiplying tumor length, width, and height. For the tumor regression study, mice (5 per

group) were first bilaterally inoculated with 2x105 RM-1-NT and RM-1-erbB2tr cells.

Three days later, mice were immunized i.p with 4x105 non-transduced or transduced

DCs. Another three days later, a second round of immunizations was performed. Mice

were sacrificed approximately two weeks post-tumor inoculation.

3.3.7 Measurement of anti-erbB2tr antibody from mouse plasma

Anti-erbB2tr antibody levels were determined using the plasma of each mouse as

previously described [317]. Briefly, about 200 ul of blood were collected from each

mouse using EDTA-coated tubes (Sarstedt, Montreal, Canada) and then tubes were

centrifuged (18,000xg at 4°C for 20 min) to isolate the plasma. We used a modified flow

cytometry-based assay [287] to determine the plasma anti-erbB2 levels relative to naïve

control mice.

3.3.8 Cytokine secretion assays

RBC-depleted splenocytes were prepared from the spleens of mice at two-weeks

post immunization or approximately two weeks post-tumor challenge. Cells were

counted, diluted, and plated in 24-well plates at a final concentration of 1x106

splenocytes per ml for IL-12 experiments or 2x106 splenocytes per ml for RANKL

experiments. Cells were cultured in a total volume of 1 ml of RPMI medium containing

10% FBS, 1% penicillin/streptomycin, 1% minimal essential amino acids (Invitrogen),

5x10-5 M 2-mercaptoethanol, and 60U/ml of rhIL-2. Splenocytes were co-cultured with

70 Gy-irradiated RM-1-NT or RM-1-erbB2tr cells at a 10:1 splenocyte:tumor cell ratio.

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As controls, splenocytes were also cultured alone or with 100ng/ml of PMA/ionomycin.

Supernatants were harvested 36 hours later and kept at -20°C until thawed and analyzed

for IFN-γ, IL-2, TNF-α, IL-4, and IL-10 levels by Bio-Plex multiplex sandwich

immunoassays according to the manufacturer’s instructions (Bio-Rad Laboratories,

Hercules, CA).

3.3.9 Analysis of tumor-infiltrating lymphocytes

RM-1-NT and RM-1-erbB2tr tumors were harvested from each mouse in our tumor

protection studies. Tumors were snap frozen in liquid nitrogen using Tissue-Tek optimal

cutting temperature (OCT) compound (Sakura Finetek USA Inc., Torrance, CA) and

stored at -20°C. Frozen sections approximately 5 um in thickness were mounted on slides

and stored at -80°C until use. Two sections per tumor were fixed in a 1:1 solution of

methanol and acetone (both from Sigma). Slides were put at -80°C for 30 minutes and

washed in PBS. Slides were blocked, stained first with either goat anti-mouse CD4

(L3T4) (clone GK1.5), goat anti-mouse CD8a (Ly-2) (clone 53-6.7) primary antibodies

(both from eBioscience), or appropriate isotype controls, and then stained with

AlexaFluor488 goat anti-rat IgG (H+L) secondary antibody (Invitrogen). Sections were

also co-stained with DAPI using a final concentration of 1 ug/ml. Up to 10 images of

each stained tumor section were captured on a Zeiss Axioskop 2 microscope using

Axiovision 3.1 and images were analyzed using ImageJ version 1.37v.

3.3.10 Statistical analysis

Student’s t tests were used to perform pairwise comparisons. Differences in means

were considered statistical significant at P< 0.05 or better as otherwise indicated.

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3.4 Results

3.4.1 DC populations can be simultaneously transduced with two

independent LVs to efficiently co-express a TAA and an immunomodulatory gene

To obtain DC populations that efficiently co-express a TAA and either IL-12 or

RANKL as an immunomodulatory gene, we sought to develop a lentiviral transduction

method that utilized two separate LVs. Instead of creating a single bicistronic LV as we

have done before [284, 304, 332, 333], we rationalized that separate transductions would

facilitate the testing of DCs that are genetically-engineered to express different

combinations of TAAs and immunomodulatory genes. The optimized transduction

protocol involved simultaneously exposing cultured DCs to pre-mixed concentrated LVs

at functional MOI values typically less than 2 for a relatively short duration of 5 hours at

37°C.

We used combinations of four monocistronic LV constructs to demonstrate the

feasibility and outcomes of our co-transduction protocol. Previously constructed

pHR’EF-erbB2tr-W-SIN [317], which encodes a kinase-deficient version of erbB2, and

the enhanced green fluorescent protein (enGFP)-encoding pHR’EF-enGFP-W-SIN [282]

were used to make LV/erb and LV/enGFP viruses, respectively. In addition, we

employed pHR’EF-IL-12-W-SIN (LV/IL-12) [331] and newly constructed pHR’EF-RL-

W-SIN (LV/RL), which carry the cDNA sequences for murine IL-12 and murine

RANKL, respectively. After producing concentrated virus from all four constructs, we

performed a single tandem transduction of day 3 BM-derived DCs with either LV/erb or

LV/enGFP alone or in combination with LV/IL-12 or LV/RL. As shown in Figure 3.1a,

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Figure 3.1. Efficiency of transducing DCs with LVs to express enGFP or erbB2tr

alone, or in combination with IL-12 or RANKL. BM cells were cultured in the presence of GM-CSF and IL-4. DC maturation was induced with TNF-α on culture day 8. Shown are transgene expression levels in DCs on culture day 5 following transductions with either LV/enGFP or LV/erb alone, or in combination with either LV/IL-12 (a,b) or LV/RL (c). Flow cytometry plots are representative of two independent experiments. Plotted IL-12 concentrations are the mean ± SEM of n=3. *P<0.005, vs DC-NT vaccination.

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after transducing DC populations with LV/enGFP, 53.2% of the population was enGFP-

positive in this representative analysis from one of two independent experiments.

Performing co-transductions with LV/enGFP and LV/IL-12 yielded a population of cells

that were 50.3% enGFP-positive. In the case of DC transductions with LV/erb, both

single transductions and dual co-transductions of DCs generated a population of cells that

expressed similar levels of erbB2tr - around 29.4% and 29.7%, respectively. Although the

addition of LV/IL-12 did not appear to affect the levels of enGFP or erbB2tr expressed

by DC populations compared to single transductions (Fig. 3.1a), there were differences in

the levels of IL-12 produced among these different conditions (Fig. 3.1b). Less than 100

pg/ml of IL-12 was measured in control culture supernatants of non-transduced DCs or

DCs that were transduced only with LV/erb or LV/enGFP alone. The addition of LV/IL-

12 to the co-transduction protocol increased the levels of IL-12 to ~800 pg/ml. By

contrast, DC populations transduced only with LV/IL-12 secreted far higher amounts of

IL-12, reaching a concentration of >2000 pg/ml in culture supernatants.

When DC populations were transduced to express either enGFP or erbB2tr alone

or in combination with RANKL, transgene levels were measured by flow cytometry in

two independent experiments. As shown in Figure 3.1c, transducing DCs with LV/enGFP

alone generated a population of cells that were measured to be 81.3% enGFP-positive.

Co-transducing DCs with LV/enGFP and LV/RL yielded a population of cells that was

23.9% enGFP-positive and 47.8% enGFP- and RANKL-positive, respectively. When

LV/erb was used to transduce DCs, 52.9% of the population expressed erbB2tr. Adding

LV/RL to the transduction procedure kept the levels of erbB2tr-expressing cells to

46.3%. DCs that were genetically engineered to express RANKL alone were 43.5%

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transgene-positive, compared to 37.3% for those that were engineered to co-express

erbB2tr and RANKL.

Having established the feasibility of co-transducing DC populations with LVs

encoding a TAA and an immunomodulatory protein, we aimed to verify that the IL-12

and RANKL proteins being expressed from DCs were functional. One of the signaling

effects that IL-12 expression elicits in DCs is the phosphorylation of transcription factor

Stat4 [334, 335]. We performed Western blot analyses to confirm that lysates of LV/IL-

12-transduced DCs contained higher levels of Stat4-P than lysates of DCs transduced

with LV/enGFP or LV/erb alone or left non-transduced (Fig. 3.2a). We also performed

Western blot analyses to determine the relative levels of the anti-apoptotic protein Bcl-

xL, which has been reported to become upregulated in cultured DCs in the presence of

exogenous RANKL [328]. As expected, Bcl-xL levels were elevated in the lysates of all

DC groups that were transduced with LV/RL compared to those transduced with

LV/enGFP or LV/erb alone or left non-transduced (Fig. 3.2b).

3.4.2 DCs expressing erbB2tr and IL-12 or RANKL exhibit varying levels of

DC markers

Here we aimed to monitor the potential differences in DC marker expression due

to the influence of IL-12 and RANKL expression. For this, we measured levels of

CD11c, co-stimulatory molecules CD80 and CD86, and the MHC class II molecule I-Ab.

Although we expected IL-12 to mediate enhancement of DC marker expression [336,

337], we actually observed the opposite effect according to flow cytometric analyses. As

shown in Figure 3.3a, decreases in the percentages of cells expressing these molecules

ranged from about 10% to 30%. By contrast, transducing DCs with LV/RL

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Figure 3.2. Effect of IL-12 and RANKL on DC signaling. Lysates were prepared from non-transduced and transduced DCs on culture day 8 (at 24 hours post-TNF-α addition). (a) Lysates from DCs transduced with LV/IL-12 were evaluated for Stat4 phosphorylation. (b) Lysates from DCs transduced with LV/RL were evaluated for Bcl-xL upregulation. Optical density (OD) values shown were normalized to β-actin OD values.

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Figure 3.3. Effect of transducing DCs with (a) LV/IL-12 or (b) LV/RL on the cell

surface expression of DC markers. BM-derived DCs were transduced on culture day 3 and matured on culture day 8. Transduced and non-transduced (NT) mature DCs were stained with antibodies against CD11c, I-Ab, CD80, and CD86 and analyzed by flow cytometry.

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either led to an enhancement in DC purity and maturation state or offered no clear

modulation (Fig. 3.3b).

3.4.3 Vaccination using DC-erbB2tr/IL-12, but not DC-erbB2tr/RANKL,

elicits erbB2tr-specific anti-tumor protection

We have previously demonstrated that immunizing mice with 2x105 DCs

transduced with LV/erb could completely protect against the development of the

aggressive RM-1-erbB2tr prostate tumors in an antigen-specific manner [317]. In that

study, we also tested the 100-fold lower dose of 2x103 DC-erbB2tr cells and discovered

that complete tumor protection could still be achieved in some animals (in 2 of 6 mice

with 2 other animals demonstrating reduced tumor volumes). To evaluate whether this

low dose could be made more effective, we tested the immunomodulatory effects of IL-

12 and RANKL when co-expressed with erbB2tr on DCs.

Mice were immunized twice, two weeks apart with 2x103 DCs, and then six

weeks later inoculated bilaterally with RM-1-erbB2tr cells in one hind flank and with

wild-type RM-1-NT cells in the opposite flank. We first compared the vaccination

efficacies of DCs transduced with enGFP or erbB2tr with and without IL-12 (Fig. 3.4a).

The best inhibition of RM-1-erbB2tr tumor development was achieved with DC-

erbB2tr/IL-12 vaccinations. In this cohort, all 5 mice displayed robust protection

specifically against RM-1-erbB2tr, but not RM-1-NT tumors. Mice that were

administered DC-IL-12 also exhibited a moderate degree of RM-1-erbB2tr tumor

protection over the 13 days of observation (Fig. 3.4a), but vaccinations with DC-

enGFP/IL-12 offered no significant erbB2tr-specific tumor protection. As expected, all

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Figure 3.4. Immunizing mice with low doses of DC-erbB2tr/IL-12 leads to robust tumor protection. Following two DC immunizations of 2x103 cells given two weeks apart, mice were inoculated with RM-1-erbB2tr and RM-1-NT. Tumor volumes were calculated (LxWxH) from caliper measurements for each RM-1-erbB2tr (a,c) and RM-1-NT (b,d) tumor to assess the effects of IL-12 and RANKL on tumor protection, respectively. Plotted values are the mean ± SEM of 4 or 5 mice per group. *P<0.05, vs ‘No DCs’ group; **P<0.01, vs ‘No DCs’ group.

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vaccinated and naïve mice developed RM-1-NT tumors in an uninhibited manner (Fig.

3.4b), showing that the immune response generated is specific towards RM-1-erbB2tr

tumors.

In separate experiments, we next assessed the immunotherapeutic impact of co-

expressing erbB2tr and RANKL in DCs. Although mice that were immunized with DC-

RL or DC-erbB2tr/RL seemed to exhibit the most resistance to RM-1-erbB2tr tumor

growth at 9 days, this trend was not sustained to the time when mice had to be sacrificed

(Fig. 3.4c). By that point, none of the experimental test groups displayed any significant

tumor protection. As a control, we had included a cohort of mice that were immunized

with a combination of Complete Freund’s Adjuvant (CFA) and 2x103 DC-erbB2tr cells.

We observed that this vaccination group yielded the most robust protection from RM-1-

erbB2tr growth and was sustainable over the 11 days of observation, thus it is unlikely

that there were problems with the DC cultures themselves that led to the lack of

protection in the mice vaccinated with DC-RL or DC-erbB2tr/RL. As before, mice in all

vaccination cohorts allowed RM-1-NT tumors to grow without restraint (Fig. 3.4d).

3.4.4 Tumor protection was not associated with increased anti-erbB2tr

antibody response

To determine whether anti-tumor immunity correlated with an erbB2tr-specific humoral

response, we evaluated the levels of anti-erbB2tr antibody in the plasma of all vaccinated

mice relative to the baseline levels in mice that were not injected with DCs. As shown in

Figure 3.5a, vaccinations using DC-NT and DC-enGFP did not produce significant levels

of anti-erbB2tr antibodies, as one might expect, and neither did immunizing with the low

dose of DC-erbB2tr cells with or without the aid of CFA. In accordance with the well-

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Figure 3.5. Assessment of humoral immune response against erbB2 in vaccinated and non-vaccinated mice. The presence of anti-erbB2 antibodies in mouse plasma were determined using a flow cytometry-based ELISA. Levels of antibodies in IL-12 (a) or RANKL (b) experiments were normalized to ‘No DC’ treatment cohort. Plotted values are the mean ± SEM of 4 or 5 mice per group.

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established role of IL-12 in skewing immune responses towards a Th1 type [338], we did

not measure an appreciable relative level of anti-erbB2tr antibodies in any of the IL-12-

immunized cohorts. Although the vaccinations themselves did not seem to lead to

humoral immunity, challenging the vaccinated mice with RM-1-erbB2tr tumors lead to a

slight rise in titers in most cohorts, particularly in the DC-erbB2tr cohort (Fig. 3.5a).

Similarly, in our assessment of anti-erbB2tr antibody titers that could result from

RANKL immunomodulation, we did not find a significant change in antibody titers

throughout the time course of the study (Fig. 3.5b). Following the second vaccination,

there seemed to be small titer increases in the majority of cohorts, but these were not

found to be statistically significant.

3.4.5 DC-erbB2/IL-12 vaccinated mice show strong erbB2tr-specific Th1

immunity following tumor challenge

Next we were interested in evaluating which cytokines were upregulated as a result of

introducing IL-12 into our low-dose prime/boost immunization strategy. We examined

the Th1 (IL-2, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokine concentrations

secreted from splenocytes of sacrificed mice, which were cultured with either RM-1-

erbB2tr cells or RM-1-NT cells for 36 hours. First, 5 of 10 mice from each cohort were

sacrificed two-weeks post DC vaccination and we calculated the average relative

increases in cytokine concentrations upon stimulation to assess the levels of erbB2tr-

specific immunity. Here we found that neither Th1 nor Th2 cytokines were appreciably

upregulated (less than 2-fold increases) (Fig. 3.6a,b). However, after measuring cytokine

levels in all remaining mice two weeks post-tumor challenge, we found that all three Th1

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Figure 3.6. Vaccinations with DCs transduced with LV/erb and LV/IL-12 offers erbB2tr-specific Th1 immunity. Splenocytes obtained from naïve and immunized mice (n=4 or 5) were co-cultured with either RM-1-erbB2tr or RM-1-NT cells for 36 hours. Using Bio-Plex multiplex sandwich immunoassays, levels of IL-2, IFN-γ, and TNF-α were assessed two weeks post-immunizations (a) or post-tumor inoculations (b). Similarly, levels of IL-4 and IL-10 were determined two weeks post-immunizations (c) or post-tumor inoculations (d). Cytokine concentrations from RM-1-erbB2tr co-cultures were normalized to those from RM-1-NT co-cultures. Plotted values are the mean ± SEM of 4 or 5 mice per group. *P<0.10, vs. DC-NT treatment group.

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cytokines were strongly upregulated (approximately 10- to 30-fold) in the supernatant of

stimulated splenocytes in mice that were immunized with DC-erbB2tr/IL-12, but not in

any other vaccination group (Fig. 3.6c). Interestingly, even DC-IL-12 mice that displayed

tumor protection did not show this Th1 response. In contrast, post-tumor challenge Th2

cytokine levels as measured from stimulated splenocytes in mice from all vaccination

cohorts were generally very low (~2-fold) similar to the cytokine levels measured in the

stimulated splenocytes from the naïve control cohort (Fig. 3.6d).

We also tested whether immunizing mice with RANKL had any detectable effects

on cytokine secretion profiles from stimulated splenocytes. Two weeks post-vaccination,

only one cohort, DC-erbB2tr, showed considerable IFN-γ upregulation, having an

average fold-increase of 7.8 over background (Fig. 3.7a). The levels of Th2 cytokines

from stimulated splenocyte cultures were also very low, averaging less than 2.5-fold

increases over background (Fig. 3.7b). Post-tumor challenge, both Th1 and Th2 cytokine

levels were also low (all less than 3.3-fold over background), similar to levels from cells

of naïve mice (Figs. 3.7c,d).

To further investigate possible reasons that IL-12 offered tumor protection while

RANKL did not, we compared the levels of CD4+ and CD8+ tumor infiltrating

lymphocytes (TILs) in the RM-1-NT and RM-1-erbB2tr tumors of mice in select cohorts.

In mice immunized with DC-enGFP/IL-12 or DC-erbB2tr/IL-12, no significant

differences in CD4+ TIL levels were measured between tumors within either

immunization group. In addition, the CD8+ T cell levels in RM-1-erbB2tr tumors were

similar in both cohorts, but RM-1-NT tumors of mice vaccinated with DC-erbB2tr/IL-12

displayed higher percentage of CD8+ TILs (Fig. 3.8a). We also assessed levels of TILs in

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Figure 3.7. Neither Th1 nor Th2 immune responses against erbB2tr was upregulated in mice following immunizations with DC-erbB2tr/RANKL. Secretion of IL-2, IFN-γ, and TNF-α from splenocytes stimulated with tumor cells for 36 hrs was assessed two weeks post-immunizations (a) or post-tumor challenge (b). Concentrations of IL-4 and IL-10 were also measured from stimulated splenocyte cultures at two weeks post-immunizations (c) or post-tumor challenge (d). Cytokine concentrations from RM-1-erbB2tr co-cultures were normalized to those from RM-1-NT co-cultures. Plotted values are the mean ± SEM of 4 or 5 mice per group.

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Figure 3.8. Mice vaccinated with DC-erbB2tr exhibited systemically high tumor-

infiltrating CD8+ lymphocytes. Shown are the percentages of CD4+ and CD8+ TILs in mice immunized with DCs co-expressing (A) IL-12 or (B) RANKL with enGFP or erbB2tr.

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mice immunized with either DC-enGFP/RL or DC-erbB2tr/RL. Mice immunized with

DC-erbB2tr/RL neither had elevated levels of CD4+ nor CD8+ TILs compared to DC-

enGFP/RL mice (Fig. 3.8b).

3.4.6 DC-erbB2tr/IL-12 immunizations fail to reduce established aggressive

tumor growth

Having shown the potential for vaccination with DC-erbB2tr/IL-12 in protecting mice

from developing erbB2tr-expressing tumors, we further tested the ability of a dose of

4x105 transduced DCs to cause rejection of aggressive tumors already present in vivo at

the time of immunization. Given the aggressive nature of RM-1 cells, we rationalized that

this moderately high treatment dose might hedge against the possibility of an

overwhelming tumor burden from developing before the administered immunotherapy

could elicit an anti-erbB2 adaptive immune response. We began by inoculating mice

bilaterally with RM-1-NT and RM-1-erbB2tr tumors and then 3 days later, we

immunized mice with 4x105 transduced DCs, followed by a second immunization another

3 days later. As expected, RM-1-NT tumors grew well in all immunization cohorts,

however even this higher dose of DC-erbB2tr/IL-12 cells did not reduce RM-1-erbB2tr

tumors and neither did any other immunization types tested (Fig. 3.9a,b). Thus, it was

not surprising when we measured splenocyte secretion of IFN-γ and IL-4 by ELISA that

we saw no appreciable changes in production of these cytokines in any of the tested

immunization groups relative to control, mock-immunized mice (Fig. 3.9c,d).

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Figure 3.9. Immunization of tumor-bearing mice with DCs transduced with LV/erb

and LV/IL-12 does not delay tumor growth. Mice were inoculated bilaterally and immunized with 4x105 DCs. Tumor volumes were measured for RM-1-NT (a) and RM-1-erbB2tr (b). Levels of IFN-γ (c) and IL-4 (d) were determined by ELISA from 36-hour cultured splenocytes. Plotted values are the mean ± SEM of 5 mice per group.

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3.5 Discussion

The prevention and treatment of aggressive and metastatic cancers remains an

elusive task. DC-based immunotherapy is one modality that has shown considerable

promise in recent years in pre-clinical models and many clinical trials [277]. Although

the efficacy of DCs in eliciting anti-tumor immunity has been well established, one

hindrance to their use in clinical trials is the difficulty in generating modified DCs in the

required large numbers [164, 339]. We were the first to demonstrate that when DCs were

transduced with LV/erb, a DC dose as low as 2x103 cells could be effective in

vaccinating mice against RM-1-erbB2tr growth [317]. Using the same calculation as

discussed, 2x103 murine DCs would be equivalent to 5x106 human DCs, which is less

than the average immunization dose of ~107 DCs, and thus our work models a clinically

feasible protocol. Continuing our goal to develop an effective low-DC-dose vaccination,

we hypothesized that co-transducing DCs with LV/erb and an immunomodulatory gene

such as IL-12 or RANKL could enhance the efficacy of our strategy towards mounting an

antigen-specific anti-tumor response. To test our hypothesis, we first established the

feasibility of simultaneously transducing murine BM-derived DCs with two different

LVs, in order to engineer expression of both a TAA and an immunomodulatory protein.

To our knowledge, we are the first to show the feasibility of co-transducing DCs with two

separate LVs; we developed a protocol that not only achieves efficient transduction, but

also avoids the cell death that can be associated with transducing DCs with high viral

loads [340].

One advantage to employing separate vectors to encode the antigen and

immunomodulatory cDNAs of choice is that in principle, it would allow us to control the

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ratio of each vector and hence factor expression levels in transduced cells. Importantly,

IRES elements often used in bicistronic vectors may have reduced downstream transgene

expression, thus generating lower levels of the desired co-factor.

In performing our DC transductions, we noticed that transducing DCs with

LV/IL-12 alone yielded much higher IL-12 production than with LV/IL-12 combined

with LV/erb or LV/enGFP. This phenomenon occurred despite the fact that we

consistently used the same relative number of LV/IL-12 viral particles for each

transduction. It may reflect the possibility that we have simply reached a ceiling in the

number of viral particles that can productively enter the target cells. Despite this

discrepancy in IL-12 production, we demonstrated the ability of DC populations that

express both erbB2tr and IL-12 to protect mice from developing aggressive ectopic

tumors.

Tumor protection in our DC-erbB2tr/IL-12 treated mice was specifically against

erbB2tr, as demonstrated by the unrestricted growth of RM-1-NT tumors in comparison

to reduced growth of RM-1-erbB2tr tumors. We further showed that expressing erbB2tr

and IL-12 together in DCs offered strong antigen-specific Th1 immunity. In addition,

DCs expressing the higher level of IL-12 alone also offered significant tumor protection,

without evidence of a Th1 response. Thus, it appears that the combination of erbB2tr and

IL-12 provided a synergistic effect. The efficacy of these strategies is underscored by the

lack of both tumor protection and significant immune responses following vaccination

with DC-erbB2tr alone or with DC-enGFP/IL-12, which expressed similar IL-12 levels as

DC-erbB2tr/IL-12. It was not surprising that the relatively high IL-12 production from

DC-IL-12 therapy alone was sufficient to protect against RM-1-erbB2tr tumor

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development; others have shown that IL-12 alone can have anti-tumor effects [249, 251].

To achieve RM-1-erbB2tr tumor protection with a DC vaccine that produced less IL-12

than the DC-IL-12 vaccine itself required co-expression of erbB2tr. Thus, the lack of

specific protection offered by DC-enGFP/IL-12 may be due to the fact that it lacked

sufficient IL-12 expression or the directed combination of erbB2tr and IL-12.

The finding that Th1 immunity was the predominant immune response towards

erbB2tr following immunizations with DCs co-transduced to express erbB2tr and IL-12

is in agreement with the role of IL-12 in promoting Th1 responses. Based on the

mechanism of Th1 differentiation, the ligation of IL-12 receptor on T cells by IL-12

triggers the release of IFN-γ, which in turn leads to the activation of transcription factor

T-bet [341]. Activated T-bet not only modulates the opening of chromatin around the

IFN-γ locus to increase IFN-γ production for further Th1 development, but it also inhibits

the activation of transcription factor GATA-3, which is a main regulator of Th2

development [342]. Our finding that the presence of IL-12 l primarily lead to Th1

immunity without evidence of significant Th2 immunity supports this model of Th1/Th2

differentiation.

Although the same DC generation and transduction protocols were applied to all

DC cultures used in our study, we found non-transduced control DCs did not consistently

exhibit the same upregulation of maturation markers in our studies. In addition, we found

that transducing DCs with RANKL alone increased expression of DC markers relative to

non-transduced DCs; yet marker expression was not altered when RANKL was combined

with another LV. This held true for co-transduction of LV/RL with either LV/enGFP or

LV/erb. Due to these differences in DC maturation status, it is not clear whether this

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could have partly accounted for the lack of immunotherapy enhancement by RANKL.

However, similar to a recent report that demonstrated the inability of RANKL to improve

anti-tumor immunity [343], we did not find that it aided anti-erbB2 immunity in our

prophylactic tumor model as neither tumor protection nor enhanced Th1/Th2 immunity

towards erbB2tr were detected.

It is important to note that while we have previously reported the ability of our

low-dose DC-erbB2tr immunization strategy alone to offer partial protection against RM-

1-erbB2tr in a prophylactic tumor model [317], we did not find that it faired as well in

our current study. This finding may point to the possibility that in this current system

with this antigen and tumor model, the dose of 2x103 transduced DCs may represent a

threshold value around which immune effects are measurable and adequate enough to

offer benefit. It follows that slight variations in different preparations of DC cultures at

this threshold dose may determine whether the critical level of anti-erbB2tr immunity can

be achieved. Indeed our present data emphasizes the need for addition of immunity-

enhancing IL-12, which is sufficient to tip the balance towards tumor protection. On the

other hand, similar to a recent report that indicated the lack of benefit that RANKL had

on improving anti-tumor immunity [343], we did not find that it aided anti-erbB2

immunity in our prophylactic tumor model as neither tumor protection nor enhanced

Th1/Th2 immunity towards erbB2tr were detected.

Although DC-erbB2tr/IL-12 vaccinations effectively prevented tumor growth in

an antigen-specific manner, this immunization strategy failed when used to regress

established tumors, despite using the dose of 4x105 DCs per immunization. Reasons for

this finding may relate to the highly aggressive nature of RM-1 cells [289]; suggesting

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that analyses using a slower-growing tumor model may show greater efficacy.

Alternatively, specific inhibitory mechanisms due to the presence of these tumor cells

themselves may have dampened the immune response [344], especially since significant

differences in cytokine levels were not found between groups.

When developing anti-tumor immunotherapy strategies in a mouse model, it is

essential that the therapies themselves are clinically practical. Since obtaining large

quantities of DCs from cancer patients for manipulations carries inherent limitations, we

aimed to develop a DC vaccine that would be effective at a dose low enough to be

actually achievable on a human scale. Testing the low dose of 2x103 DCs sets an

important precedent in the field of cancer immunotherapy, as current studies of this kind

typically use 500-fold higher doses. We are also the first to demonstrate the feasibility of

co-transducing DCs with two separate LVs, an approach that facilitates fine-tuning of the

dosing of key components. In our current study, we measured the effects of combining

erbB2tr and either IL-12 or RANKL expression on LV-transduced DCs. The synergistic

effects of co-expressing erbB2tr and IL-12 on DCs led to strong erbB2tr-specific Th1

immunity and tumor protection. By contrast, we did not find that RANKL was a valuable

immunomodulator. Overall, our study provides a basis for the development of clinically

feasible and effective immunotherapy strategies using low doses of LV-transduced DCs.

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Chapter 4:

Discussion

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4.1 Summary of findings

The role of DCs in eliciting potent antigen-specific immunity is well recognized

[103, 345]. Their use in generating anti-tumor effects in mouse models remains an

important area of research, as the knowledge gained from these studies can be useful in

developing therapies for cancer patients. Unfortunately, given the difficulties associated

with obtaining and generating DCs from cancer patients, the use of typical murine DC

doses on the order of 106 cells is not realistically translatable to the human scale. The

goal of the research described in this thesis was to develop an effective immunotherapy

strategy using low doses of DCs to target erbB2-expressing prostate tumors in a mouse

model. It was hypothesized that if DCs could be engineered to efficiently express erbB2

via lentiviral transduction, then the combined intrinsic potency of DCs could allow lower

numbers of DCs to be sufficient to produce appreciable anti-tumor effects. The TAA

erbB2 was chosen as the target antigen due to its upregulation in many cancers, including

prostate cancer.

While many techniques exist for generating DCs that present TAA-derived

peptides [316, 346], viral transduction remains one of the most efficient methods of

engineering expression of TAAs in DCs for subsequent peptide presentation [313].

Transduction of cells using LVs carries many advantages over other methods including

the ability to transduce slowly-dividing cells, stable transgene expression, and low

toxicity for primary cells [313, 347]. Taken together, LV transduction of DCs is an ideal

approach to efficiently generate DCs that express TAA-derived peptides and has been the

method of choice for my thesis projects described herein.

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The utility of DCs in directing TAA-specific immune responses has been

established for many years. However, the efficacy of low doses of DCs that have been

LV-transduced to express a TAA in the prevention of tumor formation is unknown. In

Chapter 2, I have shown that LV-transduction of DCs is efficient, since one overnight

transduction could yield a population of DCs that was approximately 47% positive for

kinase-truncated murine erbB2, erbB2tr. Use of the truncated version of this TAA

reduced the likelihood of obtaining oncogenic signaling events in transduced DCs. In

addition, LV transduction of DCs did not extensively alter either the cell surface marker

profile on DCs, or their ability to induce an allogeneic mixed lymphocyte reaction. The

efficacy of low doses of LV/erb-transduced DCs in protecting mice from developing

ectopic tumors was also demonstrated in Chapter 2. The dose of 2x105 cells in a

prime/boost vaccination strategy completely protected mice from developing erbB2tr+

RM-1 tumors, but not control tumors. This antigen-specific anti-tumor effect correlated

with an anti-erbB2tr antibody response as well as an upregulation in Th1 immunity.

Using the 100-fold lower dose of 2x103 LV/erb-transduced DCs also offered some mice

antigen-specific tumor protection, but evidence of either humoral or cellular immunity

was not found.

The finding that partial and complete tumor protection could be achieved with

2x103 and 2x105 LV/erb-transduced DCs suggested that there might be a lower threshold

dose of complete protection between the tested doses. In Chapter 3, I have investigated

this possibility by testing whether co-transducing DCs to express erbB2tr and either IL-

12 or RANKL could create an enhanced DC vaccination such that the low dose of 2x103

DCs could actually lead to robust tumor protection and detectable immune responses. IL-

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12 is well known for it ability to skew immunity towards Th1 [324, 325] and was chosen

with the hypothesis that it could enhance immunity against erbB2tr. RANKL is better

known for its ability to enhance DC function by enhancing its life-span and persistence in

draining lymph nodes [330]. The hypothesis was that combining the antigen-specific

factor of expressing erbB2tr on DCs with the immunomodulatory factor of expressing

either IL-12 or RANKL on DCs would augment the ability of the 2x103 DC dose in the

context of tumor protection and possibly even regression. In Chapter 3, the feasibility of

co-transducing DCs was shown and that combining LV/erb and LV/IL-12, but not

LV/RL, offered Th1 upregulation and strong tumor protection, but not regression. As

with the findings of Chapter 2, the 2x103 low-dose DC immunotherapy did not lead to

any significant humoral responses.

Although the DC doses used in our experiments are markedly lower than those

used by others investigating DC-mediated cancer immunotherapy, the concept of low

immunization doses in the context of infectious diseases is well studied. Animal models

of mycobacterial or Leishmania infection have been used to demonstrate that low antigen

doses can lead to Th1 polarized immune responses [121, 122]. Thus, the finding that low

numbers of DCs expressing TAA can also lead to a Th1 phenotype may reflect a similar

mechanism of T cell activation. Parallels between the two systems may be useful in

understanding the mechanism of inducing TAA-specific cellular immunity at low antigen

doses.

Overall, investigating the efficacy of low DC vaccine doses sets a good example

for developing clinically-relevant immunotherapy approaches. In the case of erbB2, the

use of IL-12 as an immunomodulatory adjuvant appears to be advantageous and

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underscores the feasibility and importance of finding potent adjuvants to combine with

TAAs. To my knowledge, this is the first reported use of such low doses of DCs. Its

successful application in a prophylactic setting provides a platform for further low-dose

immunotherapy advancements.

4.2 Future directions

4.2.1 Modifying the immunization strategy

To carry forward the research described in this thesis, one could modify the

immunization strategy to potentially improve its anti-tumor potency. Whereas I tested the

effects of 1 prime and boost immunization set, one could evaluate whether performing

multiple boost immunizations could strengthen a recall response against erbB2tr.

Designing this type of study would provide useful information, since injecting DCs

several times is an approach that is used in the clinic [316, 346].

An alternative way to modifying the immunization strategy is to engineer

expression of multiple TAAs on DCs via LV transduction. Our lab has already

demonstrated the prophylactic and therapeutic benefits of administering DCs expressing

TAAs such as PSA and PSMA [191]. Testing a combinatorial approach with multiple

antigens may prove more effective against prostate tumors than one TAA alone. In my

thesis, the rationale for using just one TAA, erbB2, was to establish the feasibility of LV

transduction methods and to demonstrate anti-tumor effects as a proof-of-principle.

Choosing additional TAAs to target should take into account its expression profile on

normal tissues and tumors, as well as its inherent immunogenicity. Ideally, the TAA(s) of

choice would be tumor-specific.

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Instead of or in addition to expressing two or more TAAs on DCs, one could also

investigate the effects of incorporating one or more immunomodulatory components into

the immunization strategy. The validity of such an approach has been affirmed in clinical

trials against prostate and colon cancer using immunizations with a TRIad of

COstimulatory Molecules (TRICOM; B7-1, ICAM-1 and LFA-3) [348] and supports the

rationale for developing this area of research. The positive results from incorporating IL-

12, but not RANKL, into the immunization strategy developed in my thesis emphasizes

the importance of carefully choosing which immunomodulatory components should be

used. While in these experiments the enhancement effects associated with IL-12 were

investigated, it would be interesting to test the effects of several other cytokines including

IFN-α and other type I interferons, which have proven to be helpful in enhancing DC

function [190, 349]. Since the induction of potent immune responses is dependent on

using mature DCs, factors to enhance their maturation status could also be considered.

The effects of fusing HSP70 with human papilloma antigen E7, for example, was shown

to protect mice from developing tumors [350]. The clinical benefit of using heat-shock

protein gp96 was also demonstrated in melanoma clinical trials [351, 352]. Finally, one

could knockdown specific proteins to achieve a potentially stronger vaccine. One

example is knocking down SOCS1 in DCs, since its expression in DCs has been

attributed to maintaining immune tolerance [334, 353].

Taking into consideration that the peripheral tolerance mechanisms need to be

overcome for DCs to work more effectively, studies have shown that injecting anti-CD25

antibodies into mice to deplete Treg cells can enhance the effects of DC-mediated

immunotherapy [354]. Thus, an interesting experiment would be to test the efficacy of an

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approach using low doses of TAA-presenting DCs to determine whether such a strategy

can produce stronger protection or even tumor regression without the aid of a cytokine

adjuvant.

4.2.2 Modifying the prostate tumor model

To better evaluate the efficacy of the vaccines developed in this thesis, one could

use a more stringent and more clinically-relevant tumor model. For example, one

approach would be to factor in the complication of age into the tumor model by using

mice that are near the end of their life expectancy; prostate cancer often occurs in elderly

men [355] and their weakened immune system [356] may best be modeled with an old

mouse [357]. Another way of altering the tumor model to make it more stringent and

clinically-relevant would be to use a slower-growing tumor cell line that causes immune

suppression, such as via TGF-β secretion, since prostate cancer is generally slow-

growing and immunosuppressive [358]. RM-1 tumor cells do secrete this cytokine [359],

but grow very rapidly, thus making it difficult to assess whether tumor protection is

sustained in a bilateral model. Nevertheless, as already mentioned, the aggressive nature

of this cell line provided a stringent model for evaluating the potency of the

immunotherapy strategies developed in this thesis. Although injecting tumor cell lines to

generate ectopic tumors is a popular approach for modeling cancer having mice strains,

such as the TRAMP mouse [29] that develop orthotopic prostate tumors, may serve as a

more accurate model, depending on the expression levels of erbB2 in TRAMP mice. It is

not clear whether the prostate tumors that develop in these transgenic mice are erbB2+,

but it has been recently established that at least their premalignant dorsolateral prostates

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are erbB2+ [360]. Thus, it would be worth testing the prostate tumors of TRAMP mice

for erbB2 expression by Western blot analysis, for example. If the naturally growing

orthotopic tumors are indeed erbB2+, then the next logical step would be to use TRAMP

mice to evaluate erbB2-targeted immunotherapy strategies.

4.2.3 Testing new applications

Although I performed experiments using a prostate cancer model, other cancer

models could be used instead, since human HER2/neu is overexpressed in ovarian, lung,

and breast cancer, for example. Tumors originating from any of these other organs may

express unexpected levels of immune-evading factors or display other properties that

would make them inherently different from prostate-derived tumors. Thus, using other

tumor models may give different results than those described in this thesis.

In the case of breast cancer, HER2/neu is already a successful therapeutic target;

the monoclonal antibody Herceptin is currently in clinical use [361]. However, in cases

where Herceptin is ineffective against HER2/neu-positive breast tumors, perhaps the use

of a DC-based immunotherapy strategy could be worth investigating, since it is not clear

why some patients respond to Herceptin better than others. If, for example, Herceptin is

unable to bind to HER2/neu due to changes in HER2/neu conformational states following

binding with other EGFR family members, then a DC-mediated approach could perhaps

overcome this issue. DCs prime T cells using peptides derived from whole antigen, and

could therefore be effective even in situations of altered HER2/neu conformations.

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Towards the goal of applying LV-transduced DCs in immunization strategies

against other tumors, our laboratory is already working with a colon cancer mouse model

and targeting the TAA carcinoembryonic antigen (CEA).

An LV/erb-transduced DC immunization strategy could also be valuable in

combination with other treatment modalities. For example, to combine immunotherapy

with tumor resection, one could first allow tumors to form in mice, then resect the tumors

and inject LV/erb-transduced DCs to help eliminate any remaining tumor cells. This

approach would mirror the clinical setting of surgical tumor resection and subsequent

attempts to prevent relapse. Alternatively, DC immunizations could be administered with

chemotherapy or radiation therapy, as these may lead to enhancement of anti-tumor

effects [362, 363]. Thus, developing a hybridized approach using novel DC

immunotherapy and standard therapies for the prevention and treatment of cancer may

prove to be a valuable direction of research to pursue.

4.3 Conclusions

DC-mediated immunotherapy is a rapidly-expanding field of research. Many

studies using animal models have shown that tumors can be effectively targeted and

eliminated using TAA-presenting DCs. Achieving the same success has been more

difficult in clinical settings. Reasons for this discrepancy are unclear, but may point to the

shortcomings of experimental vaccines in their translatability from mouse to human, or

the animal models themselves in their ability to truly mimic cancer patients.

Alternatively, it may be that clinical trials are being performed on patients with a disease

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state that is simply too advanced for DC-mediated immunotherapy to consistently

provide objective clinical responses.

The thesis work presented herein contributes to the growing field of

immunotherapy. Unlike many of the currently published mouse model studies, however,

my main goal was to develop a clinically-feasible immunotherapy approach. To this end,

lentiviruses were employed to efficiently transduce primary DCs without causing cellular

toxicity. In addition, I tested relatively low doses of murine DCs that would translate to

reasonable human DC doses. Choosing erbB2 as the TAA of interest was an important

component of the immunotherapy developed here; since it represents a true self-antigen

and would thus require peripheral tolerance mechanisms to be overcome in order to

achieve positive anti-tumor results. We are the first to report the successful use of such

low doses of DCs. Moreover, using IL-12 as an immunomodulatory protein, we were

able to boost the efficacy of the 2x103 DC dose to a level where strong erbB2-specific

Th1 immunity tumor protection was achieved. It is hoped that the work presented in this

thesis can provide the basis for developing even more potent immunization strategies

worthy of being translated to the clinic.

A great proportion of cancer immunotherapy research is focused on the

optimization of the DC therapy. A retrospective look at how DC vaccines have evolved

since they were first used in the clinic over a decade ago shows that many significant

changes have been made. Whereas early vaccines often consisted of DCs that were

pulsed with a single type of tumor antigen-derived peptide [364], many of the more

recent vaccines utilize transfected or transduced DCs [200, 365] in an effort to achieve

more efficient antigen presentation of a wider variety of peptides. Furthermore, many

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recent DC immunotherapy clinical protocols use DCs that not only present peptides from

multiple TAAs, but also express co-stimulatory molecules or adjuvant cytokines [348,

366].

Although the DC vaccines themselves have become more sophisticated, the

immune status of cancer patients also need to be appropriately addressed. It seems that

the next step for the field of cancer immunotherapy is to expand in scope to address the

role of the host. The patient’s immune system is likely as important in determining the

success of an immunotherapy strategy as the therapy itself. If one considers the DC

vaccine to be a key and the patient to be a lock, then it becomes clear that both must be in

a good working condition; if either component is rusty, then the key may not fit into the

lock. Thus, if the host is not able to receive or respond to the DC therapy, then the patient

cannot be adequately treated. Recently, attention has been given to specifically tackling

the host’s immune tolerance status. Methods to overcome peripheral tolerance may create

a better setting for administered DCs to prime antigen-specific anti-tumor responses. For

example, the Treg-depletion protocol using recombinant IL-2 diphtheria toxin conjugate

DAB(389)IL-2 has resulted in encouraging clinical outcomes when combined with a DC

vaccine [367]. Thus, designing feasible immunotherapy approaches in animal models and

patients that can be combined with methods that simultaneously address the tolerant state

of the patient’s immune systems would be useful contributions to the field of cancer

research.

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References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer statistics, 2008. CA Cancer J Clin 58: 71-96. 2. Marrett L, Dryer D, Ellison L, Logan H, Mery L, Morrison H, Schacter B, Xie L, Semenciw R (2008) Canadian Cancer Society/National Cancer Institute of Canada: Canadian Cancer Statistics 2008, Toronto, Canada, 2008. : . 3. Sarma AV, McLaughlin JC, Wallner LP, Dunn RL, Cooney KA, Schottenfeld D, Montie JE, Wei JT (2006) Sexual behavior, sexually transmitted diseases and prostatitis: the risk of prostate cancer in black men. J Urol 176: 1108-1113. 4. Norrish AE, Ferguson LR, Knize MG, Felton JS, Sharpe SJ, Jackson RT (1999) Heterocyclic amine content of cooked meat and risk of prostate cancer. J Natl Cancer Inst 91: 2038-2044. 5. Samanta M, Harkins L, Klemm K, Britt WJ, Cobbs CS (2003) High prevalence of human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma. J Urol 170: 998-1002. 6. De Marzo AM, Marchi VL, Epstein JI, Nelson WG (1999) Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis. Am J Pathol 155: 1985-1992. 7. Parsons JK, Nelson CP, Gage WR, Nelson WG, Kensler TW, De Marzo AM (2001) GSTA1 expression in normal, preneoplastic, and neoplastic human prostate tissue. Prostate 49: 30-37. 8. He WW, Sciavolino PJ, Wing J, Augustus M, Hudson P, Meissner PS, Curtis RT, Shell BK, Bostwick DG, Tindall DJ, Gelmann EP, Abate-Shen C, Carter KC (1997) A novel human prostate-specific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer. Genomics 43: 69-77. 9. Tsihlias J, Kapusta LR, DeBoer G, Morava-Protzner I, Zbieranowski I, Bhattacharya N, Catzavelos GC, Klotz LH, Slingerland JM (1998) Loss of cyclin-dependent kinase inhibitor p27Kip1 is a novel prognostic factor in localized human prostate adenocarcinoma. Cancer Res 58: 542-548. 10. Latil A, Vidaud D, Valéri A, Fournier G, Vidaud M, Lidereau R, Cussenot O, Biàche I (2000) htert expression correlates with MYC over-expression in human prostate cancer. Int J Cancer 89: 172-176. 11. Millar DS, Ow KK, Paul CL, Russell PJ, Molloy PL, Clark SJ (1999) Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer. Oncogene 18: 1313-1324. 12. Putzi MJ, De Marzo AM (2000) Morphologic transitions between proliferative inflammatory atrophy and high-grade prostatic intraepithelial neoplasia. Urology 56: 828-832. 13. Belleannée G (2006) [The TNM system: only 3 letters for a rich but sometimes ambiguous language] Ann Pathol 26: 435-44; quiz 418. 14. Bostwick DG (1994) Grading prostate cancer. Am J Clin Pathol 102: S38-56. 15. Dearnaley DP, Khoo VS, Norman AR, Meyer L, Nahum A, Tait D, Yarnold J, Horwich A (1999) Comparison of radiation side-effects of conformal and conventional radiotherapy in prostate cancer: a randomised trial. Lancet 353: 267-272.

118

16. Han K, Cohen JK, Miller RJ, Pantuck AJ, Freitas DG, Cuevas CA, Kim HL, Lugg J, Childs SJ, Shuman B, Jayson MA, Shore ND, Moore Y, Zisman A, Lee JY, Ugarte R, Mynderse LA, Wilson TM, Sweat SD, Zincke H et al. (2003) Treatment of organ confined prostate cancer with third generation cryosurgery: preliminary multicenter experience. J Urol 170: 1126-1130. 17. Holzbeierlein JM (2006) Managing complications of androgen deprivation therapy for prostate cancer. Urol Clin North Am 33: 181-90, vi. 18. Poissonnier L, Chapelon J, Rouvière O, Curiel L, Bouvier R, Martin X, Dubernard JM, Gelet A (2007) Control of prostate cancer by transrectal HIFU in 227 patients. Eur Urol 51: 381-387. 19. Urakami S, Igawa M, Kikuno N, Yoshino T, Kishi H, Shigeno K, Shiina H (2002) Combination chemotherapy with paclitaxel, estramustine and carboplatin for hormone refractory prostate cancer. J Urol 168: 2444-2450. 20. Walsh PC (2000) Radical prostatectomy for localized prostate cancer provides durable cancer control with excellent quality of life: a structured debate. J Urol 163: 1802-1807. 21. Han M, Partin AW, Zahurak M, Piantadosi S, Epstein JI, Walsh PC (2003) Biochemical (prostate specific antigen) recurrence probability following radical prostatectomy for clinically localized prostate cancer. J Urol 169: 517-523. 22. Amling CL, Leibovich BC, Lerner SE, Bergstralh EJ, Blute ML, Myers RP, Zincke H (1997) Primary surgical therapy for clinical stage T3 adenocarcinoma of the prostate. Semin Urol Oncol 15: 215-221. 23. Soloway MS, Pareek K, Sharifi R, Wajsman Z, McLeod D, Wood DPJ, Puras-Baez A (2002) Neoadjuvant androgen ablation before radical prostatectomy in cT2bNxMo prostate cancer: 5-year results. J Urol 167: 112-116. 24. Crook JM, Perry GA, Robertson S, Esche BA (1995) Routine prostate biopsies following radiotherapy for prostate cancer: results for 226 patients. Urology 45: 624-31; discussion 631-2. 25. Bolla M, Collette L, Blank L, Warde P, Dubois JB, Mirimanoff R, Storme G, Bernier J, Kuten A, Sternberg C, Mattelaer J, Lopez Torecilla J, Pfeffer JR, Lino Cutajar C, Zurlo A, Pierart M (2002) Long-term results with immediate androgen suppression and external irradiation in patients with locally advanced prostate cancer (an EORTC study): a phase III randomised trial. Lancet 360: 103-106. 26. Merrick GS, Butler WM, Galbreath RW, Lief JH (2001) Five-year biochemical outcome following permanent interstitial brachytherapy for clinical T1-T3 prostate cancer. Int J Radiat Oncol Biol Phys 51: 41-48. 27. Ross RW, Xie W, Regan MM, Pomerantz M, Nakabayashi M, Daskivich TJ, Sartor O, Taplin M, Kantoff PW, Oh WK (2008) Efficacy of androgen deprivation therapy (ADT) in patients with advanced prostate cancer: association between Gleason score, prostate-specific antigen level, and prior ADT exposure with duration of ADT effect. Cancer 112: 1247-1253. 28. Oh WK, Tay M, Huang J (2007) Is there a role for platinum chemotherapy in the treatment of patients with hormone-refractory prostate cancer? Cancer 109: 477-486. 29. Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ, Finegold MJ, Angelopoulou R, Rosen JM, Greenberg NM (1996) Metastatic prostate cancer in a transgenic mouse. Cancer Res 56: 4096-4102.

119

30. Majumder PK, Yeh JJ, George DJ, Febbo PG, Kum J, Xue Q, Bikoff R, Ma H, Kantoff PW, Golub TR, Loda M, Sellers WR (2003) Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: the MPAKT model. Proc Natl Acad Sci U S A 100: 7841-7846. 31. Han G, Buchanan G, Ittmann M, Harris JM, Yu X, Demayo FJ, Tilley W, Greenberg NM (2005) Mutation of the androgen receptor causes oncogenic transformation of the prostate. Proc Natl Acad Sci U S A 102: 1151-1156. 32. Thompson TC, Kadmon D, Timme TL, Merz VW, Egawa S, Krebs T, Scardino PT, Park SH (1991) Experimental oncogene induced prostate cancer. Cancer Surv 11: 55-71. 33. Thompson TC, Park SH, Timme TL, Ren C, Eastham JA, Donehower LA, Bradley A, Kadmon D, Yang G (1995) Loss of p53 function leads to metastasis in ras+myc-initiated mouse prostate cancer. Oncogene 10: 869-879. 34. Foster BA, Gingrich JR, Kwon ED, Madias C, Greenberg NM (1997) Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res 57: 3325-3330. 35. Baley PA, Yoshida K, Qian W, Sehgal I, Thompson TC (1995) Progression to androgen insensitivity in a novel in vitro mouse model for prostate cancer. J Steroid Biochem Mol Biol 52: 403-413. 36. Stone KR, Mickey DD, Wunderli H, Mickey GH, Paulson DF (1978) Isolation of a human prostate carcinoma cell line (DU 145). Int J Cancer 21: 274-281. 37. Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW (1979) Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol 17: 16-23. 38. Horoszewicz JS, Leong S S , Chu TM, Wajsman ZL, Friedman M, Papsidero L, Kim U, Chai LS,Kakati S , Arya SK, Sandberg AA (1980) The LNCaP cell line - A new model for studies on human prostatic carcinoma.. (New York: Alan R Liss,Inc., ) pp . 39. Bousso P, Bhakta NR, Lewis RS, Robey E (2002) Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science 296: 1876-1880. 40. Kwan J, Killeen N (2004) CCR7 directs the migration of thymocytes into the thymic medulla. J Immunol 172: 3999-4007. 41. Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, von Boehmer H, Bronson R, Dierich A, Benoist C, Mathis D (2002) Projection of an immunological self shadow within the thymus by the aire protein. Science 298: 1395-1401. 42. Aaltonen J, Bjorses P, Perheentupa J, Horelli-Kuitunen N, Palotie A, Peltonen L, Lee YS, Francis F, HenningSteffen, Thiel C, Leharach H, Yaspo M (1997) An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains Nat Genet 17: 399-403. 43. Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, Heino M, Krohn KJ, Lalioti MD, Mullis PE, Antonarakis SE, Kawasaki K, Asakawa S, Ito F, Shimizu N (1997) Positional cloning of the APECED gene. Nat Genet 17: 393-398. 44. Akiyama T, Maeda S, Yamane S, Ogino K, Kasai M, Kajiura F, Matsumoto M, Inoue J (2005) Dependence of self-tolerance on TRAF6-directed development of thymic stroma. Science 308: 248-251.

120

45. Boehm T, Scheu S, Pfeffer K, Bleul CC (2003) Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho-epithelial cross talk via LTbetaR. J Exp Med 198: 757-769. 46. Kajiura F, Sun S, Nomura T, Izumi K, Ueno T, Bando Y, Kuroda N, Han H, Li Y, Matsushima A, Takahama Y, Sakaguchi S, Mitani T, Matsumoto M (2004) NF-kappa B-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner. J Immunol 172: 2067-2075. 47. He L, Wu X, Siegel R, Lipsky PE (2006) TRAF6 regulates cell fate decisions by inducing caspase 8-dependent apoptosis and the activation of NF-kappaB. J Biol Chem 281: 11235-11249. 48. Yin L, Wu L, Wesche H, Arthur CD, White JM, Goeddel DV, Schreiber RD (2001) Defective lymphotoxin-beta receptor-induced NF-kappaB transcriptional activity in NIK-deficient mice. Science 291: 2162-2165. 49. Heino M, Peterson P, Sillanpää N, Guérin S, Wu L, Anderson G, Scott HS, Antonarakis SE, Kudoh J, Shimizu N, Jenkinson EJ, Naquet P, Krohn KJ (2000) RNA and protein expression of the murine autoimmune regulator gene (Aire) in normal, RelB-deficient and in NOD mouse. Eur J Immunol 30: 1884-1893. 50. Zuklys S, Balciunaite G, Agarwal A, Fasler-Kan E, Palmer E, Holländer GA (2000) Normal thymic architecture and negative selection are associated with Aire expression, the gene defective in the autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). J Immunol 165: 1976-1983. 51. Weih F, Carrasco D, Durham SK, Barton DS, Rizzo CA, Ryseck RP, Lira SA, Bravo R (1995) Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family. Cell 80: 331-340. 52. Gallegos AM, Bevan MJ (2004) Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation. J Exp Med 200: 1039-1049. 53. Buhlmann JE, Elkin SK, Sharpe AH (2003) A role for the B7-1/B7-2:CD28/CTLA-4 pathway during negative selection. J Immunol 170: 5421-5428. 54. Gao J, Zhang H, Bai X, Wen J, Zheng X, Liu J, Zheng P, Liu Y (2002) Perinatal blockade of b7-1 and b7-2 inhibits clonal deletion of highly pathogenic autoreactive T cells. J Exp Med 195: 959-971. 55. Nishikawa H, Kato T, Tanida K, Hiasa A, Tawara I, Ikeda H, Ikarashi Y, Wakasugi H, Kronenberg M, Nakayama T, Taniguchi M, Kuribayashi K, Old LJ, Shiku H (2003) CD4+ CD25+ T cells responding to serologically defined autoantigens suppress antitumor immune responses. Proc Natl Acad Sci U S A 100: 10902-10906. 56. Reddy J, Illes Z, Zhang X, Encinas J, Pyrdol J, Nicholson L, Sobel RA, Wucherpfennig KW, Kuchroo VK (2004) Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A 101: 15434-15439. 57. Fontenot JD, Rudensky AY (2005) A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat Immunol 6: 331-337. 58. Danke NA, Koelle DM, Yee C, Beheray S, Kwok WW (2004) Autoreactive T cells in healthy individuals. J Immunol 172: 5967-5972.

121

59. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi M, Bluestone JA (2004) In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 199: 1455-1465. 60. Tarbell KV, Yamazaki S, Olson K, Toy P, Steinman RM (2004) CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J Exp Med 199: 1467-1477. 61. Bell SE, Goodnow CC (1994) A selective defect in IgM antigen receptor synthesis and transport causes loss of cell surface IgM expression on tolerant B lymphocytes. EMBO J 13: 816-826. 62. Goodnow CC, Crosbie J, Jorgensen H, Brink RA, Basten A (1989) Induction of self-tolerance in mature peripheral B lymphocytes. Nature 342: 385-391. 63. Cyster JG, Goodnow CC (1995) Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling in B lymphocytes and determines thresholds for negative selection. Immunity 2: 13-24. 64. Ravetch JV, Lanier LL (2000) Immune inhibitory receptors. Science 290: 84-89. 65. Hippen KL, Tze LE, Behrens TW (2000) CD5 maintains tolerance in anergic B cells. J Exp Med 191: 883-890. 66. Smith K, Seddon B, Purbhoo MA, Zamoyska R, Fisher AG, Merkenschlager M (2001) Sensory adaptation in naive peripheral CD4 T cells. J Exp Med 194: 1253-1261. 67. Wong P, Barton GM, Forbush KA, Rudensky AY (2001) Dynamic tuning of T cell reactivity by self-peptide-major histocompatibility complex ligands. J Exp Med 193: 1179-1187. 68. D'Ambrosio D, Hippen KL, Minskoff SA, Mellman I, Pani G, Siminovitch KA, Cambier JC (1995) Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by Fc gamma RIIB1. Science 268: 293-297. 69. Takai T, Ono M, Hikida M, Ohmori H, Ravetch JV (1996) Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice. Nature 379: 346-349. 70. Thien M, Phan TG, Gardam S, Amesbury M, Basten A, Mackay F, Brink R (2004) Excess BAFF rescues self-reactive B cells from peripheral deletion and allows them to enter forbidden follicular and marginal zone niches. Immunity 20: 785-798. 71. Lesley R, Xu Y, Kalled SL, Hess DM, Schwab SR, Shu H, Cyster JG (2004) Reduced competitiveness of autoantigen-engaged B cells due to increased dependence on BAFF. Immunity 20: 441-453. 72. Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD (2002) Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 3: 991-998. 73. So T, Takenoyama M, Mizukami M, Ichiki Y, Sugaya M, Hanagiri T, Sugio K, Yasumoto K (2005) Haplotype loss of HLA class I antigen as an escape mechanism from immune attack in lung cancer. Cancer Res 65: 5945-5952. 74. Atkins D, Breuckmann A, Schmahl GE, Binner P, Ferrone S, Krummenauer F, Störkel S, Seliger B (2004) MHC class I antigen processing pathway defects, ras mutations and disease stage in colorectal carcinoma. Int J Cancer 109: 265-273. 75. Mehta AM, Jordanova ES, Kenter GG, Ferrone S, Fleuren G (2008) Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in cervical carcinoma. Cancer Immunol Immunother 57: 197-206. 76. Bladergroen BA, Meijer CJLM, ten Berge RL, Hack CE, Muris JJF, Dukers DF, Chott A, Kazama Y, Oudejans JJ, van Berkum O, Kummer JA (2002) Expression of the

122

granzyme B inhibitor, protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system? Blood 99: 232-237. 77. Medema JP, de Jong J, Peltenburg LT, Verdegaal EM, Gorter A, Bres SA, Franken KL, Hahne M, Albar JP, Melief CJ, Offringa R (2001) Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors. Proc Natl Acad Sci U S A 98: 11515-11520. 78. Strand S, Hofmann WJ, Hug H, Müller M, Otto G, Strand D, Mariani SM, Stremmel W, Krammer PH, Galle PR (1996) Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells--a mechanism of immune evasion? Nat Med 2: 1361-1366. 79. Panka DJ, Mano T, Suhara T, Walsh K, Mier JW (2001) Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells. J Biol Chem 276: 6893-6896. 80. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, Boon T, Van den Eynde BJ (2003) Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9: 1269-1274. 81. Terness P, Bauer TM, Röse L, Dufter C, Watzlik A, Simon H, Opelz G (2002) Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 196: 447-457. 82. Harizi H, Juzan M, Pitard V, Moreau J, Gualde N (2002) Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions. J Immunol 168: 2255-2263. 83. Singh S, Ross SR, Acena M, Rowley DA, Schreiber H (1992) Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells. J Exp Med 175: 139-146. 84. Yeon CH, Pegram MD (2005) Anti-erbB-2 antibody trastuzumab in the treatment of HER2-amplified breast cancer. Invest New Drugs 23: 391-409. 85. Albanell J, Codony J, Rovira A, Mellado B, Gascón P (2003) Mechanism of action of anti-HER2 monoclonal antibodies: scientific update on trastuzumab and 2C4. Adv Exp Med Biol 532: 253-268. 86. Maker AV, Yang JC, Sherry RM, Topalian SL, Kammula US, Royal RE, Hughes M, Yellin MJ, Haworth LR, Levy C, Allen T, Mavroukakis SA, Attia P, Rosenberg SA (2006) Intrapatient dose escalation of anti-CTLA-4 antibody in patients with metastatic melanoma. J Immunother 29: 455-463. 87. Small EJ, Tchekmedyian NS, Rini BI, Fong L, Lowy I, Allison JP (2007) A pilot trial of CTLA-4 blockade with human anti-CTLA-4 in patients with hormone-refractory prostate cancer. Clin Cancer Res 13: 1810-1815. 88. Grimm EA, Ramsey KM, Mazumder A, Wilson DJ, Djeu JY, Rosenberg SA (1983) Lymphokine-activated killer cell phenomenon. II. Precursor phenotype is serologically distinct from peripheral T lymphocytes, memory cytotoxic thymus-derived lymphocytes, and natural killer cells. J Exp Med 157: 884-897. 89. Grimm EA, Robb RJ, Roth JA, Neckers LM, Lachman LB, Wilson DJ, Rosenberg SA (1983) Lymphokine-activated killer cell phenomenon. III. Evidence that

123

IL-2 is sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer cells. J Exp Med 158: 1356-1361. 90. Rosenberg SA (1986) Adoptive immunotherapy of cancer using lymphokine activated killer cells and recombinant interleukin-2. (Philadelphia, JB Lippincott, ) pp . 91. Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, et al. (1987) A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 316: 889-897. 92. Muul LM, Spiess PJ, Director EP, Rosenberg SA (1987) Identification of specific cytolytic immune responses against autologous tumor in humans bearing malignant melanoma. J Immunol 138: 989-995. 93. Tomita S, Lotze MT, Rosenberg SA (1987) Clonal analysis of tumor infiltrating lymphocytes against human malignant melanoma. Fed Proc 46: 1195. 94. Rosenberg SA, Spiess P, Lafreniere R (1986) A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233: 1318-1321. 95. Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, Simon P, Lotze MT, Yang JC, Seipp CA, et al. (1988) Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med 319: 1676-1680. 96. Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME (2008) Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 8: 299-308. 97. Livingston PO, Watanabe T, Shiku H, Houghton AN, Albino A, Takahashi T, Resnick LA, Michitsch R, Pinsky CM, Oettgen HF, Old LJ (1982) Serological response of melanoma patients receiving melanoma cell vaccines. I. Autologous cultured melanoma cells. Int J Cancer 30: 413-422. 98. Livingston PO, Albino AP, Chung TJ, Real FX, Houghton AN, Oettgen HF, Old LJ (1985) Serological response of melanoma patients to vaccines prepared from VSV lysates of autologous and allogeneic cultured melanoma cells. Cancer 55: 713-720. 99. Atanackovic D, Altorki NK, Stockert E, Williamson B, Jungbluth AA, Ritter E, Santiago D, Ferrara CA, Matsuo M, Selvakumar A, Dupont B, Chen Y, Hoffman EW, Ritter G, Old LJ, Gnjatic S (2004) Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients. J Immunol 172: 3289-3296. 100. Disis ML, Grabstein KH, Sleath PR, Cheever MA (1999) Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res 5: 1289-1297. 101. Abe A, Emi N, Taji H, Kasai M, Kohno A, Saito H (1996) Induction of humoral and cellular anti-idiotypic immunity by intradermal injection of naked DNA encoding a human variable region gene sequence of an immunoglobulin heavy chain in a B cell malignancy. Gene Ther 3: 988-993. 102. Borysiewicz LK, Fiander A, Nimako M, Man S, Wilkinson GW, Westmoreland D, Evans AS, Adams M, Stacey SN, Boursnell ME, Rutherford E, Hickling JK, Inglis SC (1996) A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 347: 1523-1527. 103. Steinman RM (2007) Dendritic cells: understanding immunogenicity. Eur J Immunol 37 Suppl 1: S53-60.

124

104. Wu L, Li CL, Shortman K (1996) Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J Exp Med 184: 903-911. 105. Leenen PJ, Radosević K, Voerman JS, Salomon B, van Rooijen N, Klatzmann D, van Ewijk W (1998) Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover. J Immunol 160: 2166-2173. 106. Pulendran B, Lingappa J, Kennedy MK, Smith J, Teepe M, Rudensky A, Maliszewski CR, Maraskovsky E (1997) Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol 159: 2222-2231. 107. Inaba K, Inaba M, Deguchi M, Hagi K, Yasumizu R, Ikehara S, Muramatsu S, Steinman RM (1993) Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow. Proc Natl Acad Sci U S A 90: 3038-3042. 108. Vremec D, Zorbas M, Scollay R, Saunders DJ, Ardavin CF, Wu L, Shortman K (1992) The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med 176: 47-58. 109. Sallusto F, Cella M, Danieli C, Lanzavecchia A (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 182: 389-400. 110. Inaba K, Witmer-Pack M, Inaba M, Hathcock KS, Sakuta H, Azuma M, Yagita H, Okumura K, Linsley PS, Ikehara S, Muramatsu S, Hodes RJ, Steinman RM (1994) The tissue distribution of the B7-2 costimulator in mice: abundant expression on dendritic cells in situ and during maturation in vitro. J Exp Med 180: 1849-1860. 111. Larsen CP, Ritchie SC, Pearson TC, Linsley PS, Lowry RP (1992) Functional expression of the costimulatory molecule, B7/BB1, on murine dendritic cell populations. J Exp Med 176: 1215-1220. 112. Schweitzer AN, Sharpe AH (1998) Studies using antigen-presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production. J Immunol 161: 2762-2771. 113. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088. 114. Inaba K, Turley S, Iyoda T, Yamaide F, Shimoyama S, Reis e Sousa C, Germain RN, Mellman I, Steinman RM (2000) The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J Exp Med 191: 927-936. 115. Austyn JM, Kupiec-Weglinski JW, Hankins DF, Morris PJ (1988) Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone. J Exp Med 167: 646-651.

125

116. Larsen CP, Steinman RM, Witmer-Pack M, Hankins DF, Morris PJ, Austyn JM (1990) Migration and maturation of Langerhans cells in skin transplants and explants. J Exp Med 172: 1483-1493. 117. Ingulli E, Mondino A, Khoruts A, Jenkins MK (1997) In vivo detection of dendritic cell antigen presentation to CD4(+) T cells. J Exp Med 185: 2133-2141. 118. Unternaehrer JJ, Chow A, Pypaert M, Inaba K, Mellman I (2007) The tetraspanin CD9 mediates lateral association of MHC class II molecules on the dendritic cell surface. Proc Natl Acad Sci U S A 104: 234-239. 119. Hosken NA, Shibuya K, Heath AW, Murphy KM, O'Garra A (1995) The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model. J Exp Med 182: 1579-1584. 120. Rogers PR, Croft M (2000) CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen. J Immunol 164: 2955-2963. 121. Menon JN, Bretscher PA (1998) Parasite dose determines the Th1/Th2 nature of the response to Leishmania major independently of infection route and strain of host or parasite. Eur J Immunol 28: 4020-4028. 122. Power CA, Wei G, Bretscher PA (1998) Mycobacterial dose defines the Th1/Th2 nature of the immune response independently of whether immunization is administered by the intravenous, subcutaneous, or intradermal route. Infect Immun 66: 5743-5750. 123. Rogers PR, Dubey C, Swain SL (2000) Qualitative changes accompany memory T cell generation: faster, more effective responses at lower doses of antigen. J Immunol 164: 2338-2346. 124. Bates EE, Ravel O, Dieu MC, Ho S, Guret C, Bridon JM, Ait-Yahia S, Brière F, Caux C, Banchereau J, Lebecque S (1997) Identification and analysis of a novel member of the ubiquitin family expressed in dendritic cells and mature B cells. Eur J Immunol 27: 2471-2477. 125. Norbury CC, Chambers BJ, Prescott AR, Ljunggren HG, Watts C (1997) Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells. Eur J Immunol 27: 280-288. 126. Bevan MJ (1976) Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J Exp Med 143: 1283-1288. 127. Kovacsovics-Bankowski M, Clark K, Benacerraf B, Rock KL (1993) Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. Proc Natl Acad Sci U S A 90: 4942-4946. 128. Kovacsovics-Bankowski M, Rock KL (1995) A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267: 243-246. 129. Pfeifer JD, Wick MJ, Roberts RL, Findlay K, Normark SJ, Harding CV (1993) Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature 361: 359-362. 130. Inaba K, Turley S, Yamaide F, Iyoda T, Mahnke K, Inaba M, Pack M, Subklewe M, Sauter B, Sheff D, Albert M, Bhardwaj N, Mellman I, Steinman RM (1998) Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J Exp Med 188: 2163-2173.

126

131. Cresswell P (1996) Invariant chain structure and MHC class II function. Cell 84: 505-507. 132. Kleijmeer MJ, Ossevoort MA, van Veen CJ, van Hellemond JJ, Neefjes JJ, Kast WM, Melief CJ, Geuze HJ (1995) MHC class II compartments and the kinetics of antigen presentation in activated mouse spleen dendritic cells. J Immunol 154: 5715-5724. 133. Pierre P, Mellman I (1998) Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93: 1135-1145. 134. Bachmann MF, Sebzda E, Kündig TM, Shahinian A, Speiser DE, Mak TW, Ohashi PS (1996) T cell responses are governed by avidity and co-stimulatory thresholds. Eur J Immunol 26: 2017-2022. 135. Boise LH, Minn AJ, Noel PJ, June CH, Accavitti MA, Lindsten T, Thompson CB (1995) CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity 3: 87-98. 136. Curtsinger JM, Schmidt CS, Mondino A, Lins DC, Kedl RM, Jenkins MK, Mescher MF (1999) Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells. J Immunol 162: 3256-3262. 137. Tu L, Fang TC, Artis D, Shestova O, Pross SE, Maillard I, Pear WS (2005) Notch signaling is an important regulator of type 2 immunity. J Exp Med 202: 1037-1042. 138. Amsen D, Antov A, Jankovic D, Sher A, Radtke F, Souabni A, Busslinger M, McCright B, Gridley T, Flavell RA (2007) Direct regulation of Gata3 expression determines the T helper differentiation potential of Notch. Immunity 27: 89-99. 139. Bennett SR, Carbone FR, Karamalis F, Miller JF, Heath WR (1997) Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help. J Exp Med 186: 65-70. 140. Keene JA, Forman J (1982) Helper activity is required for the in vivo generation of cytotoxic T lymphocytes. J Exp Med 155: 768-782. 141. Wang JE, Livingstone AM (2003) Cutting edge: CD4+ T cell help can be essential for primary CD8+ T cell responses in vivo. J Immunol 171: 6339-6343. 142. Batista FD, Iber D, Neuberger MS (2001) B cells acquire antigen from target cells after synapse formation. Nature 411: 489-494. 143. Lanzavecchia A (1985) Antigen-specific interaction between T and B cells. Nature 314: 537-539. 144. Rudge EU, Cutler AJ, Pritchard NR, Smith KGC (2002) Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc gamma RII-mediated B cell suppression. J Exp Med 195: 1079-1085. 145. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136: 2348-2357. 146. Porgador A, Gilboa E (1995) Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes. J Exp Med 182: 255-260. 147. Tjoa B, Erickson S, Barren R3, Ragde H, Kenny G, Boynton A, Murphy G (1995) In vitro propagated dendritic cells from prostate cancer patients as a component of prostate cancer immunotherapy. Prostate 27: 63-69. 148. Vari F, Hart DNJ (2004) Loading DCs with Ag. Cytotherapy 6: 111-121.

127

149. Gjertsen MK, Bakka A, Breivik J, Saeterdal I, Gedde-Dahl,, T 3rd, Stokke KT, Sølheim BG, Egge TS, Søreide O, Thorsby E, Gaudernack G (1996) Ex vivo ras peptide vaccination in patients with advanced pancreatic cancer: results of a phase I/II study. Int J Cancer 65: 450-453. 150. Lesterhuis WJ, de Vries IJM, Schuurhuis DH, Boullart ACI, Jacobs JFM, de Boer AJ, Scharenborg NM, Brouwer HMH, van de Rakt MWMM, Figdor CG, Ruers TJ, Adema GJ, Punt CJA (2006) Vaccination of colorectal cancer patients with CEA-loaded dendritic cells: antigen-specific T cell responses in DTH skin tests. Ann Oncol 17: 974-980. 151. Liu K, Wang C, Chen L, Cheng A, Lin D, Wu Y, Yu W, Hung Y, Yang H, Juang S, Whang-Peng J (2004) Generation of carcinoembryonic antigen (CEA)-specific T-cell responses in HLA-A*0201 and HLA-A*2402 late-stage colorectal cancer patients after vaccination with dendritic cells loaded with CEA peptides. Clin Cancer Res 10: 2645-2651. 152. Banchereau J, Ueno H, Dhodapkar M, Connolly J, Finholt JP, Klechevsky E, Blanck J, Johnston DA, Palucka AK, Fay J (2005) Immune and clinical outcomes in patients with stage IV melanoma vaccinated with peptide-pulsed dendritic cells derived from CD34+ progenitors and activated with type I interferon. J Immunother (1997) 28: 505-516. 153. Lee W, Wang H, Hung C, Huang P, Lia C, Chen M (2005) Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial. J Immunother (1997) 28: 496-504. 154. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG (2000) New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92: 205-216. 155. Boczkowski D, Nair SK, Snyder D, Gilboa E (1996) Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med 184: 465-472. 156. Rouse RJ, Nair SK, Lydy SL, Bowen JC, Rouse BT (1994) Induction in vitro of primary cytotoxic T-lymphocyte responses with DNA encoding herpes simplex virus proteins. J Virol 68: 5685-5689. 157. Zhou Y, Bosch ML, Salgaller ML (2002) Current methods for loading dendritic cells with tumor antigen for the induction of antitumor immunity. J Immunother (1997) 25: 289-303. 158. Markiewicz MA, Kast WM (2004) Progress in the development of immunotherapy of cancer using ex vivo-generated dendritic cells expressing multiple tumor antigen epitopes. Cancer Invest 22: 417-434. 159. Strobel I, Berchtold S, Götze 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: 2028-2035. 160. Mincheff M, Zoubak S, Altankova I, Tchakarov S, Makogonenko Y, Botev C, Ignatova I, Dimitrov R, Madarzhieva K, Hammett M, Pomakov Y, Meryman H, Lissitchkov T (2003) Human dendritic cells genetically engineered to express

128

cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes. Cancer Gene Ther 10: 907-917. 161. O-Sullivan I, Ng LK, Martinez DM, Kim TS, Chopra A, Cohen EP (2005) Immunity to squamous carcinoma in mice immunized with dendritic cells transfected with genomic DNA from squamous carcinoma cells. Cancer Gene Ther 12: 825-834. 162. Rains N, Cannan RJ, Chen W, Stubbs RS (2001) Development of a dendritic cell (DC)-based vaccine for patients with advanced colorectal cancer. Hepatogastroenterology 48: 347-351. 163. Gilboa E, Vieweg J (2004) Cancer immunotherapy with mRNA-transfected dendritic cells. Immunol Rev 199: 251-263. 164. Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas CD, Dahm P, Niedzwiecki D, Gilboa E, Vieweg J (2002) Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest 109: 409-417. 165. Edelstein ML, Abedi MR, Wixon J (2007) Gene therapy clinical trials worldwide to 2007--an update. J Gene Med 9: 833-842. 166. Kikuchi T (2006) Genetically modified dendritic cells for therapeutic immunity. Tohoku J Exp Med 208: 1-8. 167. Molinier-Frenkel V, Prévost-Blondel A, Hong S, Lengagne R, Boudaly S, Magnusson MK, Boulanger P, Guillet J (2003) The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain. J Biol Chem 278: 37175-37182. 168. Tillman BW, de Gruijl TD, Luykx-de Bakker SA, Scheper RJ, Pinedo HM, Curiel TJ, Gerritsen WR, Curiel DT (1999) Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector. J Immunol 162: 6378-6383. 169. Shayakhmetov DM, Lieber A (2000) Dependence of adenovirus infectivity on length of the fiber shaft domain. J Virol 74: 10274-10286. 170. Sili U, Huls MH, Davis AR, Gottschalk S, Brenner MK, Heslop HE, Rooney CM (2003) Large-scale expansion of dendritic cell-primed polyclonal human cytotoxic T-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy. J Immunother (1997) 26: 241-256. 171. Pastoret P, Vanderplasschen A (2003) Poxviruses as vaccine vectors. Comp Immunol Microbiol Infect Dis 26: 343-355. 172. Drillien R, Spehner D, Bohbot A, Hanau D (2000) Vaccinia virus-related events and phenotypic changes after infection of dendritic cells derived from human monocytes. Virology 268: 471-481. 173. Essajee S, Kaufman HL (2004) Poxvirus vaccines for cancer and HIV therapy. Expert Opin Biol Ther 4: 575-588. 174. Chen Q, He Y, Yang K (2005) Gene therapy for Parkinson's disease: progress and challenges. Curr Gene Ther 5: 71-80. 175. Klink D, Schindelhauer D, Laner A, Tucker T, Bebok Z, Schwiebert EM, Boyd AC, Scholte BJ (2004) Gene delivery systems--gene therapy vectors for cystic fibrosis. J Cyst Fibros 3 Suppl 2: 203-212. 176. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15: 871-875.

129

177. Iwakuma T, Cui Y, Chang LJ (1999) Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 261: 120-132. 178. Miyoshi H, Blömer U, Takahashi M, Gage FH, Verma IM (1998) Development of a self-inactivating lentivirus vector. J Virol 72: 8150-8157. 179. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72: 9873-9880. 180. Lizée G, Aerts JL, Gonzales MI, Chinnasamy N, Morgan RA, Topalian SL (2003) Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther 14: 497-507. 181. Sastry L, Johnson T, Hobson MJ, Smucker B, Cornetta K (2002) Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther 9: 1155-1162. 182. Steitz J, Tormo D, Schweichel D, Tüting T (2006) Comparison of recombinant adenovirus and synthetic peptide for DC-based melanoma vaccination. Cancer Gene Ther 13: 318-325. 183. Broder H, Anderson A, Kremen TJ, Odesa SK, Liau LM (2003) MART-1 adenovirus-transduced dendritic cell immunization in a murine model of metastatic central nervous system tumor. J Neurooncol 64: 21-30. 184. Walker PR, Calzascia T, de Tribolet N, Dietrich PY (2003) T-cell immune responses in the brain and their relevance for cerebral malignancies. Brain Res Brain Res Rev 42: 97-122. 185. Okada N, Masunaga Y, Okada Y, Mizuguchi H, Iiyama S, Mori N, Sasaki A, Nakagawa S, Mayumi T, Hayakawa T, Fujita T, Yamamoto A (2003) Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice. Gene Ther 10: 1891-1902. 186. Sakai Y, Morrison BJ, Burke JD, Park J, Terabe M, Janik JE, Forni G, Berzofsky JA, Morris JC (2004) Vaccination by genetically modified dendritic cells expressing a truncated neu oncogene prevents development of breast cancer in transgenic mice. Cancer Res 64: 8022-8028. 187. Murakami T, Tokunaga N, Waku T, Gomi S, Kagawa S, Tanaka N, Fujiwara T (2004) Antitumor effect of intratumoral administration of bone marrow-derived dendritic cells transduced with wild-type p53 gene. Clin Cancer Res 10: 3871-3880. 188. Tatsumi T, Huang J, Gooding WE, Gambotto A, Robbins PD, Vujanovic NL, Alber SM, Watkins SC, Okada H, Storkus WJ (2003) Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity. Cancer Res 63: 6378-6386. 189. Montoya M, Schiavoni G, Mattei F, Gresser I, Belardelli F, Borrow P, Tough DF (2002) Type I interferons produced by dendritic cells promote their phenotypic and functional activation. Blood 99: 3263-3271. 190. Kuwashima N, Nishimura F, Eguchi J, Sato H, Hatano M, Tsugawa T, Sakaida T, Dusak JE, Fellows-Mayle WK, Papworth GD, Watkins SC, Gambotto A, Pollack IF, Storkus WJ, Okada H (2005) Delivery of dendritic cells engineered to secrete IFN-alpha

130

into central nervous system tumors enhances the efficacy of peripheral tumor cell vaccines: dependence on apoptotic pathways. J Immunol 175: 2730-2740. 191. Medin JA, Liang S, Hou JW, Kelley LS, Peace DJ, Fowler DH (2005) Efficient transfer of PSA and PSMA cDNAs into DCs generates antibody and T cell antitumor responses in vivo. Cancer Gene Ther 12: 540-551. 192. Gruber A, Kan-Mitchell J, Kuhen KL, Mukai T, Wong-Staal F (2000) Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro. Blood 96: 1327-1333. 193. Tan PH, Beutelspacher SC, Xue S, Wang Y, Mitchell P, McAlister JC, Larkin DFP, McClure MO, Stauss HJ, Ritter MA, Lombardi G, George AJT (2005) Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy. Blood 105: 3824-3832. 194. He Y, Zhang J, Mi Z, Robbins P, Falo LDJ (2005) Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity. J Immunol 174: 3808-3817. 195. Metharom P, Ellem KA, Schmidt C, Wei MQ (2001) Lentiviral vector-mediated tyrosinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma. Hum Gene Ther 12: 2203-2213. 196. Cui Y, Kelleher E, Straley E, Fuchs E, Gorski K, Levitsky H, Borrello I, Civin CI, Schoenberger SP, Cheng L, Pardoll DM, Whartenby KA (2003) Immunotherapy of established tumors using bone marrow transplantation with antigen gene--modified hematopoietic stem cells. Nat Med 9: 952-958. 197. Zhang X, Zhao P, Kennedy C, Chen K, Wiegand J, Washington G, Marrero L, Cui Y (2008) Treatment of pulmonary metastatic tumors in mice using lentiviral vector-engineered stem cells. Cancer Gene Ther 15: 73-84. 198. Shen L, Evel-Kabler K, Strube R, Chen S (2004) Silencing of SOCS1 enhances antigen presentation by dendritic cells and antigen-specific anti-tumor immunity. Nat Biotechnol 22: 1546-1553. 199. Antonia SJ, Mirza N, Fricke I, Chiappori A, Thompson P, Williams N, Bepler G, Simon G, Janssen W, Lee J, Menander K, Chada S, Gabrilovich DI (2006) Combination of p53 cancer vaccine with chemotherapy in patients with extensive stage small cell lung cancer. Clin Cancer Res 12: 878-887. 200. Di Nicola M, Carlo-Stella C, Mortarini R, Baldassari P, Guidetti A, Gallino GF, Del Vecchio M, Ravagnani F, Magni M, Chaplin P, Cascinelli N, Parmiani G, Gianni AM, Anichini A (2004) Boosting T cell-mediated immunity to tyrosinase by vaccinia virus-transduced, CD34(+)-derived dendritic cell vaccination: a phase I trial in metastatic melanoma. Clin Cancer Res 10: 5381-5390. 201. Morse MA, Clay TM, Hobeika AC, Osada T, Khan S, Chui S, Niedzwiecki D, Panicali D, Schlom J, Lyerly HK (2005) Phase I study of immunization with dendritic cells modified with fowlpox encoding carcinoembryonic antigen and costimulatory molecules. Clin Cancer Res 11: 3017-3024. 202. Rosenberg SA, Yang JC, Restifo NP (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med 10: 909-915. 203. Casalini P, Iorio MV, Galmozzi E, Ménard S (2004) Role of HER receptors family in development and differentiation. J Cell Physiol 200: 343-350.

131

204. Shih C, Padhy LC, Murray M, Weinberg RA (1981) Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature 290: 261-264. 205. Tzahar E, Waterman H, Chen X, Levkowitz G, Karunagaran D, Lavi S, Ratzkin BJ, Yarden Y (1996) A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 16: 5276-5287. 206. Hsieh AC, Moasser MM (2007) Targeting HER proteins in cancer therapy and the role of the non-target HER3. Br J Cancer 97: 453-457. 207. Vijapurkar U, Kim M, Koland JG (2003) Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling. Exp Cell Res 284: 291-302. 208. Iwamoto R, Mekada E (2006) ErbB and HB-EGF signaling in heart development and function. Cell Struct Funct 31: 1-14. 209. Jones FE, Stern DF (1999) Expression of dominant-negative ErbB2 in the mammary gland of transgenic mice reveals a role in lobuloalveolar development and lactation. Oncogene 18: 3481-3490. 210. Lee KF, Simon H, Chen H, Bates B, Hung MC, Hauser C (1995) Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378: 394-398. 211. Ozcelik C, Erdmann B, Pilz B, Wettschureck N, Britsch S, Hübner N, Chien KR, Birchmeier C, Garratt AN (2002) Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy. Proc Natl Acad Sci U S A 99: 8880-8885. 212. Crone SA, Negro A, Trumpp A, Giovannini M, Lee K (2003) Colonic epithelial expression of ErbB2 is required for postnatal maintenance of the enteric nervous system. Neuron 37: 29-40. 213. Osman I, Scher HI, Drobnjak M, Verbel D, Morris M, Agus D, Ross JS, Cordon-Cardo C (2001) HER-2/neu (p185neu) protein expression in the natural or treated history of prostate cancer. Clin Cancer Res 7: 2643-2647. 214. Iglehart JD, Kraus MH, Langton BC, Huper G, Kerns BJ, Marks JR (1990) Increased erbB-2 gene copies and expression in multiple stages of breast cancer. Cancer Res 50: 6701-6707. 215. Ito K, Sasano H, Ozawa N, Sato S, Silverberg SG, Yajima A (1992) Immunolocalization of epidermal growth factor receptor and c-erbB-2 oncogene product in human ovarian carcinoma. Int J Gynecol Pathol 11: 253-257. 216. Shi D, He G, Cao S, Pan W, Zhang HZ, Yu D, Hung MC (1992) Overexpression of the c-erbB-2/neu-encoded p185 protein in primary lung cancer. Mol Carcinog 5: 213-218. 217. Hung MC, Lau YK (1999) Basic science of HER-2/neu: a review. Semin Oncol 26: 51-59. 218. Paik S, Bryant J, Tan-Chiu E, Yothers G, Park C, Wickerham DL, Wolmark N (2000) HER2 and choice of adjuvant chemotherapy for invasive breast cancer: National Surgical Adjuvant Breast and Bowel Project Protocol B-15. J Natl Cancer Inst 92: 1991-1998. 219. Zhou BP, Hu MC, Miller SA, Yu Z, Xia W, Lin SY, Hung MC (2000) HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway. J Biol Chem 275: 8027-8031.

132

220. Press MF, Pike MC, Chazin VR, Hung G, Udove JA, Markowicz M, Danyluk J, Godolphin W, Sliwkowski M, Akita R, et al. (1993) Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease. Cancer Res 53: 4960-4970. 221. Seshadri R, Firgaira FA, Horsfall DJ, McCaul K, Setlur V, Kitchen P (1993) Clinical significance of HER-2/neu oncogene amplification in primary breast cancer. The South Australian Breast Cancer Study Group. J Clin Oncol 11: 1936-1942. 222. Wang SE, Narasanna A, Perez-Torres M, Xiang B, Wu FY, Yang S, Carpenter G, Gazdar AF, Muthuswamy SK, Arteaga CL (2006) HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell 10: 25-38. 223. McKeage K, Perry CM (2002) Trastuzumab: a review of its use in the treatment of metastatic breast cancer overexpressing HER2. Drugs 62: 209-243. 224. Tokunaga E, Kimura Y, Mashino K, Oki E, Kataoka A, Ohno S, Morita M, Kakeji Y, Baba H, Maehara Y (2006) Activation of PI3K/Akt signaling and hormone resistance in breast cancer. Breast Cancer 13: 137-144. 225. Austin CD, De Mazière AM, Pisacane PI, van Dijk SM, Eigenbrot C, Sliwkowski MX, Klumperman J, Scheller RH (2004) Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol Biol Cell 15: 5268-5282. 226. Delord JP, Allal C, Canal M, Mery E, Rochaix P, Hennebelle I, Pradines A, Chatelut E, Bugat R, Guichard S, Canal P (2005) Selective inhibition of HER2 inhibits AKT signal transduction and prolongs disease-free survival in a micrometastasis model of ovarian carcinoma. Ann Oncol 16: 1889-1897. 227. Lane HA, Motoyama AB, Beuvink I, Hynes NE (2001) Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling. Ann Oncol 12 Suppl 1: S21-2. 228. Ziada A, Barqawi A, Glode LM, Varella-Garcia M, Crighton F, Majeski S, Rosenblum M, Kane M, Chen L, Crawford ED (2004) The use of trastuzumab in the treatment of hormone refractory prostate cancer; phase II trial. Prostate 60: 332-337. 229. Valabrega G, Montemurro F, Aglietta M (2007) Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol 18: 977-984. 230. Moasser MM (2007) Targeting the function of the HER2 oncogene in human cancer therapeutics. Oncogene 26: 6577-6592. 231. Spector NL, Xia W, Burris H3, Hurwitz H, Dees EC, Dowlati A, O'Neil B, Overmoyer B, Marcom PK, Blackwell KL, Smith DA, Koch KM, Stead A, Mangum S, Ellis MJ, Liu L, Man AK, Bremer TM, Harris J, Bacus S (2005) Study of the biologic effects of lapatinib, a reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, on tumor growth and survival pathways in patients with advanced malignancies. J Clin Oncol 23: 2502-2512. 232. D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G (1993) Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med 178: 1041-1048.

133

233. Du C, Sriram S (1998) Mechanism of inhibition of LPS-induced IL-12p40 production by IL-10 and TGF-beta in ANA-1 cells. J Leukoc Biol 64: 92-97. 234. Schulz O, Edwards AD, Schito M, Aliberti J, Manickasingham S, Sher A, Reis e Sousa C (2000) CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 13: 453-462. 235. Bacon CM, McVicar DW, Ortaldo JR, Rees RC, O'Shea JJ, Johnston JA (1995) Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12. J Exp Med 181: 399-404. 236. Bacon CM, Petricoin EF3, Ortaldo JR, Rees RC, Larner AC, Johnston JA, O'Shea JJ (1995) Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes. Proc Natl Acad Sci U S A 92: 7307-7311. 237. Jacobson NG, Szabo SJ, Weber-Nordt RM, Zhong Z, Schreiber RD, Darnell JEJ, Murphy KM (1995) Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J Exp Med 181: 1755-1762. 238. Smyth MJ, Taniguchi M, Street SE (2000) The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J Immunol 165: 2665-2670. 239. Chiodoni C, Stoppacciaro A, Sangaletti S, Gri G, Cappetti B, Koezuka Y, Colombo MP (2001) Different requirements for alpha-galactosylceramide and recombinant IL-12 antitumor activity in the treatment of C-26 colon carcinoma hepatic metastases. Eur J Immunol 31: 3101-3110. 240. Di Carlo E, Rovero S, Boggio K, Quaglino E, Amici A, Smorlesi A, Forni G, Musiani P (2001) Inhibition of mammary carcinogenesis by systemic interleukin 12 or p185neu DNA vaccination in Her-2/neu transgenic BALB/c mice. Clin Cancer Res 7: 830s-837s. 241. Gollob JA, Mier JW, Veenstra K, McDermott DF, Clancy D, Clancy M, Atkins MB (2000) Phase I trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-gamma induction is associated with clinical response. Clin Cancer Res 6: 1678-1692. 242. Cebon J, Jäger E, Shackleton MJ, Gibbs P, Davis ID, Hopkins W, Gibbs S, Chen Q, Karbach J, Jackson H, MacGregor DP, Sturrock S, Vaughan H, Maraskovsky E, Neumann A, Hoffman E, Sherman ML, Knuth A (2003) Two phase I studies of low dose recombinant human IL-12 with Melan-A and influenza peptides in subjects with advanced malignant melanoma. Cancer Immun 3: 7. 243. Mortarini R, Borri A, Tragni G, Bersani I, Vegetti C, Bajetta E, Pilotti S, Cerundolo V, Anichini A (2000) Peripheral burst of tumor-specific cytotoxic T lymphocytes and infiltration of metastatic lesions by memory CD8+ T cells in melanoma patients receiving interleukin 12. Cancer Res 60: 3559-3568. 244. Dunn GP, Koebel CM, Schreiber RD (2006) Interferons, immunity and cancer immunoediting. Nat Rev Immunol 6: 836-848. 245. Kanegane C, Sgadari C, Kanegane H, Teruya-Feldstein J, Yao L, Gupta G, Farber JM, Liao F, Liu L, Tosato G (1998) Contribution of the CXC chemokines IP-10 and Mig to the antitumor effects of IL-12. J Leukoc Biol 64: 384-392. 246. Sgadari C, Angiolillo AL, Tosato G (1996) Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10. Blood 87: 3877-3882.

134

247. Mitola S, Strasly M, Prato M, Ghia P, Bussolino F (2003) IL-12 regulates an endothelial cell-lymphocyte network: effect on metalloproteinase-9 production. J Immunol 171: 3725-3733. 248. Rüegg C, Yilmaz A, Bieler G, Bamat J, Chaubert P, Lejeune FJ (1998) Evidence for the involvement of endothelial cell integrin alphaVbeta3 in the disruption of the tumor vasculature induced by TNF and IFN-gamma. Nat Med 4: 408-414. 249. Younes A, Pro B, Robertson MJ, Flinn IW, Romaguera JE, Hagemeister F, Dang NH, Fiumara P, Loyer EM, Cabanillas FF, McLaughlin PW, Rodriguez MA, Samaniego F (2004) Phase II clinical trial of interleukin-12 in patients with relapsed and refractory non-Hodgkin's lymphoma and Hodgkin's disease. Clin Cancer Res 10: 5432-5438. 250. Parihar R, Nadella P, Lewis A, Jensen R, De Hoff C, Dierksheide JE, VanBuskirk AM, Magro CM, Young DC, Shapiro CL, Carson WE3 (2004) A phase I study of interleukin 12 with trastuzumab in patients with human epidermal growth factor receptor-2-overexpressing malignancies: analysis of sustained interferon gamma production in a subset of patients. Clin Cancer Res 10: 5027-5037. 251. Sangro B, Melero I, Qian C, Prieto J (2005) Gene therapy of cancer based on interleukin 12. Curr Gene Ther 5: 573-581. 252. Mazzolini G, Alfaro C, Sangro B, Feijoó E, Ruiz J, Benito A, Tirapu I, Arina A, Sola J, Herraiz M, Lucena F, Olagüe C, Subtil J, Quiroga J, Herrero I, Sádaba B, Bendandi M, Qian C, Prieto J, Melero I (2005) Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas. J Clin Oncol 23: 999-1010. 253. Heinzerling L, Burg G, Dummer R, Maier T, Oberholzer PA, Schultz J, Elzaouk L, Pavlovic J, Moelling K (2005) Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy. Hum Gene Ther 16: 35-48. 254. Lum L, Wong BR, Josien R, Becherer JD, Erdjument-Bromage H, Schlöndorff J, Tempst P, Choi Y, Blobel CP (1999) Evidence for a role of a tumor necrosis factor-alpha (TNF-alpha)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival. J Biol Chem 274: 13613-13618. 255. Wong BR, Josien R, Lee SY, Sauter B, Li HL, Steinman RM, Choi Y (1997) TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 186: 2075-2080. 256. Josien R, Li HL, Ingulli E, Sarma S, Wong BR, Vologodskaia M, Steinman RM, Choi Y (2000) TRANCE, a tumor necrosis factor family member, enhances the longevity and adjuvant properties of dendritic cells in vivo. J Exp Med 191: 495-502. 257. Chen A, Xu H, Choi Y, Wang B, Zheng G (2004) TRANCE counteracts FasL-mediated apoptosis of murine bone marrow-derived dendritic cells. Cell Immunol 231: 40-48. 258. Wiethe C, Dittmar K, Doan T, Lindenmaier W, Tindle R (2003) Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B (RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen. J Immunol 171: 4121-4130.

135

259. Hofbauer LC, Schoppet M (2004) Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases. JAMA 292: 490-495. 260. Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, Tan HL, Elliott G, Kelley MJ, Sarosi I, Wang L, Xia XZ, Elliott R, Chiu L, Black T, Scully S, Capparelli C, Morony S, Shimamoto G, Bass MB et al. (1999) Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A 96: 3540-3545. 261. Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, Capparelli C, Li J, Elliott R, McCabe S, Wong T, Campagnuolo G, Moran E, Bogoch ER, Van G, Nguyen LT, Ohashi PS, Lacey DL, Fish E, Boyle WJ et al. (1999) Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402: 304-309. 262. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V et al. (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93: 165-176. 263. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Lüthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L et al. (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89: 309-319. 264. Thomas GP, Baker SU, Eisman JA, Gardiner EM (2001) Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts. J Endocrinol 170: 451-460. 265. Eghbali-Fatourechi G, Khosla S, Sanyal A, Boyle WJ, Lacey DL, Riggs BL (2003) Role of RANK ligand in mediating increased bone resorption in early postmenopausal women. J Clin Invest 111: 1221-1230. 266. Standal T, Seidel C, Hjertner Ø, Plesner T, Sanderson RD, Waage A, Borset M, Sundan A (2002) Osteoprotegerin is bound, internalized, and degraded by multiple myeloma cells. Blood 100: 3002-3007. 267. Malyankar UM, Scatena M, Suchland KL, Yun TJ, Clark EA, Giachelli CM (2000) Osteoprotegerin is an alpha vbeta 3-induced, NF-kappa B-dependent survival factor for endothelial cells. J Biol Chem 275: 20959-20962. 268. Jono S, Ikari Y, Shioi A, Mori K, Miki T, Hara K, Nishizawa Y (2002) Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease. Circulation 106: 1192-1194. 269. Makino M, Wakamatsu S, Shimokubo S, Arima N, Baba M (2000) Production of functionally deficient dendritic cells from HTLV-I-infected monocytes: implications for the dendritic cell defect in adult T cell leukemia. Virology 274: 140-148. 270. Yang L, Carbone DP (2004) Tumor-host immune interactions and dendritic cell dysfunction. Adv Cancer Res 92: 13-27. 271. Pedersen AE, Thorn M, Gad M, Walter MR, Johnsen HE, Gaarsdal E, Nikolajsen K, Buus S, Claesson MH, Svane IM (2005) Phenotypic and functional characterization of clinical grade dendritic cells generated from patients with advanced breast cancer for therapeutic vaccination. Scand J Immunol 61: 147-156.

136

272. Boczkowski D, Nair SK, Nam JH, Lyerly HK, Gilboa E (2000) Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells. Cancer Res 60: 1028-1034. 273. Nair SK, Boczkowski D, Morse M, Cumming RI, Lyerly HK, Gilboa E (1998) Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat Biotechnol 16: 364-369. 274. Breckpot K, Dullaers M, Bonehill A, van Meirvenne S, Heirman C, de Greef C, van der Bruggen P, Thielemans K (2003) Lentivirally transduced dendritic cells as a tool for cancer immunotherapy. J Gene Med 5: 654-667. 275. Firat H, Zennou V, Garcia-Pons F, Ginhoux F, Cochet M, Danos O, Lemonnier FA, Langlade-Demoyen P, Charneau P (2002) Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy. J Gene Med 4: 38-45. 276. He Y, Zhang J, Donahue C, Falo LDJ (2006) Skin-derived dendritic cells induce potent CD8(+) T cell immunity in recombinant lentivector-mediated genetic immunization. Immunity 24: 643-656. 277. Mossoba ME, Medin JA (2006) Cancer immunotherapy using virally transduced dendritic cells: animal studies and human clinical trials. Expert Rev Vaccines 5: 717-732. 278. Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, Wolter JM, Paton V, Shak S, Lieberman G, Slamon DJ (1999) Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 17: 2639-2648. 279. Hellström I, Goodman G, Pullman J, Yang Y, Hellström KE (2001) Overexpression of HER-2 in ovarian carcinomas. Cancer Res 61: 2420-2423. 280. Hirsch FR, Franklin WA, Veve R, Varella-Garcia M, Bunn PAJ (2002) HER2/neu expression in malignant lung tumors. Semin Oncol 29: 51-58. 281. Scott GK, Robles R, Park JW, Montgomery PA, Daniel J, Holmes WE, Lee J, Keller GA, Li WL, Fendly BM, et al. (1993) A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol Cell Biol 13: 2247-2257. 282. Siatskas C, Underwood J, Ramezani A, Hawley RG, Medin JA (2005) Specific pharmacological dimerization of KDR in lentivirally transduced human hematopoietic cells activates anti-apoptotic and proliferative mechanisms. FASEB J 19: 1752-1754. 283. Naldini L, Blömer U, Gage FH, Trono D, Verma IM (1996) Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A 93: 11382-11388. 284. Yoshimitsu M, Sato T, Tao K, Walia JS, Rasaiah VI, Sleep GT, Murray GJ, Poeppl AG, Underwood J, West L, Brady RO, Medin JA (2004) Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors. Proc Natl Acad Sci U S A 101: 16909-16914. 285. Takenaka T, Qin G, Brady RO, Medin JA (1999) Circulating alpha-galactosidase A derived from transduced bone marrow cells: relevance for corrective gene transfer for Fabry disease. Hum Gene Ther 10: 1931-1939.

137

286. Lutz MB, Kukutsch N, Ogilvie AL, Rössner S, Koch F, Romani N, Schuler G (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 223: 77-92. 287. Piechocki MP, Pilon SA, Wei W (2002) Quantitative measurement of anti-ErbB-2 antibody by flow cytometry and ELISA. J Immunol Methods 259: 33-42. 288. Maecker HT, Moon J, Bhatia S, Ghanekar SA, Maino VC, Payne JK, Kuus-Reichel K, Chang JC, Summers A, Clay TM, Morse MA, Lyerly HK, DeLaRosa C, Ankerst DP, Disis ML (2005) Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunol 6: 17. 289. Chhikara M, Huang H, Vlachaki MT, Zhu X, Teh B, Chiu KJ, Woo S, Berner B, Smith EO, Oberg KC, Aguilar LK, Thompson TC, Butler EB, Aguilar-Cordova E (2001) Enhanced therapeutic effect of HSV-tk+GCV gene therapy and ionizing radiation for prostate cancer. Mol Ther 3: 536-542. 290. Dullaers M, Breckpot K, Van Meirvenne S, Bonehill A, Tuyaerts S, Michiels A, Straetman L, Heirman C, De Greef C, Van Der Bruggen P, Thielemans K (2004) Side-by-side comparison of lentivirally transduced and mRNA-electroporated dendritic cells: implications for cancer immunotherapy protocols. Mol Ther 10: 768-779. 291. Levine BL, Humeau LM, Boyer J, MacGregor R, Rebello T, Lu X, Binder GK, Slepushkin V, Lemiale F, Mascola JR, Bushman FD, Dropulic B, June CH (2006) Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci U S A 103: 17372-17377. 292. Tarantal AF, McDonald RJ, Jimenez DF, Lee CCI, O'Shea CE, Leapley AC, Won RH, Plopper CG, Lutzko C, Kohn DB (2005) Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery. Mol Ther 12: 87-98. 293. Will E, Bailey J, Schuesler T, Modlich U, Balcik B, Burzynski B, Witte D, Layh-Schmitt G, Rudolph C, Schlegelberger B, von Kalle C, Baum C, Sorrentino BP, Wagner LM, Kelly P, Reeves L, Williams DA (2007) Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials. Mol Ther 15: 782-791. 294. Worsham DN, Schuesler T, von Kalle C, Pan D (2006) In vivo gene transfer into adult stem cells in unconditioned mice by in situ delivery of a lentiviral vector. Mol Ther 14: 514-524. 295. Chan T, Sami A, El-Gayed A, Guo X, Xiang J (2006) HER-2/neu-gene engineered dendritic cell vaccine stimulates stronger HER-2/neu-specific immune responses compared to DNA vaccination. Gene Ther 13: 1391-1402. 296. Chen Z, Huang H, Chang T, Carlsen S, Saxena A, Marr R, Xing Z, Xiang J (2002) Enhanced HER-2/neu-specific antitumor immunity by cotransduction of mouse dendritic cells with two genes encoding HER-2/neu and alpha tumor necrosis factor. Cancer Gene Ther 9: 778-786. 297. zum Büschenfelde CM, Metzger J, Hermann C, Nicklisch N, Peschel C, Bernhard H (2001) The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells. J Immunol 167: 1712-1719. 298. Garnache-Ottou F, Chaperot L, Biichle S, Ferrand C, Remy-Martin J, Deconinck E, de Tailly PD, Bulabois B, Poulet J, Kuhlein E, Jacob M, Salaun V, Arock M, Drenou B, Schillinger F, Seilles E, Tiberghien P, Bensa J, Plumas J, Saas P (2005) Expression of

138

the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells. Blood 105: 1256-1264. 299. Nigro LL, Sainati L, Leszl A, Mirabile E, Spinelli M, Consarino C, Di Cataldo A, Magro S, Felix CA, Basso G (2007) Acute differentiated dendritic cell leukemia: a variant form of pediatric acute myeloid leukemia with MLL translocation. Leukemia 21: 360-362. 300. Bueno C, Almeida J, Lucio P, Marco J, Garcia R, de Pablos JM, Parreira A, Ramos F, Ruiz-Cabello F, Suarez-Vilela D, San Miguel JF, Orfao A (2004) Incidence and characteristics of CD4(+)/HLA DRhi dendritic cell malignancies. Haematologica 89: 58-69. 301. Garnache-Ottou F, Feuillard J, Saas P (2007) Plasmacytoid dendritic cell leukaemia/lymphoma: towards a well defined entity? Br J Haematol 136: 539-548. 302. Reichard KK, Burks EJ, Foucar MK, Wilson CS, Viswanatha DS, Hozier JC, Larson RS (2005) CD4(+) CD56(+) lineage-negative malignancies are rare tumors of plasmacytoid dendritic cells. Am J Surg Pathol 29: 1274-1283. 303. Rossi JG, Felice MS, Bernasconi AR, Ribas AE, Gallego MS, Somardzic AE, Alfaro EM, Alonso CN (2006) Acute leukemia of dendritic cell lineage in childhood: incidence, biological characteristics and outcome. Leuk Lymphoma 47: 715-725. 304. Sato T, Neschadim A, Konrad M, Fowler DH, Lavie A, Medin JA (2007) Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy. Mol Ther 15: 962-970. 305. Chinnasamy N, Treisman JS, Oaks MK, Hanson JP, Chinnasamy D (2005) Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination. Gene Ther 12: 259-271. 306. Freeman T, Dixon A, Cambell E, Tait T, Richardson P, Rice K, et al. (1998) Expression mapping of mouse genes. MGI Direct Data submission : . 307. Bronte V, Apolloni E, Ronca R, Zamboni P, Overwijk WW, Surman DR, Restifo NP, Zanovello P (2000) Genetic vaccination with "self" tyrosinase-related protein 2 causes melanoma eradication but not vitiligo. Cancer Res 60: 253-258. 308. Morgan DJ, Kreuwel HT, Fleck S, Levitsky HI, Pardoll DM, Sherman LA (1998) Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity. J Immunol 160: 643-651. 309. Schreurs MW, Eggert AA, de Boer AJ, Vissers JL, van Hall T, Offringa R, Figdor CG, Adema GJ (2000) Dendritic cells break tolerance and induce protective immunity against a melanocyte differentiation antigen in an autologous melanoma model. Cancer Res 60: 6995-7001. 310. Gong J, Chen D, Kashiwaba M, Li Y, Chen L, Takeuchi H, Qu H, Rowse GJ, Gendler SJ, Kufe D (1998) Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells. Proc Natl Acad Sci U S A 95: 6279-6283. 311. Sherman LA, Theobald M, Morgan D, Hernandez J, Bacik I, Yewdell J, Bennink J, Biggs J (1998) Strategies for tumor elimination by cytotoxic T lymphocytes. Crit Rev Immunol 18: 47-54. 312. Vierboom MP, Nijman HW, Offringa R, van der Voort EI, van Hall T, van den Broek L, Fleuren GJ, Kenemans P, Kast WM, Melief CJ (1997) Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes. J Exp Med 186: 695-704.

139

313. Verma IM, Weitzman MD (2005) Gene therapy: twenty-first century medicine. Annu Rev Biochem 74: 711-738. 314. Savary CA, Grazziutti ML, Melichar B, Przepiorka D, Freedman RS, Cowart RE, Cohen DM, Anaissie EJ, Woodside DG, McIntyre BW, Pierson DL, Pellis NR, Rex JH (1998) Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients. Cancer Immunol Immunother 45: 234-240. 315. Yannelli JR, Sturgill J, Foody T, Hirschowitz E (2005) The large scale generation of dendritic cells for the immunization of patients with non-small cell lung cancer (NSCLC). Lung Cancer 47: 337-350. 316. Tuyaerts S, Aerts JL, Corthals J, Neyns B, Heirman C, Breckpot K, Thielemans K, Bonehill A (2007) Current approaches in dendritic cell generation and future implications for cancer immunotherapy. Cancer Immunol Immunother 56: 1513-1537. 317. Mossoba ME, Walia JS, Rasaiah VI, Buxhoeveden N, Head R, Ying C, Foley JE, Bramson JL, Fowler DH, Medin JA (2008) Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2. Mol Ther 16: 607-617. 318. Moasser MM (2007) The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene 26: 6469-6487. 319. Bedrosian I, Roros JG, Xu S, Nguyen HQ, Engels F, Faries MB, Koski GK, Cohen PA, Czerniecki BJ (2000) Granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-12 synergize with calcium ionophore to enhance dendritic cell function. J Immunother (1997) 23: 311-320. 320. Luft T, Pang KC, Thomas E, Hertzog P, Hart DN, Trapani J, Cebon J (1998) Type I IFNs enhance the terminal differentiation of dendritic cells. J Immunol 161: 1947-1953. 321. Pirtskhalaishvili G, Shurin GV, Gambotto A, Esche C, Wahl M, Yurkovetsky ZR, Robbins PD, Shurin MR (2000) Transduction of dendritic cells with Bcl-xL increases their resistance to prostate cancer-induced apoptosis and antitumor effect in mice. J Immunol 165: 1956-1964. 322. Wang Y, Whittall T, McGowan E, Younson J, Kelly C, Bergmeier LA, Singh M, Lehner T (2005) Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells. J Immunol 174: 3306-3316. 323. Tatsumi T, Takehara T, Kanto T, Miyagi T, Kuzushita N, Sugimoto Y, Jinushi M, Kasahara A, Sasaki Y, Hori M, Hayashi N (2001) Administration of interleukin-12 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines in mouse hepatocellular carcinoma. Cancer Res 61: 7563-7567. 324. Watford WT, Moriguchi M, Morinobu A, O'Shea JJ (2003) The biology of IL-12: coordinating innate and adaptive immune responses. Cytokine Growth Factor Rev 14: 361-368. 325. Trinchieri G, Pflanz S, Kastelein RA (2003) The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 19: 641-644. 326. Villinger F (2003) Cytokines as clinical adjuvants: how far are we? Expert Rev Vaccines 2: 317-326. 327. Green EA, Flavell RA (1999) TRANCE-RANK, a new signal pathway involved in lymphocyte development and T cell activation. J Exp Med 189: 1017-1020.

140

328. Walsh MC, Choi Y (2003) Biology of the TRANCE axis. Cytokine Growth Factor Rev 14: 251-263. 329. Wong BR, Josien R, Choi Y (1999) TRANCE is a TNF family member that regulates dendritic cell and osteoclast function. J Leukoc Biol 65: 715-724. 330. Josien R, Wong BR, Li HL, Steinman RM, Choi Y (1999) TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells. J Immunol 162: 2562-2568. 331. Megan Elizabeth Nelles, Alain Labbe, Jagdeep Walia, Lintao Jia, Caren Furlonger, Takahiro Nonaka, Jeffrey A. Medin and Christopher J. Paige (2007) Interleukin-12 initiates an immune response that leads to tumour rejection The Journal of Immunology 178: B151. 332. Ramsubir S, Yoshimitsu M, Medin JA (2007) Anti-CD25 targeted killing of bicistronically transduced cells: a novel safety mechanism against retroviral genotoxicity. Mol Ther 15: 1174-1181. 333. Silvertown JD, Symes JC, Neschadim A, Nonaka T, Kao JCH, Summerlee AJS, Medin JA (2007) Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth. FASEB J 21: 754-765. 334. Evel-Kabler K, Song X, Aldrich M, Huang XF, Chen S (2005) SOCS1 restricts dendritic cells' ability to break self tolerance and induce antitumor immunity by regulating IL-12 production and signaling. J Clin Invest 116: 90-100. 335. Tokumasa N, Suto A, Kagami S, Furuta S, Hirose K, Watanabe N, Saito Y, Shimoda K, Iwamoto I, Nakajima H (2007) Expression of Tyk2 in dendritic cells is required for IL-12, IL-23, and IFN-gamma production and the induction of Th1 cell differentiation. Blood 110: 553-560. 336. Ahuja SS, Reddick RL, Sato N, Montalbo E, Kostecki V, Zhao W, Dolan MJ, Melby PC, Ahuja SK (1999) Dendritic cell (DC)-based anti-infective strategies: DCs engineered to secrete IL-12 are a potent vaccine in a murine model of an intracellular infection. J Immunol 163: 3890-3897. 337. Kuipers H, Heirman C, Hijdra D, Muskens F, Willart M, van Meirvenne S, Thielemans K, Hoogsteden HC, Lambrecht BN (2004) Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization. J Leukoc Biol 76: 1028-1038. 338. Macatonia SE, Hosken NA, Litton M, Vieira P, Hsieh CS, Culpepper JA, Wysocka M, Trinchieri G, Murphy KM, O'Garra A (1995) Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 154: 5071-5079. 339. Schwarer AP, Healey G, Hammett M (2004) Improved detection of clinically significant host-reactive antigens prior to HLA-identical sibling peripheral blood stem cell transplantation using a dendritic cell-based helper T-lymphocyte precursor assay. Bone Marrow Transplant 33: 367-375. 340. Dyall J, Latouche JB, Schnell S, Sadelain M (2001) Lentivirus-transduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes. Blood 97: 114-121. 341. Murphy KM, Reiner SL (2002) The lineage decisions of helper T cells. Nat Rev Immunol 2: 933-944.

141

342. Usui T, Preiss JC, Kanno Y, Yao ZJ, Bream JH, O'Shea JJ, Strober W (2006) T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription. J Exp Med 203: 755-766. 343. Herd KA, Wiethe C, Tindle RW (2007) Co-immunization with DNA encoding RANK/RANKL or 4-1BBL costimulatory molecules does not enhance effector or memory CTL responses afforded by immunisation with a tumour antigen-encoding DNA vaccine. Vaccine 25: 5209-5219. 344. Rabinovich GA, Gabrilovich D, Sotomayor EM (2007) Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol 25: 267-296. 345. Palucka AK, Ueno H, Fay JW, Banchereau J (2007) Taming cancer by inducing immunity via dendritic cells. Immunol Rev 220: 129-150. 346. Osada T, Clay TM, Woo CY, Morse MA, Lyerly HK (2006) Dendritic cell-based immunotherapy. Int Rev Immunol 25: 377-413. 347. Breckpot K, Aerts JL, Thielemans K (2007) Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics. Gene Ther 14: 847-862. 348. Garnett CT, Greiner JW, Tsang K, Kudo-Saito C, Grosenbach DW, Chakraborty M, Gulley JL, Arlen PM, Schlom J, Hodge JW (2006) TRICOM vector based cancer vaccines. Curr Pharm Des 12: 351-361. 349. Walker J, Tough DF (2006) Modification of TLR-induced activation of human dendritic cells by type I IFN: synergistic interaction with TLR4 but not TLR3 agonists. Eur J Immunol 36: 1827-1836. 350. Hauser H, Shen L, Gu Q, Krueger S, Chen S (2004) Secretory heat-shock protein as a dendritic cell-targeting molecule: a new strategy to enhance the potency of genetic vaccines. Gene Ther 11: 924-932. 351. Belli F, Testori A, Rivoltini L, Maio M, Andreola G, Sertoli MR, Gallino G, Piris A, Cattelan A, Lazzari I, Carrabba M, Scita G, Santantonio C, Pilla L, Tragni G, Lombardo C, Arienti F, Marchianò A, Queirolo P, Bertolini F et al. (2002) Vaccination of metastatic melanoma patients with autologous tumor-derived heat shock protein gp96-peptide complexes: clinical and immunologic findings. J Clin Oncol 20: 4169-4180. 352. Pilla L, Patuzzo R, Rivoltini L, Maio M, Pennacchioli E, Lamaj E, Maurichi A, Massarut S, Marchianò A, Santantonio C, Tosi D, Arienti F, Cova A, Sovena G, Piris A, Nonaka D, Bersani I, Di Florio A, Luigi M, Srivastava PK et al. (2006) A phase II trial of vaccination with autologous, tumor-derived heat-shock protein peptide complexes Gp96, in combination with GM-CSF and interferon-alpha in metastatic melanoma patients. Cancer Immunol Immunother 55: 958-968. 353. Aldrich M, Sanders D, Lapteva N, Huang XF, Chen S (2008) SOCS1 downregulation in dendritic cells promotes memory T-cell responses. Vaccine 26: 1128-1135. 354. Steitz J, Brück J, Lenz J, Knop J, Tüting T (2001) Depletion of CD25(+) CD4(+) T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8(+) T-cell-dependent immune defense of B16 melanoma. Cancer Res 61: 8643-8646. 355. Hankey BF, Feuer EJ, Clegg LX, Hayes RB, Legler JM, Prorok PC, Ries LA, Merrill RM, Kaplan RS (1999) Cancer surveillance series: interpreting trends in prostate

142

cancer--part I: Evidence of the effects of screening in recent prostate cancer incidence, mortality, and survival rates. J Natl Cancer Inst 91: 1017-1024. 356. Hakim FT, Gress RE (2007) Immunosenescence: deficits in adaptive immunity in the elderly. Tissue Antigens 70: 179-189. 357. Linton PJ, Dorshkind K (2004) Age-related changes in lymphocyte development and function. Nat Immunol 5: 133-139. 358. Elkord E (2007) Immunology and immunotherapy approaches for prostate cancer. Prostate Cancer Prostatic Dis 10: 224-236. 359. Gantt KR, Schultz-Cherry S, Rodriguez N, Jeronimo SMB, Nascimento ET, Goldman TL, Recker TJ, Miller MA, Wilson ME (2003) Activation of TGF-beta by Leishmania chagasi: importance for parasite survival in macrophages. J Immunol 170: 2613-2620. 360. Wang J, Eltoum I, Lamartiniere CA (2004) Genistein alters growth factor signaling in transgenic prostate model (TRAMP). Mol Cell Endocrinol 219: 171-180. 361. Lin A, Rugo HS (2007) The role of trastuzumab in early stage breast cancer: current data and treatment recommendations. Curr Treat Options Oncol 8: 47-60. 362. Akutsu Y, Matsubara H, Urashima T, Komatsu A, Sakata H, Nishimori T, Yoneyama Y, Hoshino I, Murakami K, Usui A, Kano M, Ochiai T (2007) Combination of direct intratumoral administration of dendritic cells and irradiation induces strong systemic antitumor effect mediated by GRP94/gp96 against squamous cell carcinoma in mice. Int J Oncol 31: 509-515. 363. Liu J, Wu Y, Zhang X, Yang J, Li H, Mao Y, Wang Y, Cheng X, Li Y, Xia J, Masucci M, Zeng Y (2007) Single administration of low dose cyclophosphamide augments the antitumor effect of dendritic cell vaccine. Cancer Immunol Immunother 56: 1597-1604. 364. Thurner B, Haendle I, Röder C, Dieckmann D, Keikavoussi P, Jonuleit H, Bender A, Maczek C, Schreiner D, von den Driesch P, Bröcker EB, Steinman RM, Enk A, Kämpgen E, Schuler G (1999) Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 190: 1669-1678. 365. Nair SK, Morse M, Boczkowski D, Cumming RI, Vasovic L, Gilboa E, Lyerly HK (2002) Induction of tumor-specific cytotoxic T lymphocytes in cancer patients by autologous tumor RNA-transfected dendritic cells. Ann Surg 235: 540-549. 366. Di Pucchio T, Pilla L, Capone I, Ferrantini M, Montefiore E, Urbani F, Patuzzo R, Pennacchioli E, Santinami M, Cova A, Sovena G, Arienti F, Lombardo C, Lombardi A, Caporaso P, D'Atri S, Marchetti P, Bonmassar E, Parmiani G, Belardelli F et al. (2006) Immunization of stage IV melanoma patients with Melan-A/MART-1 and gp100 peptides plus IFN-alpha results in the activation of specific CD8(+) T cells and monocyte/dendritic cell precursors. Cancer Res 66: 4943-4951. 367. Dannull J, Su Z, Rizzieri D, Yang BK, Coleman D, Yancey D, Zhang A, Dahm P, Chao N, Gilboa E, Vieweg J (2005) Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest 115: 3623-3633.

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