University of Southern California Dissertations and Theses

Dendritic cell-specific vaccine utilizing antibody-mimetic ligand and lentivector system

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Dendritic cell-specific vaccine utilizing antibody-mimetic ligand and lentivector system

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Content DENDRITIC CELL-SPECIFIC VACCINE UTILIZING ANTIBODY-MIMETIC
LIGAND AND LENTIVECTOR SYSTEM

by

Liang Xiao

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)

May 2013

Copyright 2013 Liang Xiao
ii
Dedication

This thesis is dedicated to my parents Yingchun Xiao and Yuming Gu. Their
persistent support thoughout the entire Ph.D study period has been enormous motion for
me.

iii
Acknowledgements

I’d like to thank the people who made this work possible.
First of all, I want to express my heartfelt gratitude to my Ph.D advisor Dr. Pin
Wang. He recruited me to USC and has always been there to help me to overcome all
sorts of difficulties. He is very enthusiastic and strict about scientific questions;
discussion with him has been very inspirational for me to continue my research.
Meanwhile, I’d like to thank Dr. Lili Yang from Dr. David Baltimore’s lab at Caltech.
Her help and advice is the premise of several of my research topics.
Besides, I want to show my gratitude to the rest of my committee member: Dr.
Katherine Shing and Dr. Si-Yi Chen.
Also, I would like to thank all the members of the Wang’s lab at RTH 515. It is
very lucky to be a member of this group and to work with so many talented people.
Thank you Dr. Haiguang Yang for your guide as a senior graduate student to help me out
the struggle of my first research project; Thank you Dr. Kyeil Joo, Dr. Alex Lei and Dr.
Bingbing Dai for the discussion on many interesting scientific questions; Thank you Dr.
Steve Froelich, Dr. April Tai and Dr. Paul Bryson for critical reading of my research
manuscripts; Thank you Jessy Fang, Biliang Hu, Chupei Zhang and Natnaree Siriwon for
collaboration on the study; Thank you Matthew Lim for persistent help on the
experiments, it is impossible to finish all the experiments by myself without your
contribution. Also thank all the other lab members and other collaborators in both USC
and Caltech who has given me help throughout my Ph.D study.
iv
Last but not the least, I would like to thank my entire family and my girlfriend
Ying Li for their support and care.

v
Table of Contents
Dedication ii
Acknowledgements iii
List of Figures viii
Abstract x
Chapter 1. INTRODUCTION 1
1.1 Monoclonal antibody and cell based immunotherapy 1
1.2 Immune adjuvant, cytokines and immune modulating proteins 5
1.3 Cancer vaccines 8
1.3.1 First category of cancer vaccines 8
1.3.2 Second category of cancer vaccine 9
1.4. Summary and Thesis work 12
Chapter 2. ANTIBODY-MIMETIC LIGAND SELECTED BY mRNA DISPLAY
TARGETS DC-SIGN FOR DENDRITIC CELL-DIRECTED ANTIGEN DELIVERY 14
2.1 Introduction 15
2.2 Materials and Methods 18
2.2.1 Cloning, Expression, and Purification of hDC-SIGN and mDC-SIGN 18
2.2.3 mRNA Display 21
2.2.4 In Vitro Radiolabeled Binding Assay 23
2.2.5 Cloning, Expression, and Purification of Wild-Type e10Fn3 (eFn-WT) and
Selected eFn-DC Clones with C-terminal HA Tag or Influenza Antigen Peptide 25
2.2.6 Cell Functional Assay – Binding, Internalization 25
2.2.7 Confocal Imaging 26
2.2.8 Generation of Human DCs from Human PBMCs and Mouse DCs from Mouse
Bone Marrow 27
2.2.9 Enzyme-Linked-Immunospot (ELISPOT) Assay of DCs Co-cultured with
Autologous PBMCs 28
2.3 Results 30
2.3.1 Identification of DC-SIGN-specific ligands via mRNA display 30
2.3.2 Functional binding of e10Fn3 DC-SIGN ligands to cells expressing DC-SIGN
33
2.3.3 eFn-DC6-induced internalization into DC-SIGN-expressing cells 35
vi
2.3.4 Binding and internalization of eFn-DC6 into DCs 37
2.3.5 Capacity of eFn-DC6 to mediate antigen delivery to DCs in vitro 39
2.4 Discussion 41
2.5 Acknowledgements 44
Chapter 3. DENDRITIC CELL-DIRECTED VACCINATION WITH A LENTIVECTOR
ENCODING PSCA FOR PROSTATE CANCER IN MICE 45
3.1 Introduction 46
3.2 Materials and Methods 49
3.2.1 Mice and cell lines 49
3.2.2 Construction and production of lentiviral vectors 50
3.2.3 BMDC generation and staining 51
3.2.4 In vivo depletion of CD4
+
or CD8
+
T cells 51
3.2.5 IFN-γ intracellular cytokine staining (ICCS) 52
3.2.6 Enzyme-linked immunosorbent spot (ELISPOT) assay 52
3.2.7 Histological analysis 53
3.2.8 Statistics 54
3.3 Results 55
3.3.1 Generation of DCLV-PSCA and its ability to target DC-SIGN-expressing cells
in vitro 55
3.3.2 Induction of PSCA-specific CD8
+
and CD4
+
T cell immune responses in vivo 57
3.3.3 Generation of anti-prostate tumor immunity in both prophylactic and therapeutic
models 60
3.3.4 Dependence of vaccine-elicited antitumor immunity on infiltrated CD8
+
and/or
CD4
+
T cells 62
3.3.5 Protection against lung metastasis of B16-PSCA cells 64
3.4 Discussion 65
3.5 Acknowledgements 69
Chapter 4. A TLR4 AGONIST SYNERGIZES WITH DENDRITIC CELL-DIRECTED
LENTIVIRAL VECTORS FOR INDUCING ANTIGEN-SPECIFIC IMMUNE
RESPONSES 71
4.1 Introduction 72
4.2 Materials and Methods 76
4.2.1 Mice and reagents 76
4.2.2 Lentiviral vector construct and production 76
4.2.3 BMDC generation and activation 77
4.2.4 Flow cytometric analysis of surface markers of BMDCs 78
4.2.5 Supernatant ELISA assay 78
vii
4.2.6 Immunization procedure 78
4.2.7 Intracellular cytokine staining (ICCS) 79
4.2.8 IL-2 ELISPOT assay 80
4.2.9 Statistics 80
4.3 Results 81
4.3.1 Activation of dendritic cells by the TLR4 agonist GLA in vitro 81
4.3.2 Adjuvant effect of the TLR4 agonist GLA on DC-LV-induced T cell immunity
83
4.3.3 Adjuvant effect of the TLR4 agonist GLA on DC-LV-induced antibody
responses 85
4.3.4 Adjuvant effect of the TLR4 agonist GLA on the anti-tumor immunity delivered
by DC-LV 86
4.3.5 Role of CD4
+
T cells in GLA-augmented DC-LV immunization 88
4.3.6 Study of the role of MyD88 and TRIF pathways in GLA-mediated activation of
DCs in vitro 90
4.3.7 Study of the role of MyD88 and TRIF pathways in GLA-mediated activation of
DCs in vivo 92
4.4 Discussion 94
4.5 Acknowledgements 100
REFERENCES 101

viii
List of Figures
Figure 1. 1. The generation of anti-tumour T cells used for adoptive cell therapy. 4

Figure 1. 2. An immunosuppressive network within the tumor microenvironment. 7

Figure 2. 1. Illustration of mRNA display selecting e10Fn3 ligand to target dendritic cells
for antigen delivery. 29

Figure 2. 2. Expression and functional test of human and mouse DC-SIGN extracellular
domain (ECD). 30

Figure 2. 3. An mRNA display selection to identify e10Fn3 variants that can bind to both
human and mouse DC-SIGNs. 31

Figure 2. 4. Selection and characterization of isolated e10Fn3 variants for their binding to
293T.hDC-SIGN and 293T.mDC-SIGN cells. 34

Figure 2. 5. Internalization efficiency and intracellular trafficking of eFn-DC6 in
293T.hDC-SIGN cells. 36

Figure 2. 6. Specific binding and internalization of eFn-DC6 into DCs. 38

Figure 2. 7. Antigen-specific IFN-γ release of autologous human PBMCs after coculture
with DCs treated by eFn-DC6-fused antigens. 40

Figure 3. 1. Schematic representation of key constructs used in this study. 55

Figure 3. 2. Targeted transduction and delivery of PSCA antigen gene into dendritic cells
(DCs) by DCLV-PSCA. 56
ix

Figure 3. 3. PSCA-specific T cell response after a single dose of in vivo immunization
with DCLV-PSCA. 58

Figure 3. 4. Prophylactic and therapeutic anti-TRAMP-C1 prostate cancer immunity
elicited by in vivo immunization with DCLV-PSCA. 61

Figure 3. 5. CD8+/CD4+ T cell-dependent immune protection against TRAMP-C1
tumors induced by DC-LV-PSCA immunization. 63

Figure 3. 6. The ability of DCLV-PSCA immunization to suppress lung metastases. 64

Figure 4. 1. GLA-mediated activation of mouse BMDCs in vitro. 82

Figure 4. 2. Adjuvant effects of GLA on boosting DC-LV-based vaccine-specific T cell
immune responses in vivo. 84

Figure 4. 3. Adjuvant effects of GLA on boosting DC-LV-based vaccine-specific
antibody immune responses in vivo. 86

Figure 4. 4. Adjuvant effects of GLA on DC-LV immunization to enhance antitumor
immune responses. 87

Figure 4. 5. The role of CD4+ helper T cells in GLA-enhanced CD8+ T cell responses. 89

Figure 4. 6. Involvement of MyD88 and TRIF signaling pathways in GLA-mediated
activation of BMDCs in vitro. 91

Figure 4. 7. Involvement of MyD88 and TRIF signaling pathways in GLA-adjuvanted
DC-LV immunization in vivo. 93

x
Abstract
Dendritic cell (DC)-based vaccines have shown promise as an immunotherapeutic
modality for cancer and infectious diseases in many preclinical studies and clinical trials.
Dendritic cells (DCs) are specialized antigen presenting cells (APCs) that can uptake and
process antigens for presentation through the major histocompatibility complex (MHC)
and activate naïve T cells.

Because of this unique biological role, DCs have been widely
exploited to develop DC-based vaccines for protective immunity against bacterial, viral,
and fungal infections. The development of DC-based vaccines has also been one of the
major focuses of cancer immunotherapy. Patient-derived DCs are loaded with tumor
antigens and subsequently administered back to the patient. This type of autologous cell
therapy led to the first FDA-approved cancer vaccine, sipuleucel-T.

However, the tedious
procedure for generating the vaccine, its high cost ($93,000 USD) per patient, and only
modest improvement in survival (an average of 4.1 months) could limit its extensive
application.

A better strategy would be direct and specific loading of antigens onto DCs
in vivo, which could be achieved by targeting DC-specific cell-surface receptors that
facilitate internalization of the bound antigens for antigen presentation.

DC-specific
ICAM3-grabbing non-integrin (DC-SIGN or CD209) is a promising target for DC-
specific antigen delivery because it is predominantly expressed on DCs. In chapter 2, the
recombinant extracellular domains (ECD) of human and mouse DC-SIGN (hDC-SIGN
and mDC-SIGN) were generated as DC-specific targets for mRNA display. Accordingly,
an antibody-mimetic library was constructed by randomizing two exposed binding loops
of an expression-enhanced 10th human fibronectin type III domain (e10Fn3). After three
xi
rounds of selection against mDC-SIGN, followed by four rounds of selection against
hDC-SIGN, we were able to evolve several dual-specific ligands, which could bind to
both soluble ECD of human and mouse DC-SIGNs. Using a cell-binding assay, one
ligand, eFn-DC6, was found to have high affinity to hDC-SIGN and moderate affinity to
mDC-SIGN. When fused with an antigenic peptide, eFn-DC6 could direct the antigen
delivery and presentation by human peripheral blood mononuclear cell (PBMC)-derived
DCs and stimulate antigen-specific CD8
+
T cells to secrete inflammatory cytokines. In
chapter 3, we evaluated dendritic cell (DC)-directed lentiviral vector (DCLV) encoding
murine PSCA (DCLV-PSCA) as a novel tumor vaccine for prostate cancer in mouse
models. Direct immunization with the DCLV-PSCA in male C57BL/6 mice elicited
robust PSCA-responsive CD8
+
and CD4
+

T cells in vivo. In a transgenic adenocarcinoma
mouse prostate cell line (TRAMP-C1) synergetic tumor model, we further demonstrated
that DCLV-PSCA-vaccinated mice could be protected from lethal tumor challenge in a
prophylactic model, whereas slower tumor growth was observed in a therapeutic model.
In chapter 4, we further improved the immune response of DC-directed lentiviral vaccine
by employing a novel TLR4 agonist GLA as adjuvant. Both T cells and B cells responses
were greatly enhanced and these improved responses further suppress tumor growth. We
also looked at the relationship between the improvement of CD8
+
T cells and CD4
+
T
cells and which signaling transduction pathway, MyD88 or TRIF, plays a more important
role in adjuvant effect.
1
Chapter 1
INTRODUCTION

Cancer immunotherapy
Cancer immunotherapy is to use components from the immune system to fight
against cancer. Compared with the traditional standard chemotherapy for cancer
treatment, immunotherapy is more target directed and also has less toxic effects.
1.1 Monoclonal antibody and cell based immunotherapy
Antibodies provide an important protection from bacteria and viruses, and it can
also target and kill tumor cells. The specificity and affinity of the monoclonal antibody
for each different tumor type can be carefully designed and selected. In the beginning,
Monoclonal antibodies are produced in animal, thus it may cause hypersensitivity
reaction when they are administered into human body. Later, humanized monoclonal
antibodies were developed by replacing the constant region of animal antibody with
human version(Boulianne and others 1984) or by using engineered mice with human
antibody gene sequence.(Mendez and others 1997) By binding to their targets,
monoclonal antibodies can inhibit tumor growth through distinct pathways.

(1) Inhibiting growth factor binding to its receptor.
(2) Inhibiting blood vessel formation to tumor.
2
(3) Activating complement.
(4) Activating natural killer (NK) cells by binding to Fc receptors (FcRs) and initiate
antibody dependent cellular cytotoxicity (ADCC).

Currently, there are nine monoclonal antibodies have been approved for cancer
treatment, out of which, five monoclonal antibodies bind to highly expressed protein
antigen on hematologic tumors:
(1) Alemtuzumab targets CD52 for chronic lymphocytic leukemia (CLL).(Keating and
others 2002)
(2) Gemtuzumab targets CD33 for acute myelogenous leukemia (AML).(Bross and
others 2001)
(3) Rituximab targets CD20 for non-Hodgkin’s lymphoma (NHL).(Coiffier and others
2002)
(4) Ibritumomab tiuxetan targets CD20 for non-Hodgkin’s lymphoma (NHL).(Witzig and
others 2002)
(5) Tositumomab targets CD20 for chronic lymphocytic leukemia (CLL).(Fisher and
others 2005)

The other four monoclonal antibodies are for treatment of solid tumors:
(1) Cetuximab targets EGFR for metastatic colorectal cancer.(Jonker and others 2007)
(2) Panitumumab targets EGFR for metastatic colorectal cancer.(Giusti and others 2007)
(3) Trastuzumab targets HER2/neu for breast cancer.(Hudis 2007)
3
(4) Bevacizumab targets VEGF for nonsquamous non-small cell lung cancer (NSCLC),
metastatic colon cancer, metastatic HER2/neu negative breast cancer, renal cancer
and pancreatic cancer.(Cohen MH 2007)
The efficiency of monoclonal antibodies can be further improved by engineering
them.
(1) Single chain antibody (ScFv), Fab to have better diffusion in solid tumor.(Yokota and
others 1992)
(2) Mutation in neonatal FcR region to generate better antibody half-life.(Hinton and
others 2006)
(3) Modifying Fc region to make better FcR binding for ADCC.(Shields and others 2001)

Adoptive T cells therapy refers to harvesting T cells from cancer patients or
normal person, manipulating and expanding them for desired tumor immunity. It is
usually desired to collet the tumor infiltrating T cells, because this population of T cells
tends to have high percentage of tumor responsive cells.(Dudley and others 2003) And
then the variant TCRs from these T cells can be cloned and tested for the efficacy for
MHC-peptide recognition, out of which, some good ones can be found and introduced to
peripheral blood cells for tumor recognition. However, there is a limitation for this
method, only patient MHC compatible TCRs can be used since the TCR and MHC
recognition is MHC restricted. Chimeric antigen receptor was introduced to bypass this
TCR limitation.(J. R. Park and others 2007) Single chain antibody as extracellular
domain here can be used to redirect T cells to tumor antigen, through transmembrane
4

Figure 1. 1. The generation of anti-tumour T cells used for adoptive cell therapy.
(Rosenberg, S. A. (2008) Nat Rev Cancer. 8(4): 299-308)

domain, an intracellular signaling domain is linked to deliver signal and activate T cells.
This new method brings flexibility for antigens, as long as there is a corresponding ScFv
available. And the intracellular signaling domain can be designed to achieve different
stimulation.(Cartellieri and others 2010) Some clinical trials have already show that CAR
therapy has antitumor efficacy, however, there are still some problems need to be solved,
such as graft versus host disease (GVHD) caused by CAR redirected T lymphocytes.
5
1.2 Immune adjuvant, cytokines and immune modulating proteins
Adjuvant is any agent that can stimulate immune system and improve the
response generated by vaccine, however, adjuvant itself would not cause antigen specific
responses. Under normal body condition, tumor cells will die and the antigens from these
dead tumor cells can be up taken by antigen presenting cells (APCs). Even so, this
spontaneous antigen specific immune response can hardly cause tumor regression.
Incorporating adjuvant into this context was proven to be effective under some
circumstances.
Toll-like receptors (TLRs) are a group of membrane protein receptors that are
essential for normal function of the immune systems. This type of pattern recognition
receptors (PRR) can detect some molecules that are usually associated and shared by
pathogens and then send danger signals to the immune systems. Therefore, TLRs
functions as an alarm for the immune system. Similarly, TLRs can also send danger
signal such as tumor to the immune system. There are 13 different TLRs have been
identified up to now, each of them can bind different ligands and are distributed at
different parts of different cell types.
Bacilli Calmette-Guerin (BCG) is used as adjuvant for immunotherapy in
superficial bladder cancer post surgery.(Shelley and others 2000) This therapy was
proven to be more effective than chemotherapy. Imiquimod, TLR7 agonist, has already
been approved for basal cell carcinoma, actinic keratosis and cutaneous squamous cell
carcinoma treatment.(Geisse and others 2004) Imiquimod is now also being evaluated for
nonsurgical therapy for vulvar intraepithelial neoplasia (VIN).(van Seters and others
6
2008) TLR9 is also an interesting TLR that has been studied a lot and some clinical trials
are using TLR9 agonist as immune adjuvant for cancer treatment. TLR9 can recognize
natural unmethylated CpG DNA, and it can result in very good immune response when
delivered into the circulation system. Moreover, some synthetic TLR9 agonists are also
been developed and used in cancer clinical trials. One of them is called PF-3512676 and
it is now being evaluated in phase I/II clinical trials for intratumoral injection in basal cell
carcinoma and also metastatic melanoma.(Pashenkov and others 2006)
Not only can TLR agonists be good adjuvants candidates, but also some other
small molecules can also act as powerful adjuvant. A marine sponge lipid derivative
called α-galactosylceramide, which was first isolated and found to have anti-cancer
activity,(Kobayashi and others 1995) can be displayed by CD1d molecule and activate
NKT cells.(Smyth and others 2002) In many mouse tumor models, α-galactosylceramide
was found to be very potent.(Smyth and others 2002) Nevertheless, in human clinical
trials, this method was restricted by availability of NKT cells among different cancer
patients.
Cytokines are secreted by immune cells, and they are very important functions for
immune system. Currently, IL-2 and IFN- α have been used in some tumor treatment. IL-
2 can stimulate T cells proliferation while IFN-a is considered to be crucial for antiviral
immunity. Both of these two cytokines have been approved for advance melanoma
treatment and also advance renal cell carcinoma.
There are also some other antibodies with distinct functions and they can
modulate immune response towards anti-tumor direction:
7

(1) Blocking negative signal transductions with CTLA-4 blocking antibodies
(Ipilimumab, tremelimumab);(O'Day and others 2007) PD-1 blocking antibodies (CT-
011).(Berger and others 2008)
(2) Neutralizing immune suppressive cytokines with TGF-β antibody (CAT-192);(Group
and others 2007) IL-10 antibody.(Llorente and others 2000)
(3) Activating TNF family receptors like GITR (glucocorticoid-induced tumor necrosis
factor receptor), CD137 (4-1BB, BMS-663513), CD134 (OX-40), and CD40 (CP-
870893).(Vonderheide and others 2007)

Figure 1. 2. An immunosuppressive network within the tumor microenvironment.
(Zheng, L. (2010) Springer-Verlag Berlin Heidelberg, 2010. DOI: 10.1007)
8
1.3 Cancer vaccines
Cancer vaccines are generally divided into two different categories. Because some
cancers are cause by microbial infections, like cervical cancer and some liver cancers are
usually caused by virus infection while some stomach cancer can be cause be bacteria.
Therefore, one category of cancer vaccine is the vaccine used to fight against HPV, HBV
and stomach bacteria. Since the vaccines against these viruses or bacteria normally are
very effective, thus these categories of vaccines have very good effect. The other
category of cancer vaccine refers to vaccine used to prevent or treat cancer cells
themselves and currently a lot of researchers are studying and developing this kind of
vaccine.

1.3.1 First category of cancer vaccines
Usually, bacteria and viruses these microbes are considered to be foreign from the
body, therefore, they can elicit immune response quite easily without overcoming big
immune barrier.
(1) HBV the first vaccine provides protection against known oncogenic development of
hepatocellular carcinoma (HCC).(Davis 2005)
(2) HPV-16 and HPV-18 are associated with around 70% percent of worldwide cervical
cancer occurrences and some vagina, vulva cancers.(Parkin and others 1999) Cervarix
form GSK has been approved by FDA and was shown to have 93% percent of
cervical cancer prevention in young woman.(Harper 2008) And at the same time,
9
Gardasil from Merck was also approved as cervical cancer vaccine that was shown to
protect against types 6, 11, 16, and 18 HPV associated cancers.(Siddiqui and others
2006)
(3) Xenotropic murine leukemia virus –related virus (XMRV) originally was found to
cause leukemia and sarcomas in animals, however, recently, some researchers pointed
out that this virus was present in 27% of human prostate cancers, especially
aggressive tumors.(Schlaberg and others 2009) Although some other researchers
claimed that there is no association between this virus and prostate cancer,(Switzer
and others 2011) more evidences need to be collected to reach the final conclusion.
(4) Helicobacter pylori bacteria primary cause for stomach cancer and mucosal-
associated lymphoid tissue lymphomas (MALT).(Eslick 2006)

There are also some other viruses that were reported to cause human cancers, like
human leukemia virus.

1.3.2 Second category of cancer vaccine
Although we have seen many exciting prophylactic and therapeutic vaccine that
have been very effective for cancers like prostate cancer, breast cancer in animal models.
Yet, for human, there is still lack of good cancer vaccines that can target and eradicate
tumor itself. This is probably because when tumor grows, it has already established
tolerance for the immune system. Therefore, it would be hard for vaccine to generate
effective immune response under such tolerance circumstance.
10
Tumor antigen are usually discovered either by screening the targets of tumor
infiltrating lymphocytes or through characterization of the protein expression difference
between normal somatic cells and tumor cells. Examples of antigen from TILs are gp100,
MAGE and MART-1. By analyzing the protein expression difference, researchers have
identified many antigens. They could be overexpressed protein in the tumor (HER2/neu),
mutated protein in the tumor (P53, Ras protein) or some tissue specific antigens ( NY-
ESO-1, TRP-2, PSCA).
Currently, there are several different vaccine types in the clinical trial:
Whole tumor cells: This vaccine is the most tested in the clinical trials since it is very
straightforward for vaccine generation that is to use tumor cell itself. This type of vaccine
usually use irradiated cancer cell or cancer cell lysates administered along with some
adjuvants. This vaccine tends to have a broad antigen targets, however it may require that
the tumor cell should have the relevant antigens. And clinically, the immune response
generated very low effect. Recently, some researchers are using modified tumor cells as
vaccine, for instance, adding GM-CSF genes to the tumor cells to make them attractive to
APCs.(Soiffer and others 1998)
Plasmid DNA encoding antigens: Using a plasmid carrying relevant antigen genes, this
method can be very stable and is usually administered to the muscle or through gene
gun.(Tang and others 1992) The disadvantage of this method is that it may need a lot of
DNA plasmid, and the immune response is often towards Th2 response while for tumor
vaccine, a Th1 immune response is more favorable.
11
Antigen peptides: This vaccine type can be very stable and easy to make, at the same
time, antigen epitope from wild type can also be modified to generate higher MHC
avidity and stronger immune response.(Berzofsky 1993) However, peptides need
corresponding MHC molecule for presentation.
Viral vectors: There are many different virus types available as vectors and each of them
can be very different in terms of the immune responses generated. These engineered
viruses can deliver antigens and also some genetic adjuvant (IL-2, IL15) in vivo.
Nevertheless, preexisting anti-virus immune response (adenovirus), toxicity and safety
will be major drawbacks for viral vectors.
Ex vivo modified dendritic cells: Dendritic cells are the most powerful antigens
presenting cells, and they can present both MHC-I and MHC-II restricted epitopes to
stimulate both CD8+ and CD4+ T cells. Normally, immature dendritic cells from patient
are taken out and cultured, when at the same time, antigens (peptides, tumor lysates,
antigen protein or viral vectors) are loaded, and then reinfused back to the patient.
Recently, the first therapeutic cancer vaccine Provenge from Dendreon has been
approved for some prostate cancer cases. APCs from patient’s blood are collected and
isolated and then co-cultured with a protein antigen PAP-GM-CSF. PAP is the prostate
tumor antigen prostatic acid phosphatase while GM-CFS is fused with PAP in hope that it
will improve antigen up taken and enhance immune response.(Burch and others 2004)
Finally, these modified APCs are reinfused back to the patient.
12
1.4. Summary and Thesis work
In the study, we targeted dendritic cells, the major antigen presenting cells, to
deliver antigens for vaccine development. We applied mRNA dispaly strategy to select
out DC-SIGN specific e10Fn3 library based ligand and also used DC-directed lentiviral
vector previously developed by our lab to deliver antigen protein or gene to dendritic
cells as vaccine.
In chapter 2, which has been published (Xiao, Huang et al. 2013), the
recombinant extracellular domains (ECD) of human and mouse DC-SIGN (hDC-SIGN
and mDC-SIGN) were generated as DC-specific targets for mRNA display. Accordingly,
an antibody-mimetic library was constructed by randomizing two exposed binding loops
of an expression-enhanced 10th human fibronectin type III domain (e10Fn3). After three
rounds of selection against mDC-SIGN, followed by four rounds of selection against
hDC-SIGN, we were able to evolve several dual-specific ligands, which could bind to
both soluble ECD of human and mouse DC-SIGNs. Using a cell-binding assay, one
ligand, eFn-DC6, was found to have high affinity to hDC-SIGN and moderate affinity to
mDC-SIGN. When fused with an antigenic peptide, eFn-DC6 could direct the antigen
delivery and presentation by human peripheral blood mononuclear cell (PBMC)-derived
DCs and stimulate antigen-specific CD8
+
T cells to secrete inflammatory cytokines. In
chapter 3, which has been published (Xiao, Joo et al. 2012), we evaluated dendritic cell
(DC)-directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a
novel tumor vaccine for prostate cancer in mouse models. Direct immunization with the
DCLV-PSCA in male C57BL/6 mice elicited robust PSCA-responsive CD8
+
and CD4
+

T
13
cells in vivo. In a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1)
synergetic tumor model, we further demonstrated that DCLV-PSCA-vaccinated mice
could be protected from lethal tumor challenge in a prophylactic model, whereas slower
tumor growth was observed in a therapeutic model. In chapter 4, which has been
published (Xiao, Kim et al. 2012), we improved the immune response of DC-directed
lentiviral vaccine by employing a novel TLR4 agonist GLA as adjuvant. Both T cells and
B cells responses were greatly enhanced and these improved responses further suppress
tumor growth. We also looked at the relationship between the improvement of CD8
+
T
cells and CD4
+
T cells and which signaling transduction pathway, MyD88 or TRIF, plays
a more important role in adjuvant effect.

14
Chapter 2
ANTIBODY-MIMETIC LIGAND SELECTED BY mRNA
DISPLAY TARGETS DC-SIGN FOR DENDRITIC CELL-
DIRECTED ANTIGEN DELIVERY

Portions of this chapter are adapted from: Liang Xiao, Kuo-Chan Hung, Terry T.
Takahashi, Kye-Il Joo, Matthew Lim, Richard W. Roberts, and Pin Wang. ACS Chemical
Biology (2013), 10.1021/cb300680c.

Dendritic cell (DC)-based vaccines have shown promise as an immunotherapeutic
modality for cancer and infectious diseases in many preclinical studies and clinical trials.
Provenge (sipuleucel-T), a DC-based vaccine based on ex vivo-generated autologous DCs
loaded with antigens, has recently received FDA approval for prostate cancer treatment,
further validating the potential of DC-based vaccine modalities. However, direct antigen
delivery to DCs in vivo via DC-specific surface receptors would enable a more direct and
less laborious approach to immunization. In this study, the recombinant extracellular
domains (ECD) of human and mouse DC-SIGN (hDC-SIGN and mDC-SIGN) were
generated as DC-specific targets for mRNA display. Accordingly, an antibody-mimetic
library was constructed by randomizing two exposed binding loops of an expression-
15
enhanced 10th human fibronectin type III domain (e10Fn3). After three rounds of
selection against mDC-SIGN, followed by four rounds of selection against hDC-SIGN,
we were able to evolve several dual-specific ligands, which could bind to both soluble
ECD of human and mouse DC-SIGNs. Using a cell-binding assay, one ligand, eFn-DC6,
was found to have high affinity to hDC-SIGN and moderate affinity to mDC-SIGN.
When fused with an antigenic peptide, eFn-DC6 could direct the antigen delivery and
presentation by human peripheral blood mononuclear cell (PBMC)-derived DCs and
stimulate antigen-specific CD8
+
T cells to secrete inflammatory cytokines. Taken
together, these results demonstrate the utility of mRNA display to select protein carriers
for DC-based vaccination and offer in vitro evidence that the antibody-mimetic ligand
eFn-DC6 represents a promising candidate for the development of an in vivo DC-based
vaccine in humans.
2.1 Introduction
Dendritic cells (DCs) are specialized antigen presenting cells (APCs) that can
uptake and process antigens for presentation through the major histocompatibility
complex (MHC) and activate naïve T cells.(Mellman and others 2001) Because of this
unique biological role, DCs have been widely exploited to develop DC-based vaccines
for protective immunity against bacterial, viral, and fungal infections.(Fajardo-Moser and
others 2008) The development of DC-based vaccines has also been one of the major
focuses of cancer immunotherapy.(Boudreau and others 2011; Wei and others 2008)
Patient-derived DCs are loaded with tumor antigens and subsequently administered back
16
to the patient. This type of autologous cell therapy led to the first FDA-approved cancer
vaccine, sipuleucel-T.(Cheever and others 2011; Kantoff, Higano, and others 2010)
However, the tedious procedure for generating the vaccine, its high cost ($93,000 USD)
per patient, and only modest improvement in survival (an average of 4.1 months) could
limit its extensive application.(Hammerstrom and others 2011; Plosker 2011) A better
strategy would be direct and specific loading of antigens onto DCs in vivo, which could
be achieved by targeting DC-specific cell-surface receptors that facilitate internalization
of the bound antigens for antigen presentation.(Tacken and others 2007)
C-type lectin receptors (CLRs) constitute a well-studied family of proteins that have
specific expression pattern on APCs and can uniquely capture and endocytose
antigens.(Figdor and others 2002) Although several CLRs have been reported as targets
for antigen loading, DC-specific ICAM3-grabbing non-integrin (DC-SIGN or CD209) is
a promising target for DC-specific antigen delivery because it is predominantly expressed
on DCs, allowing for higher targeting efficiencies and fewer side effects.(Geijtenbeek
and others 2000) Furthermore, studies have shown that anti-DC-SIGN antibody-based
antigen loading can elicit both naïve and memory T cell responses in vitro.(Tacken and
others 2005) Multiple mouse homologues of human DC-SIGN (hDC-SIGN) have been
identified by their sequence similarities and genetic loci; one of these homologues,
having the highest homology to hDC-SIGN, was named mouse DC-SIGN (mDC-SIGN
or CD209A/CIRE). mDC-SIGN localizes syntenically to hDC-SIGN and is expressed on
a subset of DCs in a manner similar to hDC-SIGN.(C. G. Park and others 2001) The
ability of mDC-SIGN to internalize a bound ligand has been the subject of conflicting
17
reports;(Powlesland and others 2006; Takahara and others 2004) however, lentiviral
vectors targeting mDC-SIGN have been successfully demonstrated to specifically deliver
antigen to mouse DCs and establish antigen-specific immune responses in vivo.(Dai and
others 2009; Xiao and others 2012; H. Yang and others 2011; L. Yang and others 2008)
Thus, in an effort to establish preclinical animal models and subsequent translation to
clinical human studies for DC-SIGN-targeted vaccine development, it would be
advantageous to identify highly specific ligands that can recognize both hDC-SIGN and
mDC-SIGN.
In this chapter, we utilized mRNA display of an antibody- mimetic e10Fn3
library to identify DC-SIGN-specific ligands. This antibody-mimetic e10Fn3
molecule has several advantages over humanized antibodies. First of all, it has a
relatively small size (∼10 kDa) to be used as scaffold for mRNA display and
smaller size is better for tissue penetration that is a preferable property for vaccine
delivery. Second, it can be easily produced by bacteria cells in large amounts,
greatly reduce the cost for future medical application. mRNA display is a powerful
in vitro selection technique that can evolve antibody-mimetic ligands with high
specificity and affinity to targets of interest.(Hayashi and others 2012; Jackrel and
others 2010; Roberts and others 1997; T. T. Takahashi and others 2003; T. T. R.
Takahashi, R. W. 2009)

Theoretically, antibody-mimetic libraries based on a
scaffold derived from fibronectin (e.g., the 10Fn3 library) of >10
12
molecules can
be generated using mRNA display.(Koide and others 1998; Olson and others
18
2008; Xu and others 2002) In order to improve the expression and stability of the
10Fn3 library, a new e10Fn3 library was designed based on the wild-type
10Fn3.(Olson and others 2011) The e10Fn3 scaffold is derived from the human
fibronectin protein, thereby reducing the concern of undesirable immune responses
against targeting ligands when used to direct in vivo vaccine delivery in humans.
Herein we report our efforts to design functional ligands with dual specificity
against the extracellular domains of hDC-SIGN and mDC-SIGN. Three rounds of mDC-
SIGN-based selection followed by four rounds of hDC-SIGN-based selection resulted in
one molecule, eFn-DC6, which recognizes both hDC-SIGN and mDC-SIGN. eFn-DC6
was further shown to bind selectively to human DCs and mediate antigen presentation to
stimulate antigen-specific T cells in vitro.
2.2 Materials and Methods
2.2.1 Cloning, Expression, and Purification of hDC-SIGN and mDC-SIGN
The cDNAs for the extracellular domain (ECD) of hDC-SIGN and mDC-SIGN
were PCR-amplified from plasmids FUW-mDCSIGN and FUW-hDCSIGN.(L. Yang and
others 2008) The PCR reaction also introduced a BirA biotinylation recognition sequence
at the 5’-end. The cDNAs were then separately cloned into the pET302/NT-His vector
(Life Technologies, Grand Island, NY). In order to add biotin to the target protein,
BL21(DE3) was first transformed with plasmid pBirAcm, which was purified from E.
coli strain EVB101 (Avidity, Aurora, CO) and able to express biotin ligase under
isopropyl-β-D-thiogalactoside (IPTG) induction, followed by further transformation with
19
pET302/NT-His plasmid vectors containing either hDC-SIGN or mDC-SIGN ECD.
Bacteria were inoculated in 1 L of Luria

Broth supplemented with 100 µg/mL of
ampicillin and 10 µg/mL of chloramphenicol and grown at 37 °C with shaking. When the
growth of bacteria reached the mid-log phase at 37 °C, protein expression was induced

with IPTG at a final concentration of

1 mM, and free biotin (Sigma-Aldrich, St. Louis,
MO) was added to a final concentration of 50 mM. The induced cultures

were incubated
for another 4 h before the cells were harvested

by centrifuging at 6,000 x g for 10 min at
4 °C. The resulting

cell pellet was resuspended in 15 mL of 100 mM NaH
2
PO
4
,

pH 8.0,
10 mM Tris-HCl,

and 6 M guanidine HCl, and then lysed by French press. The lysate was

supplemented with 0.01% β-mercaptoethanol, incubated at 4

°C for 2 h and then
centrifuged at 150,000 x

g for 30 min at 4 °C in a Beckman JA-25 rotor. The

supernatant
was incubated with 1 mL of nickel-nitrilotriacetic

acid-agarose (Qiagen, Valencia, CA)
(pre-equilibrated

with denaturing buffer) at 4 °C overnight. The resins were

loaded onto a
chromatography column, and all subsequent

washes were done with a 20-fold resin-
volume excess of wash

buffer (30 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM

CaCl
2
, 6 M
urea, and 10 mM imidazole). Successive

washes using the buffer (30 mM Tris-HCl, pH
8.0, 0.5 M NaCl) with decreasing concentrations

of urea starting from 6 M urea were
performed to refold the

proteins. The renatured proteins were eluted with the elution
buffer (30 mM Tris-HCl, pH 8.0, 0.5

M NaCl, 1 mM CaCl
2
, and 1 M imidazole) and
exchanged into PBS buffer using a PD-10 column (GE Healthcare, Pittsburgh, PA).
An Fc-gp120-based ELISA was used to confirm the binding function of refolded
hDC-SIGN. ELISA plates were coated with 10 µg purified hDC-SIGN or mDC-SIGN
20
ECD in each well. After blocking, the plates were then incubated with different
concentrations of Fc-gp120 protein ranging from 0 to 300 nM. After washing, the plates
were incubated with HRP-conjugated anti-human IgG antibody, and the reaction was
visualized by chromogenic substrate (TMB) and stopped by 1 M H
2
SO
4
. Absorbance at
450 nm was measured with reduction at 650 nm using an ELISA plate reader.
2.2.2 e10Fn3 Library Construction
Construction of an antibody-mimetic e10Fn3 library using eight oligonucleotides
synthesized by the Yale Keck Oligonucleotide Synthesis facility or Integrated DNA
Technologies has been described previously.(Olson and others 2007) Briefly, the e10Fn3
library differs from the human 10th fibronectin type III domain because the e10Fn3
library is truncated at the N-terminus and contains 5 scaffold mutations that increase
expression and/or solubility (V5K, A6E, T8S, L12I, and L13Q), and the last position of
the BC loop is restricted to Leu, Ile, or Val for higher structural stability.(Olson and
others 2011)
Three oligonucleotides, eFnoligo1, 2, and 3, were newly designed for introducing
the mutations and the doped last position of BC loop into e10Fn3 library. Briely,
eFnoligo3 (5’-ACC AGC ATC CAG ATC AGC TGG 55S 55S 55S 55S 55S 55S VTT
CGC TAC TAC CGC ATC ACC TAC G-3’; 5 indicated the dNTP mixture of 20% T,
30% C, 30% A, and 20% C; S were mixed with 60% C and 40% G; V denoted the
mixture of C, A, and G equally), containing the randomized BC loop was used to
introduce two mutations (L12I and L13Q) and one doped residue. eFnoligo3 was
21
annealed to Fnoligo4, extended by Klenow DNA polymerase, and purified by agarose gel
electrophoresis. The purified product was PCR amplified using the second new primer,
eFnoligo2 (5’-CAA TTA CAA TGC TCG AGG TCA AGG AAG CAT CAC CAA CCA
GCA TCC AGA TCA GCT GG-3’) and Fnoligo5. eFnoligo2 was used to insert three
mutations, V5K, A6E, and T8S, into the N-terminus of e10FnIII library. Fnoligo6 and
Fnoligo7 containing the randomized FG loop were annealed and extended by Klenow
DNA polymerase. All PCR and Klenow products were purified by agarose gel
eletrophoresis.
Both BC and FG fragments were digested with Bsa I and purified by agarose gel
electrophoresis. Purified BC and FG fragments were ligated together using T4 DNA
ligase and purified by agarose gel electrophoresis where 13 ng/mL of ligated product was
recovered. The approximate theoretical complexity of e10FnIII library is 10
12
(1 trillion
unique sequences). The library was extended and amplified by the third new
oligonucleotide, eFnoligo1 (5’-TTC TAA TAC GAC TCA CTA TAG GGA CAA TTA
CTA TTT ACA ATT ACA ATG CTC GAG GTC AAG G-3’), and Fnoligo9 (5’-GGA
GCC GCT ACC CTT ATC GTC GTC ATC CTT GTA ATC GGA TCC GGT GCG
GTA GTT GAT GGA GAT CG-3’) in a 10-mL PCR reaction.
2.2.3 mRNA Display
The first round of selection was started by PCR amplification (1.5 mL) of the
naïve e10Fn3 library to obtain a library with ~10 copies of the theoretical 10
12

independent sequences. The PCR product was used as the template in a 1.5 mL in vitro
transcription reaction using T7 RNA polymerase at 37 °C for 2 h to generate mRNA. The
22
transcription reaction was terminated with the addition of 1/10th of the reaction volume
of 0.5 M EDTA at pH 8.0. The RNA was purified by Urea PAGE electrophoresis,
electroelution, and ethanol precipitation. Purified RNA was ligated with the DNA linker-
puromycin, pF30P (5’-phosphate-dA
21
-9
3
-dAdCdC-Pu, where 9 is phosphoramidite
spacer 9, Pu is puromycin CPG, and the 5’-end was phosphorylated using chemical
phosphorylation reagent I; Glen Research Corp.), with a splint oligo, FN-pF30P-Splint
(5’-TTT TTT TTT TTT GGA GCC GCT ACC-3’, which is complementary to the 3’ end
of the RNA library and the 5’ end of pF30P) and T4 DNA ligase in a 1.0 mL reaction at
RT for 1 h. The library of mRNA-protein fusions was created via a 2.5 mL in vitro
translation reaction in rabbit reticulocyte lysate (Green Hectares; salts and buffers from
Novagen) where purified RNA-pF30P was translated at 30 °C for an 1h. To enhance
fusion formation, 10 µL of salt mixture (2 µL of 1 M MgCl
2
and 8 µL of 2.5 M KCl) was
added to each 25 µL of translation and incubated at RT for 15 min. Fusions were purified
with Oligo(dT) Cellulose Type 7 (GE Healthcare Life Science) in dT buffer (100 mM
Tris-HCl, pH 8.0, 1 M NaCl, 0.2% Triton X-100, and 1 mM EDTA) at 4 °C for 1 h and
then eluted with room temperature ddH
2
O. The elution, containing purified fusions, was
desalted and exchanged with the first strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM
KCl, and 3 mM MgCl
2
), using a 5 mL NAP-25 column (GE Healthcare Life Science).
The purified fusions were then reverse transcribed with reverse primer Fnoligo10 (5’-
GGA GCC GCT ACC CTT ATC GTCG-3’), using Superscript II enzyme (Invitrogen,
Carlsbad, CA) at 42 °C for 1 h to generate cDNA/mRNA-protein fusions for selection.
23
N-terminal biotin-tagged mDC-SIGN proteins were immobilized on acrylamide-
streptavidin beads (Pierce, Thermo Scientific, Rockford, IL) at RT for 1 h immediately
prior to the selection. mDC-SIGN-immobilized beads were resuspended in selection
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02%(v/v) Tween-20, 0.5 mg/mL
BSA, and 0.1 mg/mL tRNA) and incubated with the cDNA/mRNA-protein fusion library
at 4 °C for 1 h. After incubation, the beads were washed 4 times with selection buffer and
subjected to PCR to amplify bound fusions. The PCR product was labeled as pool 1 and
used to make the fusions for the next round of selection. From the second round of
selection, the volume of translation reaction was reduced to 100 µL, and FLAG
purification was applied to purify full-length fusions. After reverse transcription, C-
terminal FLAG-tagged fusions were pulled down with anti-FLAG M2 agarose beads
(Sigma) at 4 °C for 1 h and eluted with the FLAG peptide (Sigma) twice at RT. A
preclear step was also introduced to exclude nonspecific bead binders immediately before
selection. The FLAG-purified fusions were flowed through 100 µL of D-biotin treated
resin packed in small column. After two rounds of selection, the beads were switched to
agarose-neutravidin beads (Pierce) to avoid bead background binding. The selection was
the same for hDC-SIGN, except that hDC-SIGN ECD was used as the target. A four-
round selection was performed against hDC-SIGN for a total of seven rounds of
selection.
2.2.4 In Vitro Radiolabeled Binding Assay
The affinity of each pool was monitored by pulldown of radiolabeled fusions
against mDC-SIGN or hDC-SIGN immobilized on beads. L-[
35
S]methionine (MP
24
Biomedicals) was used to replace cold methionine in the translation reaction. After
translation, the reaction was treated with ribonuclease A (Roche Applied Science) to
generate pF30P-linked e10Fn3s. The radiolabeled fusions were incubated with mDC-
SIGN-immobilized beads, hDC-SIGN- immobilized beads, or beads without target at 4
°C for 1 h. The beads were washed 4 times with selection buffer and resuspended with
ddH
2
O. The radioactivity present in the flow through, four washes, and beads was
detected by scintillation counting. The total input of radiolabeled fusion added to the
binding reaction was the total of flow through, wash, and bead counts. The percent
binding of each pool was calculated by the ratio of bead counts/total input counts.
The final M3H4 pool was PCR amplified and introduced with two restriction
sites, Xho I and Bam HI using eFnoligo1 and Fnoligo11 as primers. The PCR products
were cloned into vector pAO5 by restriction digestion and ligation. Twenty colonies were
cultured, sequenced, and grouped by the similarity of sequences, resulting in seven clones
where two clones has high copy numbers and five singletons. They were further tested
for the affinity using the in vitro radiolabeled binding assay. The selected clones were
first PCR amplified using eFnoligo1 and Fnoligo9 and then subjected to in vitro
transcription to make mRNA. mRNA from each clone was translated in rabbit
reticulocyte lysate supplemented with
L
-[
35
S]methionine to generate radiolabeled
proteins. The radiolabeled proteins were purified by pull down with anti-FLAG M2 beads
and elution with 3X FLAG peptides. The binding of purified radiolabeled proteins and
the data analysis were the same as pool binding assay.
25
2.2.5 Cloning, Expression, and Purification of Wild-Type e10Fn3 (eFn-WT) and
Selected eFn-DC Clones with C-terminal HA Tag or Influenza Antigen Peptide
For the cell binding assay, a HA tag (YPYDVPDYA) was cloned onto the C-
terminus of eFn-WT and selected clones from the pools (eFn-DC1, eFn-DC2, eFn-DC3,
eFn-DC4, and eFn-DC6) in the pAO5 vector. Bacterial protein expression and
purification were optimized and conducted according to the Qiagen protocol for
purification under native conditions. Influenza antigen peptide sequence was introduced
following e10Fn3 and flanked by arginines and a His tag ((e10Fn3)-RR-GILGFVFTL-
RRR-HHHHH). The influenza peptide is derived from the influenza A matrix protein,
representing the HLA-A*0201 (human MHC class I molecule)- restricted
epitope.(Nijman and others 1993) The arginines were added to increase the proteasomal
digestion during processing of peptides that will be presented via the MHC class
I.(Dakappagari and others 2006; Sundaram and others 2003) All of these constructs were
expressed and purified using the same protocols as the HA-tagged protein.
2.2.6 Cell Functional Assay – Binding, Internalization
293T.hDCSIGN and 293T.mDCSIGN cells were described previously.(L. Yang
and others 2008) Five HA-tagged eFn-DC proteins (eFn-DC1, eFn-DC2, eFn-DC3, eFn-
DC4, and eFn-DC6) and HA-tagged eFn-WT were tested. For the binding test, cells were
treated with 10 nM of HA-tagged e10Fn3s at 4 °C for 30 min. The treated cells were then
washed with PBS and incubated with rabbit anti-HA antibody (Abcam, Cambridge, MA)
at 4 °C for 10 min. After incubation, the resulting cells were immunostained with
allophycocyanin (APC)-conjugated anti-rabbit IgG (Invitrogen) and analyzed via flow
26
cytometry (BD; data were analyzed using Flowjo). The approximate K
D
of eFn-DC6 was
determined by treating 293T.hDCSIGN or 293T.mDCSIGN cells with 0.4-800 nM of
eFn-DC6, followed by analysis using flow cytometry, as described previously.(Benedict
and others 1997) The internalization induced by eFn-DC6 was tested by first incubating
the 293T.hDCSIGN cells with 100 nM of HA-tagged eFn-DC6 at 4 °C for 30 min in
duplicate. After the unbound e10Fn3s were washed off, one sample was cultured at 37 °C

for another 2 h to induce internalization, while the duplicate was kept at 4 °C as a
noninternalizing control. Then, cell- surface staining for HA tag was performed, and the
difference of fluorescence intensity between 4 °C and 37 °C was calculated to obtain the
internalization efficiency.(Dakappagari and others 2006; Takahara and others 2004) PE-
conjugated anti-human DC-SIGN antibody was obtained from BioLegend (clone
9E9A8).
2.2.7 Confocal Imaging
Fluorescence images were acquired on a Yokogawa spinning-disk confocal
scanner system (Solamere Technology Group, Salt Lake City, UT) using a Nikon eclipse
Ti-E microscope equipped with a 60×/1.49 Apo TIRF oil objective and a Cascade II: 512
EMCCD camera (Photometrics, Tucson, AZ, USA). An AOTF (acousto-optical tunable
filter)-controlled laser-merge system (Solamere Technology Group Inc.) was used to
provide illumination power at each of the following laser lines: 491, 561, and 640 nm
solid-state lasers (50mW for each laser). Image processing and data analysis were carried
out using the Nikon NIS-Elements software. To quantify the extent of colocalization,
Mander’s overlap coefficients were generated using the Nikon NIS-Elements software by
27
viewing more than 50 cells at each time point. For the colocalization study, endocytic
markers, 293T.hDC-SIGN cells or iDCs were seeded on glass bottom dishes (MatTek
Corporation, Ashland, MA) and grown at 37 °C overnight. The cells were then incubated
with 100 nM HA-tagged eFn-DC6 for 30 min at 4 °C to synchronize internalization.
After being washed with PBS, the treated cells were then warmed to 37 °C to initiate
particle internalization for the indicated time periods. The cells were fixed, permeabilized
with 0.1% Triton X-100, and then immunostained with the corresponding antibodies
specific to clathrin, caveolin-1, or EEA1 (Santa Cruz Biotech., Santa Cruz, CA), followed
by counterstaining with DAPI (Invitrogen). Secondary antibody Alexa488-conjugated
goat anti-mouse immunoglobulin G (IgG) and Alexa594-conjugated anti-rabbit IgG
antibodies were purchased from Invitrogen (Carlsbad, CA). Mander's overlap coefficient
was calculated using more than 40 areas for each experiment.
2.2.8 Generation of Human DCs from Human PBMCs and Mouse DCs from Mouse
Bone Marrow
Human PBMC-derived DCs were generated and purified as previously
described,(Romani and others 1994; Sallusto and others 1994) except that human PBMCs
were from HLA-A2
+
patients (AllCells, LLC.). Briefly, plastic-adherent cell fractions of
human PBMCs were grown in RPMI 1640 with 10% heat-inactivated FBS, 2 mM
L
-
glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 1000 IU/mL GM-CSF and
1000 IU/mL IL-4 (PeproTech, Rocky Hill, NJ) for 6 days with media carefully changed
every 2 days. For mouse BMDC, we employed a previously described procedure to
generate bone marrow-derived DCs (BMDCs) with various genetic backgrounds. Briefly,
28
bone marrow from the femurs and tibias of mice was grown in RPMI 1640 with 10%
heat-inactivated FBS, 2 mM
L
-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin,
0.05 mM 2-ME, and 20 ng/mL GM-CSF (J558L supernatant) after the red blood cells
were lysed. Cultures were initiated by placing 10
7
bone marrow cells in 10 mL of
medium onto 100-mm Petri dishes (Falcon 1029 plates; BD Labware, Franklin Lakes,
NJ). On day 3, another 10 mL of J558L-conditioned medium was added. On day 6,
suspension cells were collected.
2.2.9 Enzyme-Linked-Immunospot (ELISPOT) Assay of DCs Co-cultured with
Autologous PBMCs
On day 6 of DC development, eFn-DC6 fused influenza peptide (eFn-DC6-Ag),
eFn-WT fused influenza peptide (eFn-WT-Ag), eFn-DC6 or GILGFVFTL peptide
antigen was added to the culture for 24 h. The ELISPOT assay was performed according
to a previous report(Hoffmann and others 2000) with some modifications. Briefly, anti-
human IFN-γ antibody (10 µg/mL in PBS, clone MD-1, BioLegend) was used as the
capture antibody and plated with 100 µL/well on 96-well MultiScreen-IP plates
(Millipore) overnight at 4 °C. The plate was decanted and blocked with the RPMI
medium containing 10% FBS at 37 °C for 2 h. On day 7, DCs treated with different
e10Fn3s were washed separately and cultured with freshly thawed autologous PBMCs (2
× 10
6
PBMCs co-cultured with 4 × 10
5
DCs in each well). After 60 h incubation at 37 °C,
cells were lysed, and plates were detected by 1 µg/mL biotinylated anti-IFN-γ antibody
(clone 4S.B3, BioLegend) for 2 h at RT. Plates were further washed and incubated with
the 1,000-fold-diluted streptavidin-alkaline phosphatase conjugate for 45 min at room
29
temperature. After a final extensive wash, spots were identified by adding BCIP/NBTplus
substrate (Millipore), and the number of IFN-γ-producing cells was quantified by an
ELISPOT reader.

Figure 2. 1. Illustration of mRNA display selecting e10Fn3 ligand to target dendritic cells
for antigen delivery.

Promoter ORF 5’ UTR
Puromycin
DNA
RNA
5’
dsDNA e10Fn3 library
mRNA-protein fusion
Immobilized DC-SIGN
cDNA
5’
5’
+
antigen peptide
MHC molecule
dendritic cell
T cell
antigen specific T cells
(activated by antigen presentation)
30
2.3 Results
Figure 2. 2. Expression and functional test of human and mouse DC-SIGN extracellular
domain (ECD).
(A) Schematic representation of the whole length of human and mouse DC-SIGN protein and the
ECD of each protein and necessary components that were constructed into the expression vector.
(B) SDS-PAGE analysis of recombinant DC-SIGN proteins: Lane 1, M, protein marker (in kDa);
Lane 2, UI, uninduced control from bacteria extract; Lane 3, I, IPTG-induced hDC-SIGN ECD;
Lane 4, His, His-tag-purified and refolded hDC-SIGN ECD; Lane 5, SA, hDC-SIGN ECD pulled
down by streptavidin acrylamide beads; Lane 6, UI, uninduced control from bacteria extract;
Lane 7, I, IPTG-induced mDC-SIGN ECD; Lane 8, His, His-tag-purified and refolded mDC-
SIGN ECD; Lane 9, SA, mDC-SIGN ECD pulled down by streptavidin acrylamide beads. (C) An
ELISA analysis of binding of Fc-gp120 protein to refolded DC-SIGN proteins produced by
bacterial expression.

2.3.1 Identification of DC-SIGN-specific ligands via mRNA display

31
Figure 2. 3. Figure 2.3. An mRNA display selection to identify e10Fn3 variants that can
bind to both human and mouse DC-SIGNs.
(A) Schematic representation of the procedure for 3 rounds of mDC-SIGN-targeted selection
followed by 4 more rounds of hDC-SIGN-targeted selection from the starting of e10Fn3 library.
(B) After the 3 rounds of mDC-SIGN-targeted selection, an in vitro radiolabeled binding assay
was used to assess the binding of each pool from the individual rounds (M3H0, 0 round; M3H2,
second round, M3H3, third round; M3H4, fourth round) of hDC-SIGN-based selection towards
hDC-SIGN, mDC-SIGN and background beads. (C) An in vitro radiolabeled binding assay was
used to estimate clones isolated from the last pool (M3H4) for binding to hDC-SIGN, mDC-
SIGN and background beads. (D) Protein sequence comparison among the wild-type 10Fn3,
wild-type expression-enhanced 10Fn3 (e10Fn3) and the clones isolated from the last pool
(M3H4).

The extracellular domain of human and mouse DC-SIGN proteins were produced
in E. coli, purified under denaturing condition and further refolded (Figure 2.2A and B).
It has been reported that hDC-SIGN, rather than mDC-SIGN, recognizes HIV-1
glycoprotein gp120 by the carbohydrate recognition domain (CRD) located in the
32
extracellular domain (Figure 2.2B). In order to test whether these refolded proteins could
maintain their binding function, a gp120 binding ELISA assay was carried out (Figure
2.2C). With increasing Fc-gp120 concentration, absorbance for hDC-SIGN ECD also
increased and became saturated when the Fc-gp120 concentration reached 100 nM,
indicating that refolded hDC-SIGN ECD was able to bind gp120. On the other hand, no
obvious binding of mDC-SIGN ECD to Fc-gp120 could be observed, further validating
the inability of mDC-SIGN to confer HIV-1 gp120 binding.
In order to generate DC-SIGN-specific ligands with the ability to bind both mouse
and human DC-SIGN, we first performed 3 rounds of selection against mDC-SIGN
followed by another 4 rounds against hDC-SIGN (Figure 2.3A). We then tested the
different pools’ binding against immobilized DC-SIGN. Starting with the M3H0 pool
(pool after 3 rounds of selection against mDC-SIGN and 0 round against hDC-SIGN), we
observed some affinity to mDC-SIGN (10% binding) but almost no affinity to hDC-
SIGN. However, for the M3H4 pool, increasing affinity to both mDC-SIGN (~40%) and
hDC-SIGN (~20%) was observed (Figure 2.3B). These data indicated that the final
M3H4 pool contained e10Fn3 scaffolds that could bind to both mDC-SIGN and hDC-
SIGN. Thus, the M3H4 pool was sequenced and yielded two dominant clones that were
observed multiple times, as well as five clones that appeared only once. The two
dominant clones, eFn-DC1 and eFn-DC7, contain similar BC and FG binding
loops,;however eFn-DC7 contains an E21K mutation in the BC loop. Additionally, these
two clones displayed similar results for an in vitro radiolabeled binding assay (data not
shown). Therefore, eFn-DC1 and five singletons (eFn-DC2, eFn-DC3, eFn-DC4, eFn-
33
DC5, and eFn-DC6) were tested further for binding using an in vitro radiolabeled binding
assay, and their sequences were illustrated in Figure 2.3D. Clone binding results shows
that five out of six clones (eFn-DC1, eFn-DC2, eFn-DC3, eFn-DC4, and eFn-DC6)
bound to both mDC-SIGN and hDC-SIGN; these clones possess higher affinity to mDC-
SIGN than hDC-SIGN with very little background binding to beads without target (<
1.4%), except for eFn-DC5, which has some background binding (Figure 2.3C), and was
not studied further.
2.3.2 Functional binding of e10Fn3 DC-SIGN ligands to cells expressing DC-SIGN
From the in vitro radiolabeled binding assay, five dual-specific e10Fn3 variants
were identified that could bind to bacterially expressed human and mouse DC-SIGN
ECD. A C-terminal HA tag was then added to each of these e10Fn3 scaffolds in order to
test their binding to either mDC-SIGN (293T.mDCSIGN) or hDC-SIGN
(293T.hDCSIGN) overexpressed on 293T cells. After binding, we immunostained and
analyzed the cells by flow cytometry. Our data demonstrate that only eFn-DC6 could
bind to 293T.hDCSIGN cells (relative MFI ≈ 27) and 293T.mDCSIGN cells (relative
MFI ≈ 3). The other four clones had no binding to 293T.mDCSIGN or 293T.hDCSIGN
cells (relative MFI ≈ 1) (Figure 2.4A). This result was somewhat surprising since all five
e10Fn3 variants bound well in the radiolabeled binding assay and may indicate that these
e10Fn3 variants bind to an epitope that is blocked when DC-SIGN is displayed on a cell
surface. Nonetheless, since eFn-DC6 could recognize both human and mouse DC-SIGNs,
its binding ability was further assessed by varying the concentration of eFn-DC6 (Figure
2.4B). The binding of eFn-DC6 was specific to human or mouse DC-SIGN, as shown by
34
Figure 2. 4. Selection and characterization of isolated e10Fn3 variants for their binding to
293T.hDC-SIGN and 293T.mDC-SIGN cells.
(A) 293T, 293T.hDC-SIGN, or 293T.mDC-SIGN cells were incubated at 4 °C with HA-tagged
wild-type e10Fn3 (eFn-WT) or selected clones from the last selection pool for 30 min; after the
washing step, cell surfaces were stained by anti-HA antibody followed by fluorescence-
conjugated secondary antibody for flow cytometry analysis. Relative mean fluorescence
intensities (MFIs) were calculated by dividing MFIs by MFI of eFn-WT binding to 293T cells.
(B) Various concentrations of eFn-DC6 were incubated with 293T, 293T.hDC-SIGN, or
293T.mDC-SIGN, and relative MFIs were measured. (C, D) Determination of K
D
value of eFn-
DC6 for its binding to 293T.hDC-SIGN (C) and 293T.mDC-SIGN (D) by Lineweaver-Burk
analysis. K
D
values were determined by the following equation: 1 ⁄ (F – F
back
) ≈ 1 ⁄ F
max
+ (K
D
⁄
F
max
)(1 ⁄ [eFn-DC6]), where F =fluorescence units, F
back
=background fluorescence, and F
max
was
calculated from the plot.

the absence of significant binding to 293T cells observed even up to 800 nM of eFn-DC6
(Figure 2.4B). The K
D
of the interaction between eFn-DC6 and hDC-SIGN was then
determined by Lineweaver-Burk kinetic analysis (Figure 2.4C), and the K
D
was
35
calculated as 19 ± 6 nM. Similarly, the K
D
of the interaction between eFn-DC6 and mDC-
SIGN was calculated as 133 ± 6 nM (Figure 2.4D). Therefore, eFn-DC6 was identified as
a specific ligand bearing a high affinity to hDC-SIGN and moderate affinity to mDC-
SIGN expressed on the surface of mammalian cells.
2.3.3 eFn-DC6-induced internalization into DC-SIGN-expressing cells
Based on its high and specific affinity to hDC-SIGN, our next experiments
focused on interactions between eFn-DC6 and human DC-SIGN. A good antigen carrier
should be able to mediate specific binding to a cell-surface receptor with subsequent
efficient internalization into cells for antigen processing. Therefore, eFn-DC6
internalization efficiency was quantified by a previously reported assay.(Dakappagari and
others 2006) The MFIs at 4

and 37

°C were then compared and used to calculate about a
50.2% internalization efficiency (Figure 2.5A).
It has been shown that DC-SIGN-mediated soluble antigen uptake and HIV virion
internalization are clathrin-dependent;(Cambi and others 2009; Joo and others 2008)
however, other studies have indicated that certain antibodies targeting DC-SIGN can
enter cells through a clathrin-independent mode.(Tacken and others 2011) In order to
facilitate rational design of a targeted antigen delivery strategy, it is necessary to
determine the factors mediating the internalization of eFn-DC6 through DC-SIGN. As
shown in Figure 2.5B, significant signal colocalization was observed between eFn-DC6
and clathrin, while colocalization between eFn-DC6 and caveolin-1 was less
36
Figure 2. 5. Internalization efficiency and intracellular trafficking of eFn-DC6 in
293T.hDC-SIGN cells.
(A) Internalization of eFn-DC6 into 293T.hDC-SIGN cells upon binding to hDC-SIGN. Two
groups of 293T.hDC-SIGN cells were incubated at 4 °C with 100 nM eFn-DC6 for 30 min. After
washing with PBS, one of them was then incubated at 4 °C, while the other was incubated at
37°C for 2 h. eFn-DC6 remaining on the cell surface was then stained by anti-HA antibody and
analyzed by flow cytometry. Internalization efficiency was calculated from mean fluorescence
intensity by the formula (MFI
4 °C
–

MFI
37 °C
) ⁄ MFI
4 °C
.

(B) Endocytic routes for the cell entry of
eFn-DC6. 293T.hDC-SIGN cells were incubated with 100 nM eFn-DC6 for 30 min at 4 °C to
synchronize internalization. The cells were then shifted to 37 °C for 15 min, fixed, permeabilized,
immunostained with an antibody against HA-tag (red), clathrin (green, upper) or caveolin-1
(green, lower), and counterstained with DAPI (blue). Scale bar represents 10 µm. (C)
Quantification of eFn-DC6 colocalized with clathrin or caveolin-1 signals after 15 min of
incubation. Mander’s overlap coefficients were calculated using Nikon NIS-Elements software by
viewing more than 50 cells of each sample. Statistical analysis of p-value was calculated by the
one-way ANOVA followed by Bonferroni’s multiple comparison test. (D) Intracellular
trafficking of eFn-DC6 through early endosomes. 293T.hDC-SIGN cells were incubated with
eFn-DC6 for 30 min at 4 °C to synchronize internalization. The cells were then shifted to 37 °C
for 30 min, fixed, permeabilized, immunostained with anti-HA tag (red) and anti-EEA1
antibodies (green), and counterstained with DAPI (blue). Scale bar represents 10 µm.
37
evident. A significant overlap between eFn-DC6 and clathrin was confirmed using
Mander’s overlap coefficients and imaging software (Figure 2.5C).
An efficient vaccine requires delivery of extracellular antigen to APCs and
presentation through the MHC molecules, but an antigen must first be internalized to an
early endosome. Thus, the early internalization pathway, following the binding of eFn-
DC6 to hDC-SIGN, was measured. Accordingly, eFn-DC6 was bound to 293T.hDC-
SIGN cells and incubated at 37

°C for 1 h. The cells were then fixed and stained for eFn-
DC6, while the marker EEA-1 was used to detect the early endosomes. From the
colocalization data, it was clear that eFn-DC6 was internalized to the early endosomes
following endocytosis (Figure 2.5D).
2.3.4 Binding and internalization of eFn-DC6 into DCs
We demonstrated that eFn-DC6 could specifically bind to hDC-SIGN, mDC-
SIGN and mediate internalization into cell lines that overexpress hDC-SIGN. However, it
was unknown whether eFn-DC6 could directly target primary DCs and mediate antigen
internalization. To evaluate this, we generated DCs from human peripheral blood
mononuclear cells (PBMCs). After in vitro differentiation, human DCs were incubated
with eFn-DC6, co-stained by anti-DC-SIGN and anti-eFn-DC6 antibodies, and analyzed
by flow cytometry. More than 90% of human DCs that were derived from PBMCs had a
high level of DC-SIGN expression. These DC-SIGN-positive DCs were also recognized
by eFn-DC6, but not the original wild-type e10Fn3 (designated as eFn-WT) (Figure
2.6A). The same assay was conducted using mouse DCs derived from bone marrow. As
shown in Figure 2.6B, around 84% of cells express CD11c and around 17% cells that
38
Figure 2. 6. Specific binding and internalization of eFn-DC6 into DCs.
(A) Human PBMCs derived DCs were incubated with eFn-DC6 or eFn-WT, stained by anti-HA
and anti-DC-SIGN antibodies, and further analyzed by flow cytometry. (B) Mouse bone marrow
derived DCs were incubated with eFn-DC6 or eFn-WT, stained by anti-HA and anti-CD11c
antibody, and analyzed. (C, D) Endocytic routes for eFn-DC6 to enter DCs. Human PBMC
derived DCs were incubated with eFn-DC6 for 30 min at 4 °C to synchronize internalization. The
cells were then shifted to 37 °C for 15 min, fixed, permeabilized, and immunostained with an
antibody against HA-tag (red) and clathrin or caveolin-1 (green) (C) or EEA1 (D) and further
counterstained with DAPI (blue). Scale bar represents 10 µm.

were detected by eFn-DC6, while eFn-WT could not show any signal. We further
conducted a similar internalization assay with human DCs and found that eFn-DC6
utilized a more clathrin-dependent and less caveolin-1-dependent entry pathway into DCs
39
(Figure 2.6C). Colocalization of eFn-DC6 and early endosome marker EEA-1 was also
detected (Figure 2.6D), suggesting that eFn-DC6-induced internalization could favor
antigen presentation to DCs.
2.3.5 Capacity of eFn-DC6 to mediate antigen delivery to DCs in vitro
Next, we evaluated the capacity of eFn-DC6 to mediate antigen delivery to DCs
in vitro. A new construct was designed whereby a human MHC I restricted antigen
epitope from influenza A matrix protein (58GILGFVFTL66)(Nijman and others 1993)
was fused to the C terminus of eFn-DC6 (designated as eFn-DC6-Ag). The peptide was
flanked by 2 arginines to the N terminus and 3 arginines to the C terminus (Figure 2.7A)
based on a previous study indicating that such design could maximize proper antigen
processing by proteasomes after internalization.(Sundaram and others 2003) A similar
design using eFn-WT (designated as eFn-WT-Ag) was also constructed as a control.
Previous studies have shown that cells from HLA-A2
+
adult peripheral blood contain
around 1.6% of GILGFVFTL-responsive CD8 T cells from previous exposure to
influenza virus. Thus, if eFn-DC6 could be demonstrated to facilitate internalization,
processing, and presentation of the influenza antigen peptide by DCs to autologous
PBMCs from HLA-A2
+
adult peripheral blood, then a higher response should be
observed over the control. To make this determination, HLA-A2
+
PBMCs derived DCs
were incubated with eFn-DC6-Ag and as controls, eFn-WT-Ag, eFn-DC6 lacking any
antigen (eFn-DC6) or GILGFVFTL peptide antigen. One day later, these three groups of
DCs were incubated with autologous PBMCs for 60 h. An ELISPOT assay for human
40
Figure 2. 7. Antigen-specific IFN-γ release of autologous human PBMCs after coculture
with DCs treated by eFn-DC6-fused antigens.
(A) Schematic representation of the construction of eFn-DC6 (eFn-DC6-Ag) and eFn-WT (eFn-
WT-Ag) fused with an influenza matrix protein antigen peptide GILGFVFTL, which was flanked
by 2 arginines to the N terminus and 3 arginines to the C terminus. (B) An ELISPOT assay was
used to measure the IFN-γ production by the DC-stimulated CD8
+
T cells. DCs were first treated
with 1uM eFn-DC6-Ag, eFn-WT-Ag, eFn-DC6, GILGFVFTL peptide antigen as positive control,
or not treated as a negative control for 24 h. These treated DCs in each group were further
cocultured with autologous human PBMCs for 60 h. Spot-forming cells (SFC) were measured by
an ELISPOT reader. The experiment was performed in triplicate. (n/s: not statistically significant;
p-value was calculated by one-way ANOVA followed by Bonferroni’s multiple comparison test.
Error bars represent SD.)

IFN-γ was employed to measure the stimulated T cell responses. As shown in Figure 2.67,
DCs treated with eFn-DC6 were unable to induce observable responses, while eFn-WT-
Ag-treated DCs did generate some response, presumably caused by the random
41
internalization of the antigen. However, eFn-DC6-Ag produced a much higher response
(around 150 SFC) than that of eFn-WT-Ag (around 50 SFC). Taken together, our data
show that eFn-DC6 not only specifically delivered antigens to DCs expressing hDC-
SIGN, but also induced an antigen-specific immune response.
2.4 Discussion
In the present chapter, we exploited mRNA display and a scaffold library of
antibody mimics based on an enhanced tenth fibronectin type III domain (e10Fn3) to
design ligands able to specifically bind to the type II C-type lectin DC-SIGN and mediate
antigen delivery to DCs. Since human and mouse DC-SIGNs have a homologous
carbohydrate recognition domain (CRD),(C. G. Park and others 2001) we postulated that
this design strategy would allow us to identify novel binders with an affinity to both
mDC-SIGN and hDC-SIGN. After seven rounds of selection, we found that one ligand,
eFn-DC6, displayed dual affinity for hDC-SIGN and mDC-SIGN expressed by
mammalian cells when tested in a cell-based binding assay. eFn-DC6 demonstrated
excellent affinity to 293T.hDCSIGN cells and moderate affinity to 293T.mDCSIGN cells
(Figure 2.4).
The different binding behaviors for the selected e10Fn3 variants toward DC-
SIGNs suggest possible differences in the structural configuration between the
recombinant soluble DC-SIGNs and their cell-surface-displayed counterparts. The
recombinant DC-SIGNs were expressed in E. coli and refolded in order to obtain
functional proteins. The refolded hDC-SIGN was able to recognize the HIV-1 gp120
42
protein, indicating that hDC-SIGN was folded appropriately and formed a tetramer. It
should be noted that multimerization is necessary for the binding of HIV-1 Env to hDC-
SIGN.(Bernhard and others 2004; Takahara and others 2004) In contrast, the DC-SIGNs
expressed on the mammalian cells underwent protein modification and folding in the
endoplasmic reticulum (ER), as well as further membrane trafficking, in order to be
sorted to cell surface. Thus, it is conceivable that the structure of membrane displayed
DC-SIGN presented on the 293T surface might be different from that of soluble DC-
SIGN, thereby masking binding epitopes present in vitro and preventing several of our
selected e10Fn3 variants from binding. In this regard, it remains interesting to investigate
whether it is viable and perhaps more efficient for a selection directly against DC-SIGN
expressed on the mammalian cell surface.
Many antitumor and antiviral vaccines require the induction of cytotoxic CD8
+
T
cells.(Kagi and others 1994; Tanaka and others 1999; Wong and others 2003) In order to
establish CD8
+
T cell immunity, antigen peptides need to be presented first by the MHC I
molecules on APCs and usually require antigens from an endogenous source.(Trombetta
and others 2005) Exogenous antigens are usually internalized, processed, and presented
to CD4
+
T cells by MHC II molecules.(Trombetta and others 2005) However, the
generation of CD8
+
T cell immunity by presentation of exogenous antigen by MHC I can
be accomplished by another naturally occurring pathway: when exogenous antigens are
internalized into cells and later processed by proteasomes through a process termed cross-
presentation.(Ackerman and others 2004) Therefore, it was important to evaluate whether
eFn-DC6-mediated antigen delivery could trigger the cross-presentation function of
43
APCs and, hence, stimulate antigen-specific CD8
+
T responses. For this purpose, we
chose a MHC I-restricted epitope derived from the influenza matrix protein that is
specific for human HLA-A2
+
as a model antigen for the study.(Nijman and others 1993)
Because most humans have been previously exposed to influenza virus, most human
sources of HLA-A2
+
PBMCs contain CD8
+
T cells reactive to the chosen epitope peptide.
This antigen peptide was fused to the C terminus of eFn-DC6 or eFn-WT to generate
chimeric molecules for DC-SIGN-targeted antigen delivery (Figure 2.7A). From the co-
culture experiment, the eFn-DC6-Ag group showed much higher responses than those of
the eFn-WT-Ag group (Figure 2.7B). These data confirmed that antigen delivered via
eFn-DC6-mediated targeting of DC-SIGN could be cross-presented by DCs, resulting in
CD8
+
T cell responses. Although the mechanism of eFn-DC6-mediated cross-
presentation remains elusive, we speculate that binding of eFn-DC6 to DC-SIGN induces
internalization of antigens to endosomes. Through egress from the endosomal
compartment, antigens leak into the cytosol, where they gain access to the ER-based class
I process machinery.(Ackerman and others 2004; Guermonprez and others 2003) A
similar mechanism could be used to explain the CD8
+
T cell responses generated by
antibody-mediated antigen delivery to the type I C-type lectin mannose receptor
(MR)(Ramakrishna and others 2004) and DEC-205.(Bonifaz and others 2002)
In conclusion, we constructed a library by randomizing two loops of an enhanced
fibronectin scaffold (e10Fn3) and exploited the mRNA display method to successfully
identify an e10Fn3 variant, eFn-DC6, which possesses high specificity and affinity to
hDC-SIGN and moderate affinity to mDC-SIGN. This ligand was found to enter cells in
44
a more clathrin-dependent pathway. As an antigen carrier, we demonstrated that eFn-
DC6 was able to direct antigen delivery to DCs and mediate cross-presentation of
antigens to elicit class I-based cytotoxic CD8
+
T cell responses in vitro. Taken together,
this study demonstrates the feasibility of using mRNA display selection as a method of
identifying high-affinity ligands as vaccine carriers for targeted delivery of antigens to
DCs, thus paving the way for the design of novel molecules able to target other C-type
lectins for DC-directed immunization.
2.5 Acknowledgements
We would like to thank Dr. C. Olson Anders for his help in e10Fn3 library
construction and Jun Zhao for the gift of the wild type e10Fn3 clone. This work was
supported by grants from the National Institutes of Health (R01AI68978 and
P01CA132681) and a translational acceleration grant from the Joint Center for
Translational Medicine.

45
Chapter 3
DENDRITIC CELL-DIRECTED VACCINATION WITH A
LENTIVECTOR ENCODING PSCA FOR PROSTATE
CANCER IN MICE

Portions of this chapter are adapted from: Liang Xiao, Kye-Il Joo, Matthew Lim, and Pin
Wang. PLoS One (2012), 7(11), e48866.

Many studies have demonstrated that prostate stem cell antigen (PSCA) is an
attractive target for immunotherapy based on its overexpression in prostate tumor tissue,
especially in some metastatic tissues. In this chapter, we evaluated dendritic cell (DC)-
directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a novel
tumor vaccine for prostate cancer in mouse models. We showed that DCLV-PSCA could
preferentially deliver the PSCA antigen gene to DC-SIGN-expressing 293T cells and
bone marrow-derived DCs (BMDCs). Direct immunization with the DCLV-PSCA in
male C57BL/6 mice elicited robust PSCA-responsive CD8
+
and CD4
+
T cells in vivo. In
a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1) synergetic tumor
model, we further demonstrated that DCLV-PSCA-vaccinated mice could be protected
from lethal tumor challenge in a prophylactic model, whereas slower tumor growth was
46
observed in a therapeutic model. This DCLV-PSCA vaccine also showed efficacy in
inhibiting tumor metastases using a PSCA-expressing B16-F10 model. Taken together,
these data suggest that DCLV is a potent vaccine carrier for PSCA in delivering anti-
prostate cancer immunity.
3.1 Introduction
In 2011, the FDA approved the first therapeutic cancer vaccine for treatment of
asymptomatic or slightly symptomatic hormone refractory prostate cancer,(Cheever and
others 2011; Kantoff, Higano, and others 2010) a great encouragement for both prostate
cancer patients and scientists working on cancer immunotherapy. Immunologic therapies
can instruct the immune system to recognize and eliminate tumor cells, which, under
normal conditions, usually escape from immune surveillance by downregulating tumor
antigen presentation(Khanna 1998) or by initiating immune tolerance.(Sakaguchi and
others 2008; Swann and others 2007) Presently, several antigens have been identified as
potential immunotherapy candidates for prostate cancer vaccines. They include the
prostate-specific antigen (PSA),(Kantoff, Schuetz, and others 2010) prostate stem cell
antigen (PSCA),(Dannull and others 2000; Raff and others 2009; Ross and others 2002;
D. Yang and others 2001) prostate-specific membrane antigen (PSMA),(Tjoa and others
1998) prostatic acid phosphatase (PAP),(Kantoff, Higano, and others 2010) mucin 1
(MUC1),(Pantuck and others 2004) gonadotropin-releasing hormone (GnRH),(Junco and
others 2007) and NY-ESO-1 vaccine,(Karbach and others 2011) among others. PSCA is a
123-amino acid glycosylphosphatidylinositol (GPI)-linked cell-surface protein belonging
47
to the Ly-6 family.(Reiter and others 1998) PSCA is an attractive immunotherapeutic
target based on its overexpression in a majority of prostate cancer cells, while its
expression in other somatic tissues is highly limited.(Gu and others 2000) Although the
specific mechanism underlying the contribution of PSCA to tumor growth remains
undefined, PSCA has been found to correlate positively with tumor malignancy,
pathology grade and androgen-independence.(Gu and others 2000; Zhigang and others
2004) It was suggested that PSCA might play a role in counteracting natural immune
response.(Marra and others 2010) Moreover, PSCA expression was upregulated in
metastatic tissues.(Gu and others 2000; Lam and others 2005) Currently, antibody
directed to PSCA has been tested to inhibit prostate cancer tumor growth and suppress
metastasis formation,(Saffran and others 2001) while others have investigated chimeric
antigen receptors (CAR)-based adoptive T cells therapy targeting PSCA for its potential
in treating prostate cancer.(Morgenroth and others 2007) Experiments have also been
conducted to test PSCA as a vaccine antigen, and it has been clearly shown in animal
models that PSCA-targeted vaccines can slow down prostate cancer progression.(Ahmad
and others 2009; Garcia-Hernandez Mde and others 2008)
Lentiviral vectors (LVs) are promising vectors for cancer
immunotherapy,(Breckpot, Aerts, and others 2007; Breckpot and others 2003) and they
are currently being evaluated in many clinical trials for a wide range of human
diseases.(Escors and others 2010) One desired trait of LVs is their ability to transduce
both dividing and nondividing cells,(Naldini and others 1996) including peripheral
DCs.(Koya and others 2007; Schroers and others 2000) As a vaccine carrier, LVs can
48
simultaneously deliver antigens to DCs(Breckpot and others 2003) and activate DCs
through toll-like receptors (TLRs).(Breckpot, Emeagi, and others 2007; Breckpot and
others 2010; Brown and others 2007; Grabski and others 2011; Rossetti and others 2011;
Tan and others 2005; Vandendriessche and others 2007) To further improve LVs, much
effort has been directed toward targeting LVs to antigen-presenting cells (APCs) in vivo
to achieve better specificity and safety.(Ageichik and others 2008; Cui and others 2002;
Gennari and others 2009; Goyvaerts and others 2012) We previously reported a DC-
directed LV (DCLV), which can specifically target DCs expressing DC-specific
intercellular adhesion molecule grabbing non-integrin (DC-SIGN) and deliver antigen
genes to them. Direct in vivo vaccination using DCLV encoding chicken ovalbumin
(OVA) elicited high frequency of OVA-specific CD8
+
and CD4
+
T cell
responses.(Esslinger and others 2003; Lopes and others 2008; L. Yang and others 2008)
In this chapter, we investigated DCLV-mediated cancer vaccines in a more
clinically related setting and explored the potency of this vectored immunization to
overcome the immune tolerance to the self-tumor antigen PSCA and to generate
protective immunity against prostate cancer. We showed that DCLV encoding PSCA
(DCLV-PSCA) could target DC-SIGN-expressing cell lines and bone marrow-derived
DCs (BMDCs). Direct immunization at the base of the tail evoked strong PSCA-specific
T cell responses in a mouse prostate cancer model. Furthermore, vaccination could
significantly inhibit tumor growth upon challenge with TRAMP-C1 tumor cells in mice.
When this vaccine was utilized in a therapeutic setting, it could suppress the growth of
established TRAMP-C1 tumors. Our data showed that anti-prostate tumor immunity
49
conferred by DCLV-PSCA depends on the presence of both CD8
+
and CD4
+
T cells.
Finally, we demonstrated that immunization with DCLV-PSCA could efficiently inhibit
the metastasis of B16-PSCA cells in lung tissue.
3.2 Materials and Methods
3.2.1 Mice and cell lines
Male C57BL/6 mice (6-8 weeks old) were purchased from the Charles River
Laboratories (Wilmington, MA, USA). All mice were maintained in the animal facilities
at the University of Southern California (USC) under controlled temperature and a 12h
light/dark cycle, with free access to water and standard laboratory chow. Animal
procedures were performed in accordance with the guidelines set by the National
Institutes of Health (NIH Publication No. 85-23, revised 1996) and the animal protocol
was approved by the Institutional Animal Care and Use Committee of the USC (2010-
11450). The tumor size of 2000 mm
3
was used as a surrogate endpoint of survival, and
mice will be euthanized by CO2 inhalation from a tank source and a follow-up cervical
dislocation. TRAMP-C1 cells were obtained from ATCC (Manassas, VA, USA) and
cultured in DMEM high glucose (Cellgro, Manassas, VA, USA) with L-glutamine
supplemented with 5% FBS, 5% Nu Serum IV (BD Biosciences, San Jose, CA, USA),
bovine pancreas insulin 5 µg/mL (Sigma, St. Louis, MO, USA) and 10 nM
dehydroisoandrosterone (ChromaDex, Irvine, CA, USA). B16-F10 cells were purchased
from ATCC (Manassas, VA, USA) and cultured in DMEM high glucose (Cellgro,
Manassas, VA, USA) with L-glutamine supplemented with 10% FBS. B16-F10 cells
50
stably expressing PSCA were generated by transducing B16-F10 cells with lentivirus
(FUW-PSCA) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG), and
clonal cells were selected.
3.2.2 Construction and production of lentiviral vectors
The lentiviral backbone plasmid FUW-PSCA was constructed by insertion of the
cDNA of murine PSCA downstream of the ubiquitin promoter in FUW. FUW is a HIV-
1-derived lentiviral plasmid composed of an internal human ubiquitin-C promoter to
drive transgene expression and woodchuck responsive element to improve stability of the
RNA transcript.(Lois and others 2002) We employed a previously reported procedure of
transient transfection of 293T cells to produce the DCLV-PSCA vector.(L. Yang and
others 2008) Briefly, 293T cells cultured in a 15-cm tissue culture plate (BD Biosciences,
San Jose, CA, USA) were transfected via a standard calcium phosphate precipitation
method with the following plasmids: the lentiviral backbone plasmid FUW-PSCA (37.5
µg, Figure 3.1A), the plasmid encoding the mutant Sindbis virus glycoprotein (SVGmu,
18.75 µg, Figure 3.1B), and the packaging plasmids (pMDLg/pRRE and pRSV-Rev,
18.75 µg each). The viral supernatants were harvested twice at 48 and 72 hrs post-
transfection, pooled, and filtered through a 0.45-mm filter (Corning, Lowell, MA, USA).
The concentrated viral pellets were obtained after ultracentrifugation of the viral
supernatants at 50,000 ×g for 90 min and were then resuspended in an appropriate
volume of HBSS for in vivo administration.
51
3.2.3 BMDC generation and staining
Bone marrow-derived DCs (BMDCs) were generated according to a previously
described procedure.(L. Yang and others 2008) Briefly, bone marrow from the femurs
and tibias of male C57BL/6 mice was grown in RPMI 1640 with 10% heat-inactivated
FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-
ME, and 20 ng/mL GM-CSF (J558L supernatant) after the red blood cells were lysed.
Cultures were initiated by placing 10
7
bone marrow cells in 10 mL of medium onto 100-
mm petri dishes (Falcon 1029 plates; BD Labware, Franklin Lakes, NJ). On day 3,
another 10 mL of J558L-conditioned medium were added. On day 6, suspension cells
were collected. BMDCs were seeded at a density of 0.5 million/mL in 24-well plates (BD
Labware) and spin-transduced twice with either DC-directed LV without antigen
insertion (DCLV-Null) or DCLV-PSCA at 2500 rpm and 25°C for 90 min. Five days later,
BMDCs were collected and incubated with anti-mouse CD16/CD32 Fc blocking antibody
and then stained with rabbit anti-mouse PSCA (clone M-70, Santa Cruz Biotechnology,
Santa Cruz, CA, USA) at 4°C for 20 min. After a washing step, BMDCs were further
incubated with donkey anti-rabbit IgG-PE (Abcam, San Francisco, CA, USA) and anti-
CD11c-PE-Cy5 (BioLegend, San Diego, CA, USA) at 4°C for 10 min, followed by
washing and analysis by BD LSRII flow cytometer (BD Biosciences). Acquired data
were analyzed using FlowJo software (Tree Star, Ashland, OR).
3.2.4 In vivo depletion of CD4
+
or CD8
+
T cells
Four groups of mice were implanted with 5 × 10
5
TRAMP-C1 cells
subcutaneously at day 0. Fourteen days later, three groups of mice were injected with
52
8×10
7
transduction units (TU) of replication-defective DC-LV-PSCA at the base of tail.
At day 21, 24, 27, 30 and 33, each group of immunized tumor-bearing mice was
intraperitoneally injected with one of the following antibodies: 200 µg CD4 antibody
(clone GK1.5, BioXCell, West Lebanon, NH), 200 µg CD8 antibody (clone 53.6.72,
BioXCell), or 200 µg isotype antibody (BioXCell). Tumor growth was monitored.
3.2.5 IFN-γ intracellular cytokine staining (ICCS)
Splenocytes from immunized or control mice were pooled and incubated with the
PSCA
83-91
peptide (NITCCYSDL) (GenScript, Piscataway, NJ, USA) at final
concentration of 50 µg/L for 2 h at 37°C in a 96-well round-bottom plate in RPMI
medium supplemented with 10% FBS (Sigma), 10 U/mL penicillin, 100 µg/mL
streptomycin, and 2 mM glutamine. Brefeldin A (BFA, Sigma, St. Louis, MO) was added
(10 µg/mL) to wells to inhibit cytokine exporting for another 6 h. Surface staining was
performed by incubating restimulated cells with anti-mouse CD16/CD32 Fc blocking
antibody, followed by anti-mouse CD8 and anti-mouse CD4 antibodies. Cells were then
permeabilized in 100 µl Cytofix/Cytoperm solution (BD Biosciences) at 4°C for 10 min,
washed with Perm/Wash buffer (BD Biosciences), followed by intracellular staining with
PE-conjugated anti-mouse IFN-γ at 4°C for 15 min. Flow cytometry analysis was carried
out using the FACSort instrument from BD Biosciences.
3.2.6 Enzyme-linked immunosorbent spot (ELISPOT) assay
To measure PSCA-specific CD8
+
T cell responses, ELISPOT assays were
performed to detect IFN-γ using a kit from Millipore (Billerica, MA) according to the
53
manufacturer’s instruction. Briefly, anti-mouse IFN-γ antibody (10 µg/mL in PBS) was
used as the capture antibody and plated with 100 µl/well on 96-well MultiScreen-IP
plates overnight at 4°C. The plate was decanted and blocked with the RPMI medium
containing 10% FBS at 37°C for 2 h. Splenocytes from vaccinated mice were plated at 2
× 10
5
cells/well in 100 µl complete medium in the presence of the CD8 epitope PSCA
83-91
peptide (50 µg/mL). After 18 h incubation at 37°C, cells were lysed, and plates were
detected by 1 µg/mL biotinylated anti-IFN-γ antibody (BD Biosciences) for 2 h at room
temperature. Plates were further washed and incubated with the 1,000-fold-diluted
streptavidin-alkaline phosphate conjugate for 45 min at room temperature. After a final
extensive wash, spots were identified by adding BCIP/NBTplus substrate (Millipore), and
the number of IFN-γ-producing cells was quantified by an ELISPOT reader. An IL-2
ELISPOT assay was also performed to examine PSCA-specific CD4
+
T cell responses.
The entire procedure is similar to the IFN-γ ELISPOT assay, except that IL-2 capture and
detection antibodies were used instead; splenocytes with CD8
+
T cells depleted using
CD8 MicroBead Kit (Miltenyi Biotec, Auburn, CA, USA) were co-cultured for 40 h with
lysates from 293T cells transfected with the FUW-PSCA plasmid.
3.2.7 Histological analysis
TRAMP-C1 tumor-bearing mice were injected with DC-LV-PSCA (8×10
7
TU at
the base of tail) or untreated as a control. Twenty days later, tumors were excised,
paraffin embedded and sectioned (5 µm thickness). The following antibodies were
employed to detect tumor-infiltrating lymphocytes: anti-CD3-Alexa488 (clone 17A2
from BD Biosciences, San Jose, CA, USA), anti-CD4-Alexa488 (clone RM4-5 from BD
54
Biosciences), anti-CD8-FITC (clone 53-6.7 from BD Biosciences). TO-PRO-3
(Invitrogen, Carlsbad, CA, USA) was used for nucleus staining. For the B16-PSCA
metastasis experiment, lungs from the mice were excised, paraffin embedded, sectioned
(5 µm thickness), and H&E stained. Samples were then analyzed microscopically with a
20× objective.
3.2.8 Statistics
All the statistics were calculated by either Origin Pro 7.0 or GraphPad Prism 5 software.
Error Bars in all the figures represent SD, except for the tumor growth curves in the
prophylactic and therapeutic tumor challenge models, in which SEM was used. One-way
ANOVA followed by Bonferroni’s multiple comparison test was used to determine the
significance of difference, while animal survival curves were analyzed by log-rank
(Mantel-Cox) test, and the value of P < 0.05 was considered to be statistically significant.
55
3.3 Results
3.3.1 Generation of DCLV-PSCA and its ability to target DC-SIGN-expressing cells
in vitro
Figure 3. 1. Schematic representation of key constructs used in this study.
(A) Lentiviral backbone construct encoding murine prostate stem cell antigen. ΔU3, R, and U5
and are components of the long terminal repeat (LTR), and the ΔU3 region contains the self-
inactivating deletion; SD: splicing donor; SA: splicing acceptor; ψ and ΔGag: the encapsulation
sequence; RRE: the Rev-responsive element; Ubi: human ubiquitin-C promoter; WPRE:
woodchuck hepatitis virus post-transcriptional regulatory element. (B) The glycoprotein derived
from Sindbis virus glycoprotein consists of two membrane domains (E1 for fusion induction and
E2 for receptor recognition) and a signal peptide (E3). Three mutations were introduced to disrupt
the binding of heparin sulfate glycosaminoglycan while retaining the ability of LV to bind DC-
SIGN, including 1) deletion of amino acids 61–64 of E3 domain, 2) insertion of HA tag
(MYPYDVPDYA) between amino acids 71 and 74 of E2 domain and 3) mutations of 157KE158
to 157AA158 of E2 domain.

We constructed a lentiviral backbone encoding the full length of murine PSCA
and tested the expression of PSCA in 293T cells. 293T cells were transiently transfected
with FUW-Null or FUW-PSCA (Figure 3.1A) vector. Two days after transfection, the
56
Figure 3. 2. Targeted transduction and delivery of PSCA antigen gene into dendritic cells
(DCs) by DCLV-PSCA.
(A) 293T cells were transfected transiently with plasmids FUW-Null (mock control) or FUW-
PSCA. Two days later, cells were collected and stained for PSCA expression analyzed by flow
cytometry. 293T cells stained with the isotype antibody were included as a control. (B) 293T cells
were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral
packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to
transduce 293T cells or 293T.hDC-SIGN cells. PSCA expression was analyzed by flow
cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock
vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were
assessed by flow cytometric analysis.

cells were collected for expression of PSCA by fluorescence-activated cell sorter (FACS)
analysis. 293T cells transfected with the FUW-PSCA plasmid showed positive
expression of PSCA (22.5%), while cells transfected with the FUW-Null plasmid had
only background staining (Figure 3.2A).
We then utilized previously reported 293T.DC-SIGN cells(L. Yang and others
2008) to investigate the ability of DCLV to express PSCA. As shown in Figure 3.2B,
57
approximately 60% of the 293T.DC-SIGN cells displayed PSCA expression post-DCLV-
PSCA transduction, whereas only 6.79% were PSCA-positive in the 293T cells. The
specificity observed here is consistent with previous reports showing the ability of DCLV
to preferentially transduce DC-SIGN-expressing cells.(H. Yang and others 2011; L. Yang
and others 2008) We further investigated whether DCLV-PSCA could target and mediate
PSCA expression in bone marrow-derived DCs (BMDCs). The immature BMDCs were
derived from the murine bone marrow culture and confirmed by flow cytometric analysis
of cell surface marker CD11c (Figure 3.2C). When exposed to LVs, DCs were selectively
modified by DCLV-PSCA to express PSCA (3.65% in the CD11c
+
cells vs. 0.11% in the
CD11c
‒
cells, Figure 3.2C). Our results indicated that DCLV-PSCA could target DC-
SIGN-expressing cells and deliver the PSCA antigen to DCs in vitro.
3.3.2 Induction of PSCA-specific CD8
+
and CD4
+
T cell immune responses in vivo
To determine whether this recombinant DCLV-PSCA vector could efficiently
deliver the antigen to DCs and mount antigen-specific T cell responses in vivo, we
performed vaccination with DCLV-PSCA directly to male C57BL/6 mice. Because of the
variation of DC distribution, immunization carried out through different administration
routes may result in different numbers of DCs to be targeted, leading to different levels of
antigen presentation. As such, comparison of the immunogenic response elicited through
different routes is necessary to establish an optimal immunization protocol. Therefore,
naïve male C57BL/6 mice were immunized with a single dose of DCLV-PSCA (6×10
7

TU) at intradermal area (i.d., at the base of tail), footpad area (f.p.), subcutaneous area
(s.c.), or intraperitoneal space (i.p.). A previously reported CD8
+
epitope peptide for
58
Figure 3. 3. PSCA-specific T cell response after a single dose of in vivo immunization with
DCLV-PSCA.
(A) Male C57BL/6 mice were immunized with 6×10
7
TU of DCLV-PSCA through different
administration routes: intraperitoneal space (i.p.), subcutaneous area (s.c.), footpad (f.p.), or
intradermal (the base of tail, i.d.). One immunization group was included as a negative control.
Two weeks after immunization, splenocytes from mice were harvested and analyzed for the
presence of PSCA-specific CD8
+
T cells by restimulating splenocytes with a PSCA peptide
(PSCA
83-91
), followed by intracellular staining for IFN-γ and surface staining for CD8. Percentage
of IFN-γ-secreting CD8
+
T cells is indicated. (B) Statistical comparison of immunization elicited
by administration of DCLV-PSCA among different administration routes. (C) Male C57BL/6
mice were immunized with different doses of DCLV-PSCA vectors (0, 2, 10, 40 and 80 million
TU) at the base of tail. Two weeks post-vaccination, PSCA-specific CD8
+
T cells from the spleen
were analyzed by restimulating with the peptide PSCA
83-91
, followed by intracellular staining for
IFN-γ. (D) Production of PSCA-specific IFN-γ-secreting cells from both spleen (SP) and inguinal
lymph node (LN) was evaluated by restimulation with the PSCA
83-91
peptide, followed by
ELISPOT analysis for IFN-γ. (E) Production of PSCA-specific IL-2 from splenocytes (with CD8
+

T cells depleted) was measured by restimulation with 293T cell lysate transfected to express
59
PSCA, followed by the ELISPOT analysis for IL-2. (**: P < 0.01; *: P < 0.05; One-way ANOVA
followed by Bonferroni’s multiple comparison test. Error bars represent SD.)

PSCA(Garcia-Hernandez Mde and others 2008) was used to characterize PSCA-specific
CD8
+
T cell responses in the spleen via IFN-γ intracellular cytokine staining (ICCS). As
depicted in Figure 3.3A and B, the i.d. and f.p. administration routes resulted in the
strongest PSCA-specific CD8
+
T cell response (~2%) two weeks post-immunization,
whereas the s.c. and i.p. injections resulted in a much lower response (< 0.5%). This
response trend is consistent with results from immunization with DCLV encoding HIV-1
Gag(Dai and others 2009) or human gp100.(H. Yang and others 2011) To further assess
the antigen-specific CD8
+
T cell responses elicited by i.d. immunization, an ELISPOT
experiment measuring IFN-γ secretion of T cells from both spleen and inguinal lymph
node was conducted. Out of 1 million cells, approximately 800 and 300 cells responded
to the CD8
+
epitope peptide in the spleen and in the inguinal lymph node, respectively
(Figure 3.3C). Based on the i.d. administration route, which gave the highest CD8
+
T cell
response, different doses of DCLV-PSCA (2~80×10
6
TU) were administered. As shown
in Figure 3.3D, the CD8
+
T cell response was dose-dependent, increasing from 0.5% to
2%. Thus, an optimal immunization regimen of the i.d. injection of DCLV-PSCA with
80×10
6
TU was employed for subsequent studies.
Considering the important role of CD4
+
in tumor immunotherapy, an IL-2
ELISPOT assay was employed to examine the CD4
+
T cell response triggered by this
immunization strategy. We detected approximately 250 CD4
+
T cells per million
splenocytes capable of secreting IL-2 in response to lysates from 293T cells transfected
60
with the FUW-PSCA plasmid (Figure 3.3E). Our results demonstrated that DCLV-PSCA
was efficacious as a vaccine carrier to stimulate both CD8
+
and CD4
+
T cell responses in
mice.
3.3.3 Generation of anti-prostate tumor immunity in both prophylactic and
therapeutic models
In light of the PSCA-specific CD8
+
and CD4
+
T cell response observed, it was
necessary to evaluate the antitumor efficacy conferred by DCLV-PSCA immunization. A
transplanted mouse tumor model with the transgenic adenocarcinoma mouse prostate cell
line (TRAMP-C1)(Foster and others 1997) was used for this evaluation. Male C57BL/6
mice were vaccinated with DCLV-PSCA, DCLV-Null, or left untreated. These mice were
then challenged 10 days later by s.c. injection of 5 × 10
5
TRAMP-C1 cells (Figure 3.4A).
Tumor protection was observed in the DCLV-PSCA-vaccinated group with 8 out of 12
mice tumor-free for 45 days post-tumor challenge (Figure 3.4B). Moreover, the other 4
mice in that group exhibited a much slower rate of growth than that in the null vector
group. Notably, vaccination with DCLV-Null failed to provide any measurable tumor
suppression benefit as compared to the control group (Figure 3.4B). Overall, mice from
the DCLV-PSCA group displayed a significantly better survival rate than that of mice
from either the DCLV-Null or control group. All of the tumors from the DCLV-Null and
control group exceeded the size limit within 52 days (the tumor size of 2000 mm
3
was
used as a surrogate endpoint of survival), whereas the DCLV-PSCA group survived more
than 70 days (Figure 3.4C).

61
Figure 3. 4. Prophylactic and therapeutic anti-TRAMP-C1 prostate cancer immunity
elicited by in vivo immunization with DCLV-PSCA.
(A, B) Male C57BL/6 mice were immunized with 8×10
7
TU of DCLV-PSCA, mock vector DC-
LV-Null, or PBS control at the base of tail. Ten days post-immunization, these mice were
challenged subcutaneously with 5×10
5
of TRAMP-C1 tumor cells. Tumor growth curves were
monitored with a fine caliper, and tumor volume was calculated based on the largest
perpendicular diameters (mm
3
), according to the formula V = ab
2
π/6, where a and b are the
largest perpendicular diameters. (C) Representative Kaplan Meyer survival curve for prophylactic
tumor challenge (n=12). (D, E) Male C57BL/6 mice were implanted with 5×10
5
TRAMP-C1
tumor cells subcutaneously, and 18 days later, these tumor-bearing mice were treated with 8×10
7

TU of DCLV-PSCA (n=12) or DCLV-Null (n=12) at the base of tail. Tumor volume was
monitored and calculated as previously described. (F) Representative Kaplan Meyer survival
curve for therapeutic tumor challenge. (***: P < 0.001; Log-rank (Mantel-Cox) test. Error bars
represent SEM.)

We further investigated whether DCLV-PSCA could be potent for inhibiting
tumor growth in a therapeutic TRAMP-C1 model, in which a tumor had already been
62
established (Figure 3.4D). Tumor-bearing mice therapeutically vaccinated with DCLV-
PSCA showed significantly slower tumor growth (Figure 3.4E), and the average survival
was extended from 49.5 days to 64 days following the DCLV-PSCA immunization
(Figure 3.4F).
3.3.4 Dependence of vaccine-elicited antitumor immunity on infiltrated CD8
+
and/or
CD4
+
T cells
In an effort to further understand the roles of CD8
+
and CD4
+
T cells in antitumor
immunity, tumor tissue samples from DCLV-Null- or DCLV-PSCA-immunized mice
were collected, paraffin-embedded, and subjected to staining of nucleus and surface
markers. As shown in Figure 3.5A, the immunization resulted in infiltration of more T
cells (as identified by CD3 staining), including both CD4
+
and CD8
+
T cells in tumor
tissues harvested from DCLV-PSCA-immunized mice, than that of DCLV-Null-treated
mice. This indicates that both cytotoxic and helper T cells can infiltrate into the local
tumor tissue in response to immunization. To determine the dependency of antitumor
effect on these infiltrated T cells, an in vivo T cell depletion experiment was performed.
Four groups of mice were inoculated with the TRAMP-C1 tumors, in which three groups
were then immunized with DCLV-PSCA 14 days post-tumor challenge, while the
remaining group was immunized with DCLV-Null. For the DCLV-PSCA-immunized
groups, one group was treated with an antibody capable of depleting CD4+ T cells, and
another group was treated with an antibody capable of depleting CD8
+
T cells (Figure
3.5B). As shown earlier, DCLV-PSCA immunization could significantly slow down the
overall tumor growth. In contrast, tumors in the groups with depletion of either CD8
+
or
63
Figure 3. 5. CD8+/CD4+ T cell-dependent immune protection against TRAMP-C1 tumors
induced by DC-LV-PSCA immunization.
(A) Infiltration of T cells into tumor tissues. TRAMP-C1 tumors from tumor-bearing mice were
excised 3 weeks post-immunization, paraffin-embedded, and stained for immunofluorescence-
conjugated CD3, CD4 and CD8 antibody (green color as indicated by white arrows) together with
nuclear staining (red color). Representative images showing CD4
+
and CD8
+
T cells infiltrated to
tumor tissues from DCLV-PSCA-immunized mice as compared to those of DCLV-Null-
immunized mice. (B, C) Four groups of male C57BL/6 mice (n=8 for each group) were
transplanted with 5×10
5
TRAMP-C1 cells subcutaneously at day 0. Fourteen days later, 3 groups
were immunized with DCLV-PSCA, while the other group was immunized with mock vector
DCLV-Null. Two groups of mice from the DCLV-PSCA-immunized groups were subjected to
CD4
+
or CD8
+
T cell depletion by injecting CD4- or CD8-depletion antibody intraperitoneally.
(C) Tumor volume for each group of mice was monitored. Error bars represent SEM.

CD4
+
T cells developed a faster rate of tumor growth, although some tumor-protective
effect remained. Notably, CD8
+
T cell-depleted group had markedly larger tumors than
64
that of the CD4
+
T cell-depleted group. Our data further indicate that T cells are
responsible for the observed vaccine-induced antitumor immunity and that CD8
+
T cells
play the more indispensable role in controlling tumor growth.
3.3.5 Protection against lung metastasis of B16-PSCA cells
Figure 3. 6. The ability of DCLV-PSCA immunization to suppress lung metastases.
(A) Male C57BL/6 mice were immunized with DCLV-PSCA or DCLV-Null as a mock control.
Ten days later, mice were challenged with 0.2 million B10-F10-PSCA cells by intravenous
injection through tail vein. Two weeks later, mice were sacrificed, and macroscopic views of the
lungs were shown. (B) Microscopic H&E staining (20×) of lung tissue samples from mice
immunized with DCLV-PSCA or DCLV-Null. (C) Statistical quantification of melanoma lung
metastases (number of black nodules on the lungs) of immunized mice; similar immunization, but
with the original B16-F10 melanoma metastases included as a control. (**: P < 0.01 and n/s: not
statistically significant; One-way ANOVA followed by Bonferroni’s multiple comparison test.
Error bars represent SD, n=4)

65
Overexpression of PSCA was identified to be associated with prostate tumor
metastasis in many studies, which makes it an ideal target for immunotherapy. To
facilitate the study of the ability of DCLV-PSCA immunization to inhibit tumor
metastasis formation, wild-type B16-F10 cells stably expressing PSCA was constructed
(designated as B16-PSCA). Male C57BL/6 mice were first vaccinated with DCLV-PSCA
or DCLV-Null as a negative control. Ten days later, syngeneic B16-PSCA tumor cells
were injected intravenously to the animals. After another 14 days, animals were culled,
and lung metastatic deposits were quantified macroscopically. Compared to DCLV-Null,
DCLV-PSCA immunization markedly reduced the number of surface lung metastasis
formation (>75%, Figure 3.6A and C). Histologic lung tissue samples from the two above
groups were also examined microscopically for metastasis deposits, and a similar finding
was observed (Figure 3.6B). In contrast, the protective immunity of DCLV-PSCA was
only limited to the PSCA-expressing melanoma cells, as no significant difference was
observed when B16-F10 tumor cells were transplanted (Figure 3.6C). These results
confirmed PSCA-specific antitumor immunity conferred by DCLV-PSCA immunization
and its capacity to suppress metastasis formation.
3.4 Discussion
DC-based treatments have shown promising results for cancer
immunotherapy.(Boudreau and others 2011; Wei and others 2008) DC-directed LVs are
efficient vaccine vectors. They are able to transduce and activate DCs in vivo and mediate
durable transgene expression, which can be subsequently processed by DCs and
66
presented to T cells as antigens.(He and others 2006) Additionally, these vectors are
engineered to be non-replicable, with minimal viral proteins being expressed, and,
therefore, less anti-vector immunity was found.(H. Yang and others 2011) Furthermore,
because of DC-specific transduction, fewer safety and off-target concerns arise when
DCs are applied as vaccine vehicles in vivo.(L. Yang and others 2008) In our previous
studies, we have demonstrated that DC-directed LVs (DCLVs) can elicit strong immune
responses against OVA,(Hu and others 2010) HIV-gag,(Dai and others 2009) and hgp100
antigens.(H. Yang and others 2011) In this study, we evaluated the DCLVs carrying
PSCA, a true self-tumor antigen for prostate cancer, as a vaccine for syngeneic
transplanted prostate tumor in vivo. To the best of our knowledge, this represents the first
study to use DCLVs as a vaccine modality against a self-tumor antigen in animal models.
We showed that DCLV-PSCA vaccination could overcome the tolerance to self-antigen
PSCA and generate durable antigen-specific T cell responses in vivo. This immunization
mounts an immune response that is capable of suppressing the establishment of TRAMP-
C1 prostate tumors and slowing down tumor growth in a therapeutic model.
The envelope protein used to pseudotype LVs is an engineered form of Sindbis
virus glycoprotein (SVGmu, Figure 3.1B). The wild type of this glycoprotein has the
binding affinity to both heparin sulfate and DC-SIGN; DC-SIGN is a surface protein that
is predominantly expressed in macrophages and certain subsets of DCs.(Soilleux and
others 2002) We achieved targeting of DCs by disabling the heparin sulfate binding and
retaining the DC-SIGN binding of the Sindbis virus glycoprotein. It has been
demonstrated that the binding of SVGmu to DC-SIGN is dependent on the high mannose
67
structure on DC-SIGN. Therefore, the viral glycoprotein can be further engineered to
display a higher mannose structure to enhance transduction efficiency.(Tai and others
2011) We first confirmed that DCLV-PSCA could be efficiently produced and selectively
transduce DC-SIGN-expressing cells. The in vitro BMDC transduction assay
substantiates the observation that DCLV-PSCA can direct the delivery of the PSCA
antigen into DCs.
It has been previously shown that skin-derived DCs are the main target for LV-
based vaccination.(He and others 2006) However, the distribution and accessibility of
DCs in different parts of the body vary, so immunization through different routes might
trigger different levels of immune responses. We previously reported that the f.p. route
had a relatively higher response over other administration routes for some antigen
deliveries.(Dai and others 2009; H. Yang and others 2011) In this study, we directly
compared immune responses elicited through various vaccination routes (i.d., f.p., s.c.
and i.p.). Interestingly, we found that i.d. and f.p injections generated much higher
responses than did the s.c route, whereas i.p. administration resulted in the lowest
response. Immunization generated through the i.d. route displayed a slightly higher
response than the f.p. route. To account for this result, it is speculated that DCLV-PSCA
has a better chance of encountering DCs when administered through either the i.d. or f.p.
route.
A single dose of DCLV-PSCA was able to protect these mice from prostate tumor
challenge and improved their survival rate. This result is consistent with a previous study
using a prime/boost strategy to generate PSCA-specific immune response in a
68
prophylactic model,(Ahmad and others 2009; Garcia-Hernandez Mde and others 2008)
although DCLV-PSCA elicited a higher magnitude CD8
+
T cell response. In a TRAMP-
C1 therapeutic model, our vectored vaccine markedly slowed down tumor growth and
extended mouse survival, whereas the previous prime/boost vaccine method barely
generated satisfactory tumor protection.(Ahmad and others 2009) This result can be
explained by the time interval between tumor inoculation and tumor palpability, a period
of around 20~25 days. Also, it takes a significantly longer period of time to implement
the prime/boost immunization, which likely results in missing the most opportune time to
slow down cancer progression. Thus, one potential advantage of DCLVs is their ability to
overcome immune tolerance and establish an effective antitumor immune response within
2 weeks.
Both CD8
+
and CD4
+
T cells infiltrated into local tumor tissues following DCLV-
PSCA immunization. Although CD8
+
and CD4
+
T cells are both required for tumor
protection, the antibody depletion experiment indicates that CD8
+
T cells play a more
important role in controlling tumor growth. It has been well established that cytotoxic
CD8
+
T cells can directly kill tumor cells.(Bruno and others 2012; Graubert and others
1996; Kagi and others 1994) As for the CD4
+
T cells, several possible reasons explain
their requirement for tumor protection. First, CD8
+
T cells are dependent on CD4
+
T
cells(Bennett and others 1997; Dullaers and others 2006) to elicit robust immune
responses. Previously, we observed a CD4-dependent CD8
+
T cell response that was
elicited by DCLVs.(Xiao and others 2012) Second, at least part of the antitumor effect is
mediated by the Th1 response, which relies on CD4
+
T cells.(Haabeth and others 2011)
69
Currently, no reliable treatment exists to cure advanced metastatic prostate cancer.
PSCA is highly expressed in metastatic tissue for prostate cancer and is therefore a good
target for cancer immunotherapy. We have shown in this study that DCLV-PSCA can
generate immunity able to suppress lung metastasis in the B16-PSCA model.
Interestingly, when the same number of B16-F10 or B16-PSCA cells was injected
intravenously, B16-PSCA cells were able to generate more lung metastasis formation
than that of B16-F10 cells (Figure 3.6C). Presently, the relationship between tumor
metastasis and PSCA expression has not been thoroughly investigated, although some
studies suggest that PSCA may play a role in limiting tumor migration and
metastasis.(Moore and others 2008) Nevertheless, more studies are needed to further
understand how PSCA expression contributes to prostate cancer metastasis, and B16-
PSCA might be a suitable model for such studies.
Taken together, we have reported a novel DCLV vector system that can deliver
self-tumor antigen PSCA to antigen-presenting cells and mount vaccine-specific immune
responses. This DCLV-PSCA can overcome immune tolerance to PSCA, generate T cell
immunity that can protect mice in TRAMP-C1 prostate tumor models, and significantly
inhibit B16-PSCA lung metastasis formation. These results offer evidence to support the
use of DCLVs to deliver prostate cancer vaccines.
3.5 Acknowledgements
We thank Paul Bryson for critical reading of the manuscript. This work was
supported by grants from the National Institutes of Health (R01AI68978 and
70
P01CA132681) and a translational acceleration grant from the Joint Center for
Translational Medicine.

71
Chapter 4
A TLR4 AGONIST SYNERGIZES WITH DENDRITIC
CELL-DIRECTED LENTIVIRAL VECTORS FOR
INDUCING ANTIGEN-SPECIFIC IMMUNE RESPONSES

Portions of this chapter are adapted from: Liang Xiao, Jocelyn Kim, Matthew Lim,
Bingbing Dai, Lili Yang, Steven G. Reed, David Baltimore, and Pin Wang. Vaccine
(2012), 30(15), 2570-81.

TLR4 agonists can be used as adjuvants to trigger innate immune responses of
antigen-presenting cells (APCs) such as dendritic cells (DCs) to enhance vaccine-specific
immunity. Adjuvant effects of TLR4 agonists are mediated by downstream signaling
controlled by both MyD88 and TRIF adapter proteins. In this chapter, we investigated the
adjuvanting capacity of glucopyranosyl lipid A (GLA), a chemically synthesized TLR4
agonist, to boost antigen-specific immunity elicited by DC-directed lentiviral vectors
(DC-LV). We found that stimulation by this agonist in vitro can activate DCs in a TLR4-
dependent manner. The agonist can significantly boost DC-LV-induced humoral and
cellular immune responses, resulting in better antitumor reactions in response to tumor
challenges. We observed that the adjuvant-mediated enhancement of cytotoxic CD8
+
T
72
cell responses is CD4
+
T cell-dependent and determined that in vitro the agonist
stimulation involves the participation of both MyD88 and TRIF pathways to activate
DCs. In vivo immunization study however revealed that adjuvant effects depend more on
the MyD88 signaling as TRIF
-/-
mice but not MyD88
-/-
mice were able to maintain the
enhanced CD8
+
T cell responses upon DC-LV immunization. Thus, our study supports
the use of this TLR4 agonist as a potent adjuvant candidate for boosting DC-LV
immunization.
4.1 Introduction
There has been a growing interest in utilizing lentiviral vectors (LVs) as vaccine
carriers to elicit antigen-specific humoral and cellular immune responses.(HeFalo 2007;
He, Munn, and others 2007; Hu and others 2011; Pincha and others 2010) LVs present
several desirable features of a virus-based vaccine vector(Draper and others 2010): they
are able to transduce both dividing and non-dividing cells,(Naldini and others 1996)
capable of carrying large transgenes (up to 8 kb), and low in pre-existing anti-vector
immunity,(Kootstra and others 2003) and they are currently being evaluated in human
gene therapy trials for a wide range of human diseases.(Escors and others 2010) Many
studies have demonstrated the promise of LVs to generate vaccine-specific immunity
targeting a broad range of infectious diseases and cancer.(Breckpot, Aerts, and others
2007; Hu and others 2011) Although various routes of vaccine administration have been
investigated and compared,(Maloy and others 2001; Senti and others 2009; Sigel and
others 1983) subcutaneous injection remains the most potent and practical means for LVs
73
to stimulate transgene-specific immune responses. Recent reports have convincingly
shown that a subcutaneous injection of LVs can result in genetic modification of skin-
derived dendritic cells (DCs) to have prolonged antigen expression and
presentation.(Furmanov and others 2010; Goold and others 2011; He and others 2006)
Their subsequent migration to skin-draining lymph nodes and priming of the repertoire T
cells are the major mechanism of action for the resulting immune responses to the
delivered antigens. Because of the essential role of DCs in LV-mediated immunization,
considerable effort has been devoted to developing LVs capable of targeting DCs to
improve vaccine efficacy and safety.(Ageichik and others 2011; Ageichik and others
2008; Dresch and others 2008; Goyvaerts and others 2012; Hu and others 2011; Kimura
and others 2007; Lopes and others 2008; Verhoeyen and others 2009) We have reported a
targeting transduction system, in which the human immunodeficiency virus-1 (HIV-1)-
based LV is enveloped with a mutant Sindbis virus glycoprotein (SVGmu) that, when
injected subcutaneously into mice, can target DCs through its selective recognition of the
attachment receptor DC-SIGN, a protein predominantly expressed on the DC surface.(L.
Yang and others 2008) Immunization with this vector system resulted in durable immune
responses to several delivered immunogens and required only a modest dose of vector
administration.(Dai and others 2009; Hu and others 2010; H. Yang and others 2011; L.
Yang and others 2008)
Our previous in vitro study showed a slight maturation of bone marrow-derived
DCs (BMDCs) upon exposure to this DC-directed LV (DC-LV) system,(L. Yang and
others 2008) presumably due to the interaction between SVGmu and DC-SIGN, and the
74
transduction-mediated DC activation via Toll-like receptors.(Breckpot, Emeagi, and
others 2007; Breckpot and others 2010; Tan and others 2005) We postulated that DC-
stimulating molecular adjuvants such as agonists for TLR family proteins, when co-
administered with DC-LV, could further improve the vaccine efficacy. The mammalian
TLRs are a group of pattern recognition receptors expressed by innate immune cells and
can be stimulated by structural motifs known as pathogen-associated molecular patterns
(PAMPs) contained by bacteria, viruses, and fungi.(Akira and others 2006; Iwasaki and
others 2004; Janeway and others 2002) These stimulations can trigger downstream signal
transduction pathways such as nuclear factor (NF)-κB and interferon regulatory factor
(IRF), which will activate antigen-presenting cells (APCs) and promote inflammatory
responses.(Akira and others 2006; Iwasaki and others 2010; Lee and others 2007)
Among various known TLRs, TLR4 is the only one capable of inducing two
distinct signaling pathways(Iwasaki and others 2004; Lu and others 2008): 1) the
MyD88-dependent pathway to activate NF-κB signaling and be responsible for induction
of proinflammatory cytokines; 2) the TRIF-dependent pathway to mediate the activation
of Type I interferons. Studies have shown that the ability to induce both pathways is
essential for maximizing the immunostimulatory potentials of DCs.(Shen and others 2008)
The most widely known TLR4 agonist is lipopolysaccharide (LPS) that presents in the
outer membrane of Gram-negative bacteria. Monophosphoryl lipid A (MPL) is a
derivative of LPS exacted from Salmonella minnesota R595(Baldridge and others 1999)
and exhibits only ~0.1% of the inflammatory toxicity of LPS.(Evans and others 2003;
Qureshi and others 1997) When used as an adjuvant, MPL enhances immunogen-specific
75
immune responses by promoting the development of Th1 CD4
+
T cells.(Reed and others
2009) MPL has been approved as a component of adjuvant formulation for vaccines
against human papilloma virus (HPV) and hepatitis B virus (HBV).(Reed and others
2009) Recently a synthetic TLR4 agonist, glucopyranosyl lipid A (GLA), has emerged as
a more pure and chemically defined molecular adjuvant, in contrast to the heterogeneous
mixture of MPL extracted from bacteria.(Coler and others 2011) GLA has been
demonstrated to be potent for assisting the generation of Th1-biased immune responses in
experimental vaccines against tuberculosis,(Baldwin, Bertholet, and others 2009)
leishmaniasis,(Bertholet and others 2009) influenza,(Baldwin, Shaverdian, and others
2009) and malaria.(Lousada-Dietrich and others 2011; Lumsden and others 2011) It is
currently being evaluated as an adjuvant in phase I clinical trials of an influenza virus
vaccine.(Coler and others 2010)
In this chapter, we explore this TLR4 agonist as an adjuvant for immunization
delivered by a DC-LV encoding the chicken ovalbumin (OVA) antigen. We show that
GLA can activate BMDCs in vitro and significantly improve the immune responses in
vivo by increasing the populations of both antigen-specific CD8
+
and CD4
+
T cells and
improving the titers of various antibody isotypes specific for OVA. These enhancements
resulted in improved protection against the growth of tumors yielding better survival rates
in both prophylactic and therapeutic tumor challenge models. Moreover, we also found
that the elevated CD8
+
T cell responses provided by GLA are CD4
+
T cell-dependent.
Although the in vitro activation of DCs by GLA was observed to be mediated by both
MyD88- and TRIF-dependent pathways, our DC-LV immunization assays showed that
76
GLA is a more MyD88-biased agonist of TLR4 for augmenting vaccine-specific
immunity.
4.2 Materials and Methods
4.2.1 Mice and reagents
6−8 week old female C57BL/6 mice were purchased from the Charles River
Laboratories. The strain of B6.B10ScN-Tlr4
lps-del
/JthJ (designated as TLR4
-/-
) and
C57BL/6J-Ticam1
Lps2
/J (designated as TRIF
-/-
) mice were purchased from the Jackson
Laboratory and maintained in the animal facilities of the California Institute of
Technology (Caltech) and the University of Southern California (USC). B6.129/SvJ-
MyD88
tm1AKI
(designated as MyD88
-/-
) mice were a gift from Prof. S. Akira (Osaka
University, Osaka, Japan) and maintained at Caltech and USC. All animal procedures
were performed in accordance with the guidelines set by the National Institutes of Health,
Caltech, and USC on the Care and Use of Animals. The aqueous formulated GLA (GLA-
AF, used for in vitro experiments) and the oil-in-water stable emulsion formulated GLA
(GLA-SE, used for in vivo immunization)(Coler and others 2011) were prepared at the
Infectious Disease Research Institute (IDRI, Seattle, WA, USA).
4.2.2 Lentiviral vector construct and production
The lentiviral backbone plasmid FUW-TfROVA was constructed by insertion of
the cDNA consisting of the first 118 amino acids of the membrane-anchoring domain of
murine transferrin receptor) fused downstream with the truncated chicken ovalbumin
77
(OVA, amino acids 139-386) into FUW;(Rowe and others 2006) FUW is a HIV-1-
derived lentiviral plasmid composed of an internal human ubiqutin-C promoter to drive
transgene expression and woodchuck responsive element to improve stability of the RNA
transcript.(Lois and others 2002) We employed a previously reported procedure of
transient transfection of 293T cells to produce the DC-LV-OVA vector.(L. Yang and
others 2008) Briefly, 293T cells cultured in a 15-cm tissue culture plate (BD Biosciences,
San Jose, CA, USA) were transfected via a standard calcium phosphate precipitation
method with the following plasmids: the lentiviral backbone plasmid FUW-TfROVA
(37.5 µg), the plasmid encoding the mutant Sindbis virus glycoprotein (SVGmu) (18.75
µg), and the packaging plasmids (pMDLg/pRRE and pRSV-Rev, 18.75 µg for each). The
viral supernatants were harvested twice at 48 and 72 h post-transfection, pooled, and
filtered through a 0.45-mm filter (Corning, Lowell, MA, USA). The concentrated viral
pellets were obtained after ultracentrifugation of the viral supernatants at 50,000 ×g for
90 min, and were then resuspended in an appropriate volume of HBSS for in vivo
injection.
4.2.3 BMDC generation and activation
We employed a previously described procedure to generate bone marrow-derived
DCs (BMDCs) with various genetic backgrounds.(L. Yang and others 2008) Briefly,
bone marrow from the femurs and tibias of mice was grown in RPMI 1640 with 10%
heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin,
0.05 mM 2-ME, and 20 ng/mL GM-CSF (J558L supernatant) after the red blood cells
were lysed. Cultures were initiated by placing 10
7
bone marrow cells in 10 mL of
78
medium onto 100-mm petri dishes (Falcon 1029 plates; BD Labware, Franklin Lakes,
NJ). On day 3, another 10 mL of J558L-conditioned medium was added. On day 6,
suspension cells were collected. BMDCs were seeded at a density of 0.5 million/mL in
24-well plates (BD Labware), treated with GLA-AF (1 µg/mL) or left untreated,
supernatants were collected 24 h later for cytokine measurements. Cells were also
collected for antibody staining and flow cytometric analysis.
4.2.4 Flow cytometric analysis of surface markers of BMDCs
Single cell suspensions were incubated with anti-mouse CD16/CD32 Fc blocking
antibody and then stained with fluorophore-conjugated monoclonal antibodies against
specific BMDC surface markers, including CD80, CD86, H2-K
b
, and I-A
b
. All antibodies
were purchased from Biolegend (San Diego, CA). Stained cells were assayed using BD
LSRII flow cytometer (BD Biosciences) and acquired data was analyzed using FlowJo
software (Tree Star, Ashland, OR).
4.2.5 Supernatant ELISA assay
ELISA was used to detect cytokine/chemokine levels after BMDC activation.
Specific combination of capture and detection antibodies was purchased either from
R&D Systems (Minneapolis, MN) for assaying IL-6, IL-12p70, IL-15, RANTES/CCL5,
IP-10/CXCL10, and IL-1β, or from eBioscience (San Diego, CA) for assaying TNF-α.
We followed the manufacturer’s recommended protocols to conduct these ELISA assays.
4.2.6 Immunization procedure
For immunization with DC-LV, mice were injected with replication-defective
79
DC-LV-OVA (5×10
6
Transduction Units (TU)) at the rear footpad. For immunization
with adjuvant, GLA-SE (20 µg) was administered at the base of tail when DC-LV-OVA
was injected. For the experiment to deplete CD4
+
T cells, the monoclonal depletion
antibody GK1.5 (200 µg, BioXCell, West Lebanon, NH) was intraperitoneally injected at
days 0, 2, 5, and 8 post-immunization. The vaccinated mice were analyzed for their
immune responses 2 wk post-immunization.
4.2.7 Intracellular cytokine staining (ICCS)
Splenocytes from immunized or control mice were pooled and incubated with the
OVA
257-264
peptide (SIINFEKL) (1µg/mL) in the presence of costimulatory anti-CD28
antibody (2 µg/mL, BD Biosciences) for 2 h at 37 °C in a 96-well round-bottom plate in
RPMI medium supplemented with 10% FBS (Sigma), 10 U/mL penicillin, 100 µg/mL
streptomycin, and 2 mM glutamine. Brefeldin A (BFA, Sigma, St. Louis, MO) was added
(10 µg/mL) to wells to inhibit cytokine exporting for another 4 h. Surface staining was
performed by incubating restimulated cells with anti-mouse CD16/CD32 Fc blocking
antibody, followed by anti-mouse CD8 and anti-mouse CD4 antibodies. Cells were then
permeabilized in 100 µl Cytofix/Cytoperm solution (BD Biosciences) at 4 °C for 10 min,
washed with Perm/Wash buffer (BD Biosciences), and followed by intracellular staining
with PE-conjugated anti-mouse IFN-γ at 4 °C for 15 min. The flow cytometry analysis
was carried out using the FACSort instrument from BD Biosciences. For the multi-
parameter ICCS analysis, cells were stained with the following surface monoclonal
antibodies (Biolegend): anti-CD4-PerCP, anti-CD8-APC-Cy7, anti-CD44-Alexa488, and
with the following intracellular monoclonal antibodies (BD Biosciences): anti-IFN-γ-
80
APC, and anti-TNF-α-PE-Cy7. The ICCS data were acquired on a BD LSR II flow
cytometer (BD Biosciences).
4.2.8 IL-2 ELISPOT assay
ELISPOT assays were performed for detecting IL-2 using a kit from Millipore
(Billerica, MA) according to the manufacturer’s instruction. Briefly, anti-mouse IL-2
antibody (10 µg/mL in PBS) was used as the capture antibody and plated with 100
µl/well on 96-well MultiScreen-IP plates overnight at 4 °C. The plate was decanted and
blocked with the RPMI medium containing 10% FBS at 37 °C for 2 h. Splenocytes from
mice were plated at 5 × 10
5
cells/well in 100 µL complete medium in company with the
CD4 epitope OVA
323-339
peptide (ISQAVHAAHAEINEAGR) (10 µg/mL). After 18 h
incubation at 37 °C, cells were lysed and plates were detected by 1 µg/mL biotinylated
anti-IL-2 antibody (BD Biosciences) for 2 h at room temperature. Plates were further
washed and incubated with the 1,000-fold-diluted streptavidin-alkaline phosphate
conjugate for 45 min at room temperature. After a final extensive washing, spots were
identified by adding BCIP/NBTplus substrate (Millipore), and the number of IL-2
producing cells was quantified by an ELISPOT reader.
4.2.9 Statistics
All the statistics were calculated by either Origin Pro 7.0 or GraphPad Prism 5
software. Error Bars in all the figures represent SD except the tumor growth curve in
therapeutic tumor challenge model, SEM was used. One-way ANOVA followed by a
Bonferroni’s multiple comparison test was used to determine significance of difference
81
while animal survival curves were analyzed by log-rank (Mantel-Cox) test and the value
of P < 0.05 was considered to be statistically significant.
4.3 Results
4.3.1 Activation of dendritic cells by the TLR4 agonist GLA in vitro
Pathogens binding to TLR4 can initiate downstream signal transduction and
induce NF-κB activity, which is critical for DC activation and maturation.(Andreakos and
others 2006) To evaluate the activation status of DCs, we examined the expression of
major histocompatibility complex (MHC) and costimulatory molecules on the surface of
BMDCs after GLA stimulation. We used an aqueous formation of GLA (GLA-AF) for in
vitro studies.(Baldwin, Shaverdian, and others 2009; Coler and others 2011) As shown in
the left panel of Figure 1A, MHC I (H2-K
b
) and MHC II (I-A
b
) molecules were both
elevated after GLA treatment, which is likely to enhance DCs’ antigen presentation
capability. Expression of both CD80 and CD86 was enhanced, which should allow DCs
to provide stronger costimulatory signals for T cell stimulation. Furthermore, the surface
expression both of ICAM-1 and CD40 was also greatly increased by GLA treatment (data
not shown). To examine whether activation of DCs by GLA is TLR4-dependent, we
conducted a similar experiment using BMDCs lacking TLR4 expression (TLR4
-/-

BMDCs). All of these surface activation markers were unaltered following GLA
stimulation of TLR4
-/-
BMDCs (Figure 4.1A, right panel), confirming that GLA
activation of DCs is strictly TLR4-dependent.

82
Figure 4. 1. GLA-mediated activation of mouse BMDCs in vitro.
A. FACS analysis of surface antigen presentation and costimulatory molecules of CD11c
+

BMDCs from wild-type mouse (left) and TLR4
-/-
mouse (right) treated with aqueous GLA (solid
line) or aqueous formulation lacking GLA (shaded area) for 24 h. Representative data from
triplicate cultures is shown. B. Secretion of cytokines and chemokines of BMDC cultures from
wild-type (black bar) and TLR4
-/-
mice (white bar) treated with (+) or without (‒) GLA. Mean
secretion +/- SD of triplicate culture is shown.

We then examined cytokine and chemokine secretion from BMDCs after GLA
treatment. GLA stimulation significantly enhanced production and secretion of
interleukin (IL)-6, TNF-α, RANTES, IP-10, modestly enhanced the amount of IL-1β and
IL-12p70, but had little effect on IL-15 production (Figure 4.1B). Proinflammatory
cytokines IL-6, TNF-α and IL-12 are important for promoting the proliferation of CD8
+

and CD4
+
CD25
‒
T cells, while limiting CD4
+
CD25
+
regulatory T cell (Treg)
83
proliferation.(Song and others 2008; Stephens and others 2004; Thornton and others 2000)
RANTES is known to be involved in proper T cell function and proliferation.(Makino
and others 2002) IL-1β can drive proliferation of CD4
+
CD25
+
Foxp3
‒
effector and
memory T cells, while inhibiting CD4
+
CD25
+
Foxp3
+
Treg function.(O'Sullivan and
others 2006) IL-1β is also involved in CD4
+
T cell-dependent antibody production and
promotes the function of antigen-specific T helper cells.(Nakae and others 2001) The
chemokine IP-10 is critical for effector T cell trafficking(Dufour and others 2002) and is
involved in promoting T cell-based anti-tumor immunity.(Angiolillo and others 1995)
Such elevated secretion of cytokines and chemokines was not observed in GLA-treated
TLR4
-/-
BMDCs, confirming the essential role of TLR4 for GLA to activate DCs.
4.3.2 Adjuvant effect of the TLR4 agonist GLA on DC-LV-induced T cell immunity
We next tested the ability of GLA as an adjuvant to improve OVA-specific CD8
+

and CD4
+
T cell responses induced by subcutaneous immunization of DC-LV encoding
the model OVA antigen (DC-LV-OVA). OVA was fused with the transferrin receptor
transmembrane sequence for achieving balanced CD4 and CD8 responses.(Rowe and
others 2006) Oil-in-water stable emulsion formulation of GLA (GLA-SE) was used for
all our in vivo studies because such formulation resulted in more robust adjuvant
responses as compared to aqueous GLA.(Baldwin, Shaverdian, and others 2009; Reed
and others 2009) We observed a higher level (approximately 2-3 times higher) of OVA-
specific CD8
+
T cells capable of producing IFN-γ and/or TNF-α from GLA-treated group
than that of the untreated control group (Figure 4.2A and B). An ELISPOT assay was
employed to measure CD4
+
T cell response. We found that GLA-treated mice on average
84
Figure 4. 2. Adjuvant effects of GLA on boosting DC-LV-based vaccine-specific T cell
immune responses in vivo.
Wild-type (A, B) or TLR4
-/-
(C, D) mice were immunized at rear footpad with PBS, DC-LV-
OVA, DC-LV-OVA plus GLA-SE (base of tail). 14 d later, splenocytes were collected and OVA-
specific CD8
+
T cells were analyzed by intracellular staining for IFN-γ or TNF-α expression after
stimulation with OVA
257-264
peptide for 6 h (A, C). The FACS data shown is one representative
data of four analyzed mice. (B, D) Statistical data showing the percentage of IFN-γ
+
, TNF-α
+
, or
IFN-γ
+
TNF-α
+
cells within the CD8
+
T cell population. Splenocytes were also pooled for an
ELISPOT assay to analyze IL-2 secretion following stimulation with OVA
323-339
peptide for 18 h.
(***: P < 0.001; **: P < 0.01; *: P < 0.05 and n/s: not statistically significant; One-way ANOVA
followed by a Bonferroni’s multiple comparison test. Mean + SD is shown.)

had approximately 2-3 times more IL-2 producing cells than the control mice (Figure
4.2B). When the same immunization protocol was applied to TLR4
-/-
mice, we observed
no enhancement of OVA-specific CD8
+
and CD4
+
upon administration of GLA (Figure
4.2C and D). This in vivo immunization study is in good agreement with our in vitro DC
85
activation study, showing that GLA can bolster the response to a DC-LV-based genetic
vaccine in a TLR4-dependent manner. In addition, in the absence of GLA, we noticed
similar levels of OVA-specific CD8
+
and CD4
+
T cell responses induced by DC-LV
immunization for wild-type and TLR4
-/-
mice, suggesting that DC-LV-based
immunization generates a fraction of its vaccine-specific immune response in a manner
that is independent of TLR4.
4.3.3 Adjuvant effect of the TLR4 agonist GLA on DC-LV-induced antibody
responses
Because we observed that GLA could improve DC-LV-elicited T cell responses,
we investigated whether GLA could also facilitate vaccine-induced antibody responses.
Mice were immunized with DC-LV-OVA plus and minus GLA adjuvant treatment. Two
weeks post-immunization, sera were collected and OVA-specific titers of different
antibody isotypes (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) were measured using an
ELISA assay. GLA could broadly and greatly enhance antibody responses induced by
DC-LV immunization (Figure 4.3). The generation of elevated titers of both IgG1 (Figure
4.3A) and IgG2a (Figure 4.3B) suggests that GLA is likely able to bolster both Th1 and
Th2 immune responses. Interestingly, we observed that GLA could enhance IgA antibody
responses as well (Figure 4.3E), which may be useful for vaccine applications that
require mucosal immunity.(Fagarasan and others 2003) Notably, although GLA
improved IgM antibody titer (Figure 4.3F), the enhancment of IgM titer was not as
significant as that of IgG. This might indicate a role for GLA in facilitating more efficient
isotype switching from IgM to IgG.
86
Figure 4. 3. Adjuvant effects of GLA on boosting DC-LV-based vaccine-specific antibody
immune responses in vivo.
ELISA of OVA-specific IgG1 (A), IgG2a (B), IgG2b (C), IgG3 (D), IgA (E), IgM (F) antibody
titers in sera of mice 14 d after immunization with DC-LV-OVA (left) or DC-LV-OVA plus
GLA-SE (right). Results are shown as mean titer + SD of four mice per group. (*: P < 0.05; One-
way ANOVA followed by a Bonferroni’s multiple comparison test.)

4.3.4 Adjuvant effect of the TLR4 agonist GLA on the anti-tumor immunity
delivered by DC-LV
The promising results of GLA as an adjuvant for enhancing vaccine-specific
immune responses encouraged us to test whether GLA combined with a vector vaccine
could generate better antitumor responses in mice. We employed the EG7 tumor cell line,
which stably expresses chicken OVA, as a tumor model for this investigation.(L. Yang
and others 2008) In the tumor prophylactic model, mice were inoculated with EG7 tumor
cells 14 days post-immunization. Aggressive tumor growth was seen in non-vaccinated
87
Figure 4. 4. Adjuvant effects of GLA on DC-LV immunization to enhance antitumor
immune responses.
(A, upper) Schematic showing the immunization and tumor challenge strategy in the prophylactic
model. (A, middle and lower left) Mice were vaccinated with PBS (four mice), DC-LV-OVA (six
mice), or DC-LV-OVA plus GLA-SE (six mice) on day 0, followed by EG7 tumor challenge on
day 14. Tumor growth was quantified as tumor volume (mm
3
) and plotted as a function of days
after inoculation of EG7 cells. Three of the DC-LV-OVA plus GLA-SE mice never developed
tumors. (A, lower right) Kaplan-Meier survival plot of mice vaccinated with PBS (●), DC-LV-
OVA (■), or DC-LV-OVA plus GLA-SE (▲), P < 0.0001. The tumor size of 2000 mm
3
was
used as a surrogate endpoint of survival. (B, upper) Schematic showing the tumor inoculation on
day 0 and immunization strategy on day 3 in the therapeutic model. (B, middle) Tumor growth
was plotted as mean volume +/- SEM (n=10) as a function of days after EG7 tumor challenge. (B,
lower) Kaplan-Meier survival plot of mice vaccinated with DC-LV-OVA (■) or DC-LV-OVA
plus GLA-SE (▲), P < 0.0001.

control mice. Using the tumor size of 2000 mm
3
as a surrogate endpoint of survival, none
these mice could survive for more than 20 days. For the group immunized with DC-LV-
OVA alone, although all mice developed tumors, tumor growth was slower and mice
88
survived longer as compared to the non-vaccinated group. In contrast, for mice
vaccinated with DC-LV-OVA and treated with GLA, only half of the mice developed
tumors and those tumors grew much more slowly than did those in the vaccine-only
group (Figure 4.4A), resulting in a longer overall survival for these tumor-bearing mice
(Figure 4.4A, lower right). We also evaluated GLA in a therapeutic tumor model setting.
Mice were challenged with a lethal dose of EG7 cells with immunization and GLA
treatment administration 3 days later (Figure 4.4B). As compared to the vaccine-only
group, significantly slower tumor growth was observed for mice that received both DC-
LV-OVA and GLA treatment and median survival time was 8 days longer (Figure 4.4B,
lower panel).
4.3.5 Role of CD4
+
T cells in GLA-augmented DC-LV immunization
It is well documented that CD4
+
T helper cells play a pivotal role in orchestrating
the generation of both effector and memory CD8
+
cytotoxic T cells,(Shedlock and others
2003; Sun and others 2003) although CD8
+
T cell responses can also be produced in a
CD4
+
T cell-independent manner.(Janssen and others 2005; Janssen and others 2003;
Smith and others 2008) To investigate the cellular mechanism underlying the enhanced
vaccine responses delivered by GLA, we conducted experiments to deplete CD4
+
T cells
following the immunization. Four groups of mice were immunized with DC-LV-OVA,
within which two groups also received GLA adjuvant at the same time. Depletion of
CD4
+
T cells by the GK1.5 antibody was carried out in one vaccinated group and in a
group vaccinated along with GLA treatment. Flow cytometric analysis of the resulting
splenocytes showed that the majority of CD4
+
T cells (21.5% vs. 0.33%, Figure 4.5A)
89
Figure 4. 5. The role of CD4+ helper T cells in GLA-enhanced CD8+ T cell responses.
A. FACS analysis of intracellular staining of IFN-γ from splenocytes of mice immunized with
PBS, DC-LV-OVA, DC-LV-OVA plus GK1.5 antibody-mediated depletion of CD4
+
T cells, or
DC-LV-OVA plus GLA-SE and GK1.5. One representative data from a group of four mice is
shown. Percentage shown in the left panel is the percentage of CD4
+
T cells and CD8
+
T cells. B.
Statistical data showing OVA-specific CD8
+
T cells by intracellular staining of IFN-γ following
stimulation with OVA
257-264
peptide. C. OVA-specific CD8
+
T cell percentage by intracellular
staining of IFN-γ after OVA
257-264
peptide stimulation from mice immunized for 14 d with the
same transduction units (TUs) of either SVGmu- (left) or VSVG- (right) enveloped LV-OVA.
Black bar: isotype control antibody; white bar: GK1.5 antibody. (***: P < 0.001; **: P < 0.01; *:
P < 0.05 and n/s: not statistically significant; One-way ANOVA followed by a Bonferroni’s
multiple comparison test. Mean +/- SD is shown.)

were eliminated by the antibody treatment and these mice maintained such a depletion
condition throughout the experiment. The intracellular staining of CD8
+
T cells harvested
from immunized mice for IFN-γ (Figure 4.5B) showed higher (~19% vs. ~7%) OVA-
specific CD8
+
T cells in mice with the GLA-adjuvanted vaccination. On the contrary,
90
with the CD4
+
T cell depletion, the immune response dropped to about 2% and GLA
treatment failed to improve the CD8
+
T cell response in the CD4
+
T cell-deficient
condition (Figure 4.5A and B). This data demonstrates that CD4
+
T cells are necessary
for the adjuvanting effect of GLA on lentiviral vector immunization. We also observed
that the DC-LV-OVA-induced CD8
+
T cell response was partially dependent on the
presence of CD4
+
T cells (Figure 4.5A, DC-LV-OVA vs. DC-LV-OVA/GK1.5). To
investigate whether such dependence is unique for DC-LV, we directly compared
immunization by vectors enveloped with either SVGmu (DC-LV) or the glycoprotein
derived from vesicular stomatitis virus (VSVG), which provides a broader tropism in
both normal and CD4
+
T cell-deficient conditions. Consistent with our previous
results,(L. Yang and others 2008) when immunized with an identical dose, the SVGmu-
enveloped vector (DC-LV) elicited a markedly higher CD8
+
T cell response than that
induced by the VSVG-enveloped vector (Figure 4.5C). Depletion of CD4
+
T cells
lowered CD8
+
T cell immunity in both situations, although the drop was more significant
for SVGmu-enveloped vector than VSVG-enveloped vector (Figure 4.5C).
4.3.6 Study of the role of MyD88 and TRIF pathways in GLA-mediated activation of
DCs in vitro
It has been reported that Monophosphoryl Lipid A (MPL), another TLR4 agonist,
activates macrophage cells in a TRIF-biased rather than MyD88-biased manner when
compared with its parent molecule LPS.(Mata-Haro and others 2007) Because GLA is a
relatively novel and unstudied synthetic molecule, elucidating the downstream signaling
pathway involved for activating DCs by GLA can be very useful for understanding the
91
Figure 4. 6. Involvement of MyD88 and TRIF signaling pathways in GLA-mediated
activation of BMDCs in vitro.
A. FACS analysis of surface activation markers of CD11c
+
BMDCs from wild-type mouse
(black), MyD88
-/-
mouse (white), or TRIF
-/-
mouse (dashed line) treated with (+) or without (‒)
aqueous GLA. Data is shown based on either percentage or normalized mean fluorescence
intensity (MFI). B. Secretion of cytokines and chemokines of BMDCs derived from wild-type
(black), MyD88
-/-
(white), or TRIF
-/-
mouse (dashed line) stimulated with (+) or without (‒) GLA.
The mean secretion +/- SD of triplicate culture is shown.

mechanism of its adjuvant effect and exploring its potential applications in vaccine
research. Among various Toll-like receptors, TLR4 is the only receptor that utilizes both
TRIF- and MyD88-dependent pathways for signaling.(Iwasaki and others 2004; Lu and
others 2008) To examine which pathway GLA/TLR4 depends on to trigger DC activation,
BMDCs derived from wild-type, MyD88
-/-
and TRIF
-/-
mice were generated and treated
with GLA for 1 day individually; no GLA treatment was included as controls. Treated
BMDCs were subjected to analysis of their surface expression of costimulatory
92
molecules (CD40, CD80, CD86, OX40), MHC class I molecule H2-K
b
, and MHC class II
molecule I-A
b
. As shown in Figure 4.6A, expression of these surface markers on both
MyD88
-/-
and TRIF
-/-
BMDCs were generally lower (except CD80 which has similar
levels in all three situations) than those expressed on wild-type cells after GLA treatment,
indicating that MyD88 and TRIF both participated in the downstream signaling in
response to GLA stimulation. To further this study, we collected supernatants from these
BMDCs and assayed the secretion profile of a selective set of cytokines and chemokines
(Figure 4.6B). Surprisingly, lack of MyD88 expression almost completely abolished the
ability of GLA-treated DCs to produce cytokines TNF-α, IL-6, and IL-1β, while they
were able to maintain a modest production of chemokines RANTES and IP-10. On the
other hand, as compared to MyD88-null cells, TRIF
-/-
BMDCs better retained the ability
to secrete TNF-α, IL-6, and IL-1β in response to GLA treatment, although their levels of
secretion were lower than that of the wild-type cells. This suggests that the production of
these cytokines is less dependent on the TRIF-mediated pathway. In contrast, chemokines
RANTES and IP-10 tend to depend more on TRIF expression because TRIF
-/-
BMDCs
produced less of them compared to MyD88
-/-
cells upon GLA stimulation. Interestingly,
we observed that DCs have an equal, partial dependence on MyD88 and TRIF for the
secretion of IL-12 in response to GLA stimulation.
4.3.7 Study of the role of MyD88 and TRIF pathways in GLA-mediated activation of
DCs in vivo
To examine the in vivo role of GLA adjuvant in augmenting DC-LV-based
vaccination, we compared immune responses among vaccinated MyD88
-/-
, TRIF
-/-
, and
93
Figure 4. 7. Involvement of MyD88 and TRIF signaling pathways in GLA-adjuvanted DC-
LV immunization in vivo.
A. Wild-type (upper), MyD88
-/-
(middle), and TRIF
-/-
(lower) mice were immunized with DC-
LV-OVA, or DC-LV-OVA plus GLA-SE. 2 wk later, OVA-specific CD8
+
T cells were analyzed
by intracellular staining of IFN-γ following OVA
257-264
peptide stimulation. The FACS data
shown is representative of four mice tested. B. Statistical data showing OVA-specific CD8
+
T
cells analyzed by IFN-γ
+
(upper left), TNF-α
+
(upper right), or IFN-γ
+
TNF-α
+
(lower left)
populations for groups of mice described above with (+) or without (-) GLA treatment. (B, lower
right) Splenocytes were also pooled for an ELISA assay of IL-2 production after stimulation with
OVA
323-339
peptide. (**: P < 0.01; *: P < 0.05 and n/s: not statistically significant; One-way
ANOVA followed by a Bonferroni’s multiple comparison test. Mean +/- SD is shown.)

wild-type mice. As seen previously, intracellular cytokine staining of splenocytes
restimulated with the OVA
257-264
peptide showed enhanced CD8
+
T cell immunity in
wild-type mice treated with GLA (Figure 4.7A). Significantly lower antigen-specific
CD8
+
T cell immune responses were observed in MyD88
-/-
mice and GLA treatment did
94
not improve the magnitude of the response (Figure 4.7A and B). In sharp contrast, TRIF
-/-

mice could mount the same level of CD8
+
T cell response as that of wild-type animals
and a similarly enhanced level of immune responses was obtained upon GLA treatment.
These results indicate that the MyD88 pathway, rather than the TRIF pathway, is
essential for GLA-adjuvanted CD8
+
T cell responses in vivo. We also assessed the OVA-
specific CD4
+
T cell immunity by an IL-2 ELISPOT assay for these treated mouse groups
and found a similar trend to what was observed for CD8
+
T cell responses (Figure 4.7B,
lower right), suggesting that GLA-enhanced CD4
+
T cell immune response in vivo is also
largely dependent on the MyD88 pathway. Another notable observation from this study is
the important role of MyD88 in generating vaccine-specific immunity induced by DC-LV
immunization. Comparing to the wild-type mice, the MyD88
-/-
mice had a markedly
diminished CD8
+
and CD4
+
T cell responses after immunization, while TRIF-deficient
mice could retain the similar level of the response magnitude (Figure 4.7B). All of these
data suggest that MyD88 rather than TRIF plays an important signaling transduction role
for DC-LV vaccine to generate immune responses to immunogens in vivo.
4.4 Discussion
Antigen genes delivered to DCs by replication-deficient LVs generate both
antigen-specific T cell and B cell responses in mice and rhesus macaques.(Beignon and
others 2009; Hu and others 2011) Skin-derived DCs are the major cell targets for LV-
based immunization.(Furmanov and others 2010; Goold and others 2011; He and others
2006) However, several studies have shown that LVs are weak stimulators for activation
95
of DCs by themselves,(Escors and others 2008) and further activation is needed.(Arce
and others 2011; Breckpot and others 2009) Activation of individual TLRs or combined
TLRs could be incorporated in order to achieve stronger T cell responses.(Gautier and
others 2005; Lang and others 2005; Napolitani and others 2005; Warger and others 2006;
Y. Yang and others 2004) We have developed a DC-targeted LV vector system where
LVs are enveloped with an engineered Sindbis virus glycoprotein. This DC-LV system
can target DCs through interaction with DC-SIGN and subcutaneous immunization with
this vector has induced strong antigen-specific immunity.(L. Yang and others 2008) Our
in vitro study revealed that DC-SIGN-interacting DC-LV only modestly induces
maturation of DCs.(L. Yang and others 2008) Therefore, it is possible that further
activation of DCs would improve the immune responses induced by DC-LV. Here we
demonstrate that the synthetic TLR4 agonist GLA can be used as adjuvant to enhance
DC-LV-mounted immune responses.
Some studies reported that LV-mediated transduction efficiency towards DCs
could be reduced with prior to or simultaneous activation of DCs.(Breckpot and others
2003) Other studies showed that transduction of LVs did not hamper the subsequent
activation of DCs by TLRs.(Esslinger and others 2003) The possible explanation is that
upon maturation, internalization potency of DCs is down-regulated,(Garrett and others
2000; Reis e Sousa and others 1993) which may cause the reduced transduction. In light
of these observations, we decided to employ a protocol to deliver LVs and GLA at
different locations (footpad vs. base of tail). After administration, GLA could be
transported to lymph nodes through lymphatic circulation and encounters DCs that have
96
been modified by LVs. In addition, GLA could activate DCs that have not been
transduced by LVs; these DCs could subsequently migrate to the lymph nodes to not only
secrete inflammatory cytokines but also cross-prime antigens derived from LV-modified
DCs through cross antigen presentation, which could eventually also enhance antigen-
specific immunity.
Three signals are required for efficient stimulation of T cells by DCs: 1) an
antigen-specific signal, involving the interaction of the MHC/peptide complex with T cell
receptor (TCR); 2) a surface costimultory protein on DCs interacting with a receptor on T
cells; and 3) a cytokine signal passed from DC to T cell.(Guermonprez and others 2002;
Karwacz and others 2011) Our in vitro activation study, presented in Figure 4.1, indicated
that molecular signatures involved in all these three signals could be enhanced after
BMDCs were treated by GLA, including the upregulation of MHC molecules, elevated
expression of costimulatory molecules CD80 and CD86, and improved production of
proinflammatory cytokines (IL-6, TNF-α, IL-12, and IL-1β) and chemokines
(RANTES/CCL5 and IP-10/CXCL10); a comparable potency was observed upon LPS
stimulation (data not shown). This is consistent with prior studies, in which either murine
and human monocyte/macrophage cell lines(Baldwin, Shaverdian, and others 2009) or
murine BMDCs(Coler and others 2011) secreted TNF-α, IL-6, and IP-10 in response to
aqueous GLA stimulation. GLA-mediated DC maturation is TLR4-dependent, as the
TLR4
-/-
BMDCs failed to respond to GLA. Our use of genetically deficient cells to
directly demonstrate the TLR4 dependence of GLA complements previous use of anti-
TLR4 antibody to successfully block GLA-mediated activation.(Baldwin, Shaverdian,
97
and others 2009) To further improve DCs potency, combination strategy of TLRs with
silencing of inhibitory pathways(Karwacz and others 2011; Song and others 2008) can
also be taken into consideration.
Both CD8
+
cytotoxic and CD4
+
T helper cells act coordinately to either kill tumor
cells or clear infectious pathogens. In our study, we found that by co-administration of
GLA along with DC-LV immunization, numbers of both CD8
+
and CD4
+
T cells specific
for OVA antigen were greatly increased in mice. We confirmed that the enhancement is
strictly TLR4-dependent, as no elevated response to GLA was seen in TLR4-deficient
mice. In addition to T cell immunity, the antibody response is also important to clear
infectious viruses and initiate Fc-mediate tumor cell killing by natural killer (NK)
cells.(Clynes and others 2000) We found that GLA improved the titers of various
antibody isotypes specific for OVA, including that of both IgG1 and IgG2a, indicating
that GLA can boost both the Th1 and Th2 responses. We observed an enhanced IgA
antibody titer, suggesting that GLA can be used as an adjuvant to facilitate the
application of DC-LV vaccination for inducing mucosal immunity.
CD4
+
T cells provide essential help to CD8
+
T cell during the priming stage to
generate effector and memory CD8 T cell responses.(Dullaers and others 2006; Shedlock
and others 2003; Sun and others 2003) On the other hand, it was reported that helper-
independent cytotoxic T cell priming can be promoted by upregulating CD40L
expression on DCs, and only TLR3 and TLR9, but not TLR2 and TLR4, have the
capacity to stimulate CD40L expression.(Johnson and others 2009) This suggests that
GLA, as a TLR4 agonist, may promote CD8
+
cytotoxic T cell response in a CD4
+
T
98
helper cell-dependent manner. There are several hypotheses and models regarding how
CD4
+
T cells provide help to CD8
+
T cells through APCs. One of them requires that
activated CD4
+
T cells and CD8
+
T cells interact with the same APC.(Bennett and others
1997; Ridge and others 1998) Another model is that CD4
+
T cells can acquire the MHC-
peptide complexes and costimulatory molecules from APCs, and then these CD4
+
T cells
carrying acquired antigen presentation components can efficiently stimulate CD8
+
T
cells.(Xiang and others 2005) We conducted a simple CD4
+
T cells depletion experiment
to study their role in GLA promotion of the CD8
+
T cell response. Our results show that
without help from CD4
+
T cells, the CD8
+
T cell response cannot be augmented by GLA.
Either model could explain the involvement of CD4
+
T cells with the adjuvant effect of
GLA possibly dependent on CD4
+
T cells to indirectly pass signals to CD8
+
T cells.
Interestingly, we also found that GLA failed to improve immune responses induced by
DC-LV encoding the unmodified OVA sequence (without transferrin receptor
membrane-binding segment) (data not shown). Considering that a much lower OVA-
specific CD4
+
T cell response is elicited with such an antigen configuration, it seems that
the presence of immunogen-specific CD4
+
T cells is vital for GLA to augment DC-LV-
based vaccination. We also observed that CD4
+
T cells play a critical role in generating
primary CD8
+
T cell responses induced by either DC-LV or VSVG-enveloped LVs, and
depletion of CD4
+
T cells markedly reduced vaccine-specific CD8
+
T cell responses in
either case. This is in good agreement with a recent report showing the important role of
CD4
+
T cells in LV-mediated immunization.(Goold and others 2011)
99
Activation of TLR4 triggers two different signaling pathways controlled
respectively by MyD88/MAL, which is critical for regulating proinflammatory cytokines
production, and TRIF/TRAM, which is responsible for mediating the induction of Type I
interferons.(Iwasaki and others 2004; Lu and others 2008) Many of these signaling
studies have been conducted in macrophage cells. One recent study compared MPL with
LPS in mouse macrophages and showed that MPL is a more TRIF-biased TLR4 agonist
and preferentially induces TRIF-mediated signals for macrophage activation.(Mata-Haro
and others 2007) However, their subsequent studies in BMDCs demonstrated that both
MyD88 and TRIF pathways are involved in the MPL-mediated activation of DCs.(Cekic
and others 2009) Shen et al. found that induction of both MyD88 and TRIF signaling
pathways are critical for maximizing the capacity of DCs for T cells priming.(Shen and
others 2008) We found that GLA activates BMDCs in vitro in both MyD88- and TRIF-
dependent pathways. This represents the first study to use genetically deficient DCs
(MyD88
-/-
and TRIF
-/-
) to investigate signaling activation of GLA in DCs, and the data
corroborates the result from a mouse inflammation microarray study that showed both
MyD88- and TRIF-inducible genes responding to GLA stimulation. In contrast, in our in
vivo data we observed a more MyD88-biased signaling role for GLA to elicit adjuvant
responses to DC-LV immunization. It is unknown whether the MyD88-biased role is
unique for the interplay between GLA and DC-LV or it is more general and can be seen
in other formats of vaccine delivery; more experiments are required to address these
questions. One possible explanation is found in the behavior of the cytokines IL-6 and
TNF-α, which appears to be key cytokines for priming CD8
+
T cell responses in vivo.
100
Our in vitro study showed that MyD88
-/-
BMDCs almost completely lacked secretion of
TNF-α and IL-6, while TRIF
-/-
BMDCs could still respond to GLA treatment and secrete
a significant level of these two cytokines. An inability of MyD88
-/-
BMDCs to produce
these cytokines in response to GLA could partially explain the strong dependence on the
MyD88-dependent pathway for GLA augmentation of vaccine-specific immunity in vivo.
4.5 Acknowledgements
We thank Paul Bryson and April Tai for critical reading of the manuscript. This
work was supported by grants from the National Institutes of Health (R01AI68978 and
P01CA132681), a grant from the Bill and Melinda Gates Foundation, a translational
acceleration grant from the Joint Center for Translational Medicine and a grant from the
California HIV/AIDS Research Program.

101
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Abstract (if available)

Abstract Dendritic cell (DC)-based vaccines have shown promise as an immunotherapeutic modality for cancer and infectious diseases in many preclinical studies and clinical trials. Dendritic cells (DCs) are specialized antigen presenting cells (APCs) that can uptake and process antigens for presentation through the major histocompatibility complex (MHC) and activate naïve T cells. Because of this unique biological role, DCs have been widely exploited to develop DC-based vaccines for protective immunity against bacterial, viral, and fungal infections. The development of DC-based vaccines has also been one of the major focuses of cancer immunotherapy. Patient-derived DCs are loaded with tumor antigens and subsequently administered back to the patient. This type of autologous cell therapy led to the first FDA-approved cancer vaccine, sipuleucel-T. However, the tedious procedure for generating the vaccine, its high cost ($93,000 USD) per patient, and only modest improvement in survival (an average of 4.1 months) could limit its extensive application. A better strategy would be direct and specific loading of antigens onto DCs in vivo, which could be achieved by targeting DC-specific cell-surface receptors that facilitate internalization of the bound antigens for antigen presentation. DC-specific ICAM3-grabbing non-integrin (DC-SIGN or CD209) is a promising target for DC- specific antigen delivery because it is predominantly expressed on DCs. In chapter 2, the recombinant extracellular domains (ECD) of human and mouse DC-SIGN (hDC-SIGN and mDC-SIGN) were generated as DC-specific targets for mRNA display. Accordingly, an antibody-mimetic library was constructed by randomizing two exposed binding loops of an expression-enhanced 10th human fibronectin type III domain (e10Fn3). After three rounds of selection against mDC-SIGN, followed by four rounds of selection against hDC-SIGN, we were able to evolve several dual-specific ligands, which could bind to both soluble ECD of human and mouse DC-SIGNs. Using a cell-binding assay, one ligand, eFn-DC6, was found to have high affinity to hDC-SIGN and moderate affinity to mDC-SIGN. When fused with an antigenic peptide, eFn-DC6 could direct the antigen delivery and presentation by human peripheral blood mononuclear cell (PBMC)-derived DCs and stimulate antigen-specific CD8⁺T cells to secrete inflammatory cytokines. In chapter 3, we evaluated dendritic cell (DC)-directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a novel tumor vaccine for prostate cancer in mouse models. Direct immunization with the DCLV-PSCA in male C57BL/6 mice elicited robust PSCA-responsive CD8⁺ and CD4⁺ T cells in vivo. In a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1) synergetic tumor model, we further demonstrated that DCLV-PSCA-vaccinated mice could be protected from lethal tumor challenge in a prophylactic model, whereas slower tumor growth was observed in a therapeutic model. In chapter 4, we further improved the immune response of DC-directed lentiviral vaccine by employing a novel TLR4 agonist GLA as adjuvant. Both T cells and B cells responses were greatly enhanced and these improved responses further suppress tumor growth. We also looked at the relationship between the improvement of CD8⁺ T cells and CD4⁺ T cells and which signaling transduction pathway, MyD88 or TRIF, plays a more important role in adjuvant effect.

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Asset Metadata

Creator Xiao, Liang (author)

Core Title Dendritic cell-specific vaccine utilizing antibody-mimetic ligand and lentivector system

School Andrew and Erna Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Chemical Engineering

Publication Date 04/09/2013

Defense Date 03/22/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag antibody,cancer,DC-SIGN,Dendritic cells,immunotherapy,lentivector,ligand,OAI-PMH Harvest,vaccine

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Pin (committee chair), Chen, Si-Yi (committee member), Shing, Katherine (committee member)

Creator Email liangxia@usc.edu,liangxiaousc@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-234235

Unique identifier UC11295132

Identifier usctheses-c3-234235 (legacy record id)

Legacy Identifier etd-XiaoLiang-1531.pdf

Dmrecord 234235

Document Type Dissertation

Rights Xiao, Liang

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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