Dendritic Cell Vaccine - COSMOS

Dendritic‐Cell Vaccine:
A New and Promising Approach to Treating
Cancers
Janet La
UCD COSMOS Cluster 1: Biotechnology
Mentor: Professor Paul Feldstein
Teacher Fellow: Rebecca Sela

Vaccines
Vaccines are biological
(mostly microbial) preparations
that induce the body to
produce an immune response
and boost the immune system’s
ability against infection. The
body will recognize the foreign
agent, destroy it and memorize
it for future infections by the
same microorganism.

Stimulation of the Immune System
Leukocytes (white blood cells) play
a key role in the immune system.
They include macrophages,
neutrophils and natural killer
cells.
• Some patrol the body seeking
foreign agents, diseased and
damaged cells to destroy. (in
non‐specific defense)
• Others, called lymphocytes work
against specific threats.

Example of Lymphocytes
– B cells : make antibodies, which
bind to and mark the foreign
invaders or abnormal cells for
destruction.
– Cytotoxic T cells: kill the foreign
agents and abnormal cells by
causing apoptosis.
• Helper T cells and Dendritic cells
help out by activating B cells
and killer T cells.

Cancer Vaccines
Vaccines work by activating B cells and killer T cells and “directing”
them to recognize and attack specific types of cancer.
• Antigens, which are substances that stimulate a specific immune
response, are introduced.
• They can be a protein or other molecules that are in the surface or inside of
the invader cells.
• These antigens will promote the generation of antibodies.
The vaccines, which contain cancerous cells or antigens, will stimulate
the immune system to generate cells that kill the cancer cells and
prevent the cancer from relapsing.
There are two main categories of cancer vaccines: preventive vaccines
and treatment vaccines.

Preventive Vaccines
Preventive Vaccines are designed to prevent the development
of cancer in healthy people.
They essentially work like normal vaccines by producing
immunity against the particular virus or microbe that cause
infections that lead to cancer.
The FDA has approved two types
of preventive vaccines.
•A vaccine against the Hepatitis B
virus, which leads to liver cancer.
•A vaccine against Human
Papillomavirus types 16 and 18,
which are responsible for cervical
cancer.

Treatment Vaccines
Designed to treat existing cancers by stimulating
the immune system to attack cancer cells.
Effective vaccines are hard to develop because in many cases, cancers escape
detection by the immune system because of self antigens and the vaccines
themselves often times weaken the immune system’s natural ability
against cancer cells.
The FDA has not approved any cancer treatment vaccines.
There are five types being developed:
• Antigen Vaccines –use tumor‐specific antigens
• Anti‐idoiotype Vaccines –use antibodies that act as antigens
• Dendritic Cell Vaccines –use dendritic cells to display antigens
• DNA vaccines –insert bits of DNA to increase production of antigens
• Tumor Cell Vaccines –use killed cancer cells

Dendritic Cell Vaccines
• Dendritic Cell vaccines involve using Dendritic cells from the cancer
patient to stimulate immune responses.
• The Dendritic Cell is a scarce immune
cell which recognizes, processes and
presents foreign antigens to the T‐cells
of the immune system. They constantly
roam around the body and function as
antigen‐presenting cells when they
discover antigens, calling on other cells
for help. However, Dendritic cells are
usually not present in a large enough
quantity to produce a strong immune
response. They are now generated by
the millions in labs.

Dendritic Cell Vaccines – cont.
• To make Dendritic cell vaccines,
scientists harvest peripheral blood
mononuclear cells, such as
lymphocytes or monocytes, from the
blood of the patient and grow them
in a laboratory in large amounts
while exposing them to antigens
from the patient’s own cancer. By
doing that, autologous cancer
vaccines are created.
• After the Dendritic cell takes up the
antigens, they will be displayed on
its surface.

v
• After the generation of
Dendritic cells and the
taking up of the antigens,
the cells, with antigens
presented on their surfaces,
are injected back to the
patient.
• The injected Dendritic cells
along with the antigens
activate the production of
the cytotoxic T‐cells, which
will be programmed to
recognize and destroy the
cancer cells.

Researchable Question
Are mature dendritic cells more effective than
immature dendritic cells in inducing an immune
response when used in vaccination against glioma
in mice

Mature vs. Immature Dendritic Cells
• Immature dendritic cells are commonly called
veiled cells because of their veils instead of
dendrites on mature cells.
• They are found in their immature state in the
blood.
• When activated, dendritic cells migrate to the
lymphoid tissues and interact with T and B
cells.
• Inflammatory stimuli switch dendritic cells to
an immunostimulatory mode and they are
“matured.”
• During maturation, the cells will grow branches
called dendrites.
The specifics of the workings of dendritic cells are
not yet fully understood and the maturity level
of dendritic cells may be a very important
factor in effectiveness of immunotherapies.

What is Glioma
• A type of cancer that starts in the brain or spine. It can spread
within the nervous system but usually do not spread to other
parts of the body.
• Annually about 17,000 Americans are diagnosed with gliomas
and the median survival of patients with glioblastoma
multiforme, which is the most common form of glioma, is
around 15 months.
These tumors are categorized
by location.
Supratentorial tumors, in the
cerebrum, affects mostly adults.
Infratentorial tumors, in the
cerebellum, affects mostly
children.

Why Dendritic‐cell vaccines for glioma
• Because despite advances in chemotherapy,
radiation and surgery, for many glioma patients all
these methods are ineffective.
• The location of these tumors make the traditional
methods tricky, since using each radiation treatment
kills off many healthy and crucial brain cells.
• Dendritic‐cell vaccine seems to be the most
promising for a targeted location in this type of
cancer.

Materials
• Mice implanted with
tumor cells
• Leukapheresis Machine
• Density Gradient
Centrifuge
• Counterflow Centrifuge
• Tissue Culture Flasks
• GM‐CSF (granulocyte
monocyte‐colony
stimulating factor)
• Human Serum with X‐
VIVO medium
• Interleukin‐4
• KLH (keyhole limpet
hemocyanin)
• Glioma Antigens:
EGFRvIII, tenascin and
GP 200
• ELISPOT Assay Machine

Experimental Design
• I will obtain 7 groups of mice with brain tumors as
my testing subjects. Three of the groups will be
treated with mature dendritic cell vaccines, three
groups will be treated with immature dendritic cell
vaccines and the last group will not be given any
treatment.

Experimental Design – cont.
• For the groups that will be treated with mature dendritic cell
vaccines, they will be labeled MDC‐1, MDC‐2 and MDC‐3.
Similarly, those that would be treated with immature
dendritic cell vaccines will be labeled IDC‐1, IDC‐2 and IDC‐3.
• White blood cells will be harvest and grown in culture.
– In MDC‐1 and IDC‐1 groups, dendritic cells will be grown and not
pulsed before reinjecting back into mice.
– In MDC‐2 and IDC‐2 groups, dendritic cells will be pulsed with KLH
before reinjecting back into mice.
– In MDC‐3 and IDC‐3 groups, dendritic cells will be pulsed with glioma
antigens: EGFRvIII, tenascin and GP 240.
• The seven mice groups will be tested by ELISPOT Assay to
observe immune responses.

Material Details
• GM‐CSF (granulocyte monocyte‐colony stimulating factor)
– Promotes the development of immature dendritic cells from
monocytes
• Human Serum with X‐VIVO medium
– Medium in which human cells are cultured
• Interleukin‐4
– Activate the growth of naïve immune cells
• KLH (keyhole limpet hemocyanin)
– A large protein extracted from the giant keyhole limpet Megathura
crenulata. It can act as a carrier protein
• MCM (monocyte‐conditioned medium)
– Medium generated from white blood cells that induces the maturation
of immature dendritic cells

Techniques
• Leukapheresis is a lab procedure in
which leukocytes (the white blood
cells) are separated from the blood.
• Counter flow Centrifugation is a
method to separate cells of different
sizes.
• Density Gradient Centrifugation is a
method which scientists use to
separate intact organelles from a cell.
These centrifuge machines involve
setting up a density variation or
gradient in the tubular centrifuge
such as gradients of sucrose
solutions. In the case of this
experiment, peripheral blood
mononuclear cells are separated
from the white blood cells extracted.

Techniques – cont.
• The ELISPOT assay is a highly sensitive
assay for analysis of cell activation at
very detailed levels. It is a very common
method for monitoring immune
responses in humans and animals.
• The ELISPOT assay is very useful for
analyzing specific immune responses to
antigens as proteins or peptide. The
assay can be used to identify and
differentiate between responses by
different types of T cells according to
the cytokine secretion pattern.
• The assay quantifies the secretion of
cytokines, which are signaling
molecules used for communication by T
cells.

Procedures
• Making Vaccines
1. Take blood samples from each mice and perform
leukapheresis.
2. From all samples, isolate PBMCs (peripheral blood
mononuclear cells) using density gradient centrifugation.
3. Separate half of the PBMCs and culture them to
generate MCM (monocyte‐conditioned medium)
4. Use counterflow centrifugation to isolate monocytes
from the remaining PBMCs.
5. Grow monocytes in tissue culture flasks with GM‐CSF, HS
with X‐VIVO medium and IL‐4.
6. To groups MDC‐1, MDC‐2, and MDC‐3, add the
autologous MCM to induce the maturation of the
dendritic cells. Cells will mature in about 10 days. s

Procedures – cont.
7. To groups MDC‐1 and IDC‐1, leave unpulsed.
8. To groups MDC‐2 and IDC‐2, pulse with KLH protein.
9. To groups MDC‐3 and IDC‐3, pulse with glioma antigens
EGFRvIII, tenascin and GP 240.
MDC
MDC +
KLH
MDC +
EGFRvIII,
tenascin,
GP240
No
Treatment
IDC
IDC +
KLH
IDC +
EGFRvIII,
tenascin,
GP240

Procedures – cont.
• Reinjection of Cells – Clinical Trial
10. After 5 days, inject the cell vaccines back into each
corresponding mice.
11. 1 week after the injection, collect blood samples from
each mice for immunological monitoring.
12. Use ELISPOT assay to assess the amount of antigenspecific
T cells and the amount of antibodies against KLH
and the glioma antigens.
12. Vaccinate and evaluate all groups of mice weekly.
13. Measure tumor size to check for the halting of growth

Possible Results
I think that my results will show the mature dendritic
cells to be more effective at inducing immune
responses in mice because after maturation,
dendritic cells express high levels of co‐stimulatory
molecules, which many increase cell adhesion and
enhance T cell activation.
I think the mice injected with mature dendritic cells
plus glioma antigens will live the longest and have a
longer period of halting growth of the brain tumor.

Works Cited
• Amano, Takayuki, et al. "Antitumor effects of vaccination with dendritic cells
transfected with modified receptor for hyaluronan‐mediated motility mRNA in
a mouse glioma model." J Neurosurg 106 (Apr. 2007): 638‐645.
• Bahn, Duke K., and Haakon Ragde. "Cancer Cryo‐Immunotherapy: A Battle
Between the Immune System and Cancer." PCRI Insights 9.1 (2006). Prostate
Cancer Research Institute. 29 July 2009 .
• De Vries, Jolanda M., et al. "Maturation of Dendriic Cells is a Prerequisite for
Inducing Immune Responses in Advanced Melanoma Patients." Clinical Cancer
Research 9 (Nov. 2003): 5091‐5100.
• Dendritic Cell Therapy for Cancer. 29 July 2009 .
• Dhodapkar, Madhav V., et al. "Antigen‐specific Inhibition of Effector T cell
Function in Humans after Injection of Immature Dendritic Cells." J. Exp.
Med. 193.2 (2001): 233‐238.
• Ebben, Johnathan D., Brandon G. Rocque, and John S. Kuo. "Tumour Vaccine
Approaches for CNS Malignancies ‐ Progress to Date." Drugs 69.3 (2009):
241‐249.
• ELISPOT. 29 July 2009 .
• "Glioma." Gliomas ‐ Diagnosis and Treatment Options at Mayo Clinic. 29 July 2009
.

Works Cited – cont.
• How Cancer Vaccines will work. 29 July 2009 .
• Kochar, Preeti Gokal. "Cancer Vaccines." CSA Illumina. Jan. 2006. 29 July 2009
.
• Naylor, Mark F. "Melanoma Vaccines." Dermatology Online Journal 6.1 (2000).
Department of Dermatology, University of Oklahoma Health Sciences Center .
29 July 2009 .
• Parmiani, Giorgio, et al. "Cancer Immunotherapy with Peptide‐Based Vaccines:
What have we achieved Where are we going" Journal of the National Cancer
Institute 94.11 (2002): 805‐818.
• Vaccines Definition ‐ Medical Dictionary. 29 July 2009
.
• Yamanaka, Ryuya. "Dendritic‐Cell‐ and peptide‐based vaccination strategies for
glioma." Neurosurg Rev 32 (Mar. 2008): 265‐273.

Images
• http://looi5.files.wordpress.com/2009/04/bacteria1.jpg
• https://meyerbio1b.wikispaces.com/file/view/strep_throat_bacteria.jpg
• https://mywebspace.wisc.edu/kajacobson3/web/MS21/images/Autoimmune%20R
esponse.jpg
• http://encarta.msn.com/media_461519550_761575681_‐1_1/Lymphocyte.html
• http://www.csa.com/discoveryguides/cancer/images/fig5.jpg
• http://www.astrographics.com/GalleryPrintsIndex/GP2008.html
• http://www.aecom.yu.edu/aif/gallery/sem/sem.htm
• http://dermatology.cdlib.org/DOJvol6num1/transactions/melanoma/fig013.gif
• http://www.new‐science‐press.com/browse/immunity/illustrations/4/
• http://www.40kradio.com/wp‐content/uploads/2009/04/question‐mark‐dice.jpg
• http://www.markmillermusic.org/blog/2009/01/09/
• http://www.chiron.com/images/library/hbv.jpg
• http://davispestcontrol.com/services
• http://chemistry.umeche.maine.edu/CHY431/Antibody.jpg