By Dr. Shyamasri Biswas, Dr. Megha Agrawal, Dr.

Kim Van Vliet, Contributing Editors

We introduce another new Column on Biotechnology to the magazine this month. While not strictly related to vacuum and coating technology, biotechnology employs a wide range of thin film,
plasma and surface engineering technologies. This column reflects the growing interrelationships
of all fields of science and, to this end, this column will provide news on a new technology front
and is intended to integrate with the current Columns, articles and editorial. We welcome your
comments and suggestions on this Column. The authors of the biotechnology column are Drs.
Shyamasri Biswas, Megha Agrawal and Kim Van Vliet.
Dr. Peter M. Martin, Executive Editor

Cancer Immunotherapy:

A New Hope in Cancer Treatment

ancer is a disease that affects

many families here in the United
States and worldwide. According
to the World Health Organization (WHO),
cancer cases are expected to rise 57% in
the next 20 years and renewed efforts are
needed worldwide to combat this disease.
In simple words, cancer results when cell
growth is unregulated. This uncontrollable growth of cells leads to malignant
tumor formation that can invade nearby
parts of the body. In order to grow, cancer cells are able to turn on or off genes
that enable cells to multiply and invade
neighboring organs. This process, metastasis, usually results in a poor prognosis
for the patient since these secondary tumors often go undetected for quite some
time. Three traditional treatments for cancer have involved surgery, radiation and
chemotherapy. However, these treatments
are associated with risks and undesirable
side effects. One such example is from
the most viewed and highly cited paper in
The Journal of Experimental Medicine in
December 2013 which reports about how
drugs boost resistance (1).

It is a well-known fact that chemotherapy kills cancer cells however, in the case
of colorectal cancer it was found that it
can also stimulate cancer cell growth by
activating cells called fibroblast present
in connective tissue. Matthew Kalady
and Jeremy Rich at the Cleveland Clinic
in Ohio and their colleagues analyzed tumors from patients with colorectal cancer
before and after chemotherapy. These researchers found that proliferation of cancer-associated fibroblasts increased significantly after treatment, and that these
cells enhanced the ability of a subset of
cancer cells to initiate tumor growth. The
fibroblasts seem to do this by secreting
signaling proteins, called IL-17A or interleukin 17A. The findings suggest that chemotherapy can trigger drug resistance by
changing the tumors microenvironment.
In the future, cancer therapies can be improved by disrupting this mechanism.
A fourth form of cancer treatment called
cancer immunotherapy has emerged and
developed over the last decade. This form
of biotechnology-enabled treatment involves utilizing the bodys own immune

system to treat cancers. Cancer immunotherapy is a two-way learning process: while the immune system teaches
us about its intricacies, researchers can
in turn teach immune cells how to use
those abilities to target cancer cells. After
gaining this knowledge of the immune
system researchers can create synthetic
molecules to attack a tumor, or can help
the immune system to function more effectively (2). Cancer immunotherapy can
be divided into four major types: non specific therapies, monoclonal antibodies,
vaccines (Figure 1) and T-cell activation
therapy (Figure 2). Non-specific immunotherapy includes the use of cytokines
and chemicals that can stimulate an immune response. The second type uses a
monoclonal antibody, which are antibodies that are specific to a particular antigen
and does not exhibit any cross reactivity.
The third form, called cancer vaccine
therapy, stimulates the bodys immune
system to fight cancer. These vaccines are
derived from cancer cell antigens that are
in turn recognized by cells of the immune
system (Figure 1).

Vacuum Technology & Coating March 2014 www.vactechmag.com or www.vtcmag.com 

Figure 1. Three types of cancer immunotherapies [Credit: Elert E. 2013 (2)].

the T-cells by the major histocompatibility complex (MHC) molecules present

on the surface of the APCs. The second
signal that results after recognition activates T-cells which can then attack the tumor cells and destroy it. The activation of
T-cells is mediated by interaction with B7
and CD28. However in some cases T-cell
activation is blocked. This occurs when
B7 present on the APCs binds to CTLA-4,
which results in a lack of activation of the
T-cells. However, if the CTLA-4 receptor
is blocked by an antibody like ipilimumab, B7 becomes available to CD28 thereby activating the T-cell response (Figure
2) against cancer cells (6).
Another molecule which was discovered in the 1990s by a biologist in Japan
called programmed death 1 or PD1 can
also block the T-cell response and an anti-PD antibody has shown promise in cancer treatment. Oncologist Drew Pardoll of
Johns Hopkins University urged Medarex
to test this antibody on cancer patients.
In 2008, five of the volunteers who participated in the clinical trials noticed their
tumors shrink in size. PD1 is different
in specificity and biological function in
comparison to CTLA-4. Although PD1
like CTLA-4 is present on the surface of
the T-cells, it can recognize two different
ligands PDL1 and PDL2 present on APC.
These two ligands are immunosuppressive and can keep PD1 engaged preventing T-cell activation against cancer. This

A fourth method which has shown 20% of patients by 3 years as opposed to

great promise involves T-cells which are a 12% of patients who received the drug
type of white blood cell and can be distin- dacarbazine without any antibody (5).
Ipilimumab also known as Yervoy is
guished from other cells by the presence
of a receptor commonly known as T-cell a human IgG1 antibody that can block
receptors (TCR). This research began in CTLA-4 present on the surface of the
the late 1980s when a new protein recep- T-cell. The mechanism of action of this
tor was identified on the surface of T-cells drug can be explained in the following few
called cytotoxic T-lymphocyte antigen steps: T-cells at first recognize an antigen
4 or CTLA-4. At this time, a team of re- present on the cell surface of an antigen
searchers suggested that blocking CTLA- presenting cell (APC). These antigens are
4 on the surface of the T-cell would set proteins that the body recognizes as forthe bodys immune system free to destroy eign and which in turn can be destroyed
cancer cells. Later cancer immunologist by T-cells. The antigens are presented to
James Allison in 1996 published a report
in Science demonstrating that antibodies against CTLA-4 destroyed tumors in
mice (3). In the year 1999, Medarex, a
small biotechnology company took this
science forward and developed an antibody against CTLA-4.
In 2010 Bristol-Myers Squibb which
purchased Medarex reported that patients
with metastatic melanoma who took the
antibody to CTLA-4 lived 4 months longer on average compared to those who
did not. They also showed that more than
a quarter of the patients with advanced
melanoma survived for at least two years.
In March 2011, the US Food and Drug
Administration (FDA) approved an anti-CTLA-4 antibody named ipilimumab Figure 2. Negative signaling in T-cells blocked by Ipilimumab: T-cell response is activated
(4). In addition to this, recent clinical trials when CD 28 interacts with B7, however when B7 interacts with CTLA-4 the T-cell response
on melanoma patients demonstrated that a is inactivated. Blocking CTLA-4 with ipilimumab, B7 can interact with CD28 leading to acticombination of chemotherapy drug dacar- vation of the T-cell response. APC, antigen presenting cell; CTLA-4, cytotoxic T-lymphocyte
bazine with the antibody ipilimumab im- antigen-4; MHC, major histocompatibility complex; TCR, T-cell receptor [Credit: Lebbe et al.
proved the chances of survival in nearly 2008 ESMO oral presentation (7)].
2 vtc@vtcmag.com 
March 2014 Vacuum Technology & Coating

interaction of PD1 with its ligands also

leads to T-cell exhaustion in cancer patients; hence, blocking the ligand with anti-PD1 antibody can lead to the activation
of the T-cell response. The side effects
seen with anti PD-1 were also milder
which makes this an encouraging drug for
the treatment of cancers (4).
The effects of these antibody treatments
on the bodys immune system still remain
under investigation. In the case of both
CTLA-4 and anti-PD1 treatments, tumors
were seen to grow before disappearing.
The side effects involved with ipilimumab treatment included severe diarrhea and
colitis. Patients who were treated with
dacarbazine and ipilumumab suffered
side effects such as liver toxicity. Some
patients were seen responding to the
treatment even after the antibody was discontinued. Most of the side effects were
gastrointestinal immune related adverse
events (IRAE).
Bristol-Meyers Squibb reported in
2013 that 22% of 1800 patients with melanoma who were treated with ipilimumab
were alive 3 years later. In June of 2013
a number of researchers reported that a
combination of ipilimumab and anti-PD1
administration resulted in rapid tumor regression in one third of the patients tested.
The complex cellular mechanism
that leads to tumor cell rejection is still
a topic thats under investigation by researchers in this field. T-cells are capable of producing cytokines, chemokines
and anti-angiogenic factors after the
antigen is recognized on the tumor cell
surface thereby inhibiting their growth.
Effective antitumor responses by T-cells
also lead to cytotoxic responses like cell
death by apoptosis or the release of cytotoxic molecules.
Two types of T-cells exist, that participate in the process of the antitumor
response. These include T-cells that are
CD8+ and CD4+. The term CD is associated with the type of glycoprotein on the
surface of the T-cells. CD4+ T-cells are
helper T-cells that are activated when they
come in contact with the peptides presented by MHC class II molecules present on
the surface of APCs, while CD8+ T-cells
are cytotoxic T-cells that can destroy tumor cells or virally infected cells. The
CD8+ cells are activated by antigens presented by MHC class I molecules present

on the APC surface. Fully mature CD8+

T-cells and some CD4+ T-cells can release
interferon- (IFN- ) and tumor necrosis
factor (TNF) which enhances the immune
response by expressing more MHC class
I and Class II APC molecules that can recruit additional T-cells thereby amplifying
the immune response. Nearly every nucleated cell in the body expresses MHC class
I molecules, however neoplastic cells are
known to downregulate MHC class I expression thereby leading to the proliferation of tumor cells.
Another area of research based on T-cell
immunotherapy was reported by Steven
Rosenberg of the National Cancer Institute (8). This method is called adoptive
cell transfer (ACT). There was mounting evidence based on intravital imaging
that migration of tumor specific T-cells
are arrested upon binding to tumor antigens. These arrested T-cells also known
as tumor infiltrating lymphocytes (TIL),
can be isolated from melanoma patients
and expanded in the laboratory by adding
the T-cell growth factor IL2 (Interleukin
2). These expanded T-cells can then be
infused back into patients after preparatory lymphodepletion. The lymphodepletion in patients is achieved by using
chemotherapy or total body irradiation of
patients. Patients observed eradication of

tumors for extended periods of time when

these T-cells along with IL2 were infused
into them (Figure 3).
The ACT based therapy outlined above
should not be confused with cancer vaccines (Figure 1 panel 3); one such cancer
vaccine sipuleucel-T has been approved
by the US Food and Drug Administration
(FDA) for treatment of castration-resistant prostate cancer. Although these vaccines are easy to use and have been found
to be less toxic, they merely enhance the
patients immune system. So far these
vaccines have been unable to prolong the
lives of cancer patients, even when combined with the long term effects of other
treatments. Moreover, these vaccines have
been unable to cure or improve conditions
of metastatic forms of cancer.
TIL-based immunotherapy has offered
promise in curing metastatic melanoma.
Some patients who participated in clinical trials have lived for 8 years and have
remained disease free. The efficacy of
TILs along with IL2 infusion in patients
have been restricted to melanoma patients
mainly due to the fact that this type of cancer has more mutations associated with it
than any other type of cancer. However,
TIL therapy also has some limitations.
Not all patients responded well to the
treatment and the reason for this is still

Figure 3. TIL (Tumor infiltrating lymphocyte) based immunotherapy [Credit: Restifo NP et. al
2012 (8)].

Vacuum Technology & Coating March 2014 www.vactechmag.com or www.vtcmag.com 

unknown. Additionally, this treatment

could only be administered to a subset of
patients who could withstand the lymphodepletion process.
In 2010, Rosenberg and colleagues
published reports that genetically engineered T-cells can be produced in the lab
to attack and destroy tumor cells. These
T-cells can be produced by three different
methods: (a) T-cells can be isolated from
patients who have shown a good antitumor response. The T-cell receptors of
these cells can be cloned into retrovirus
and lentiviral vectors which can in turn
infect the cancer patients T-cells. This
T-cell population can be expanded in the
lab and then infused into patients to treat
cancer. (b) The second process is known
as chimeric antigen receptor therapy or
CAR therapy. In this method the variable
region of the antibody which is specific
to a tumor antigen is identified and then
grafted into T-cell receptors which are
also capable of activating T-cells. These

CAR grafted T-cells are specific to certain antigens and can recognize MHC
non-restricted structures present in tumor
cells and can be directly given to patients
for treating cancer. (c) The third process
involves isolating T-cell receptors from
humanized mice. These transgenic mice
expressing human MHC class I or MHC
class II molecules can be treated with human tumor antigens. The T-cells generated in this manner can be isolated from the
mice and their TCR genes can be cloned
into retroviral vectors. These vectors can
then be used to generate tumor specific
T-cell receptors compatible to the patients immune system and then used for
treating cancer (Figure 4) (8).
The advantages of using genetically engineered T-cells include the ability by the
researcher to develop T-cells against various types of cancer. The T-cells with high
affinity and high selectivity can be identified to treat patients and then produced
by genetic engineering. In addition, the

T-cell population at various differentiation

stages can be selected. The highly specific
TCR genes are generated using retroviral or lentiviral vectors. This method has
shown promise with a range of cancer
histologies like neuroblastoma, synovial
cell sarcoma, leukemia and lymphoma.
Genetic engineering has enabled inserting
genes that can improve the efficacy of the
T-cells by introducing genes for costimulation as well as preventing inflammation
and apoptosis.
The chimeric antigen receptor or CARs
offer great promise in future cancer immunotherapy as they can combine the antigen binding site with the signal activating machinery of the T-cell. The binding
site of the monoclonal antibody (mFab) is
combined with the signaling portion of the
T-cell. Since the Fab is the product of two
genes the corresponding heavy and light
chain genes are combined with a linker,
which allows them to fold over each other in a native like conformation creating

Figure 4. Three ways to generate genetically engineered T-cells specific to tumor antigens [Credit: Restifo NP et. al 2012 (8)].

vtc@vtcmag.com 

March 2014 Vacuum Technology & Coating

Figure 5. Development of chimeric antigen receptor (CAR). Part of the monoclonal antibody
shown in circle that recognizes the cancer antigen is combined together to form a single chain via
a linker which is then combined to the signaling portion of the T-cell receptor. These genetically
engineered T-cells can then recognize the tumor cell effectively. [Credit: Ramos et. al 2011 (9)].

a single chain also known as scFv or single-chain fragment variable. This single
chain derivative has high affinity for cancer antigens. Using genetic engineering
techniques, the single chain antibody is
combined with the cytoplasmic domain of
immune receptors that can initiate signal
transduction thereby activating the T-cell
response (Figure 5) (9).
Treatment of tumors using genetically
engineered T-cells also has certain disadvantages. Often times mispairing of and
TCR chains can occur which leads to
reactivity against unintended antigens. Instead of recognizing tumor antigens other
unintended healthy cells can be attacked
due to antigen mimicry. This process
can lead to unnecessary side effects like
inflammation and release of cytokines.
Clinically this can lead to various side
effects like poor pulmonary function, renal failure and even death. However, the
side effects can be brought under control
by rapidly eliminating these T-cells from
the patients body. One such process involves the use of T-cells which are able to
express caspase 9 and are capable of killing toxic T-cells when activated by a small
molecule or drug.
Currently, there are over 1000 clinical
trials to evaluate immunotherapy for the
treatment of a variety of cancers, including prostate cancer and malignant melanoma. Several of the clinical trials will
test currently approved therapies (radiation; chemotherapy) combined with im-

munotherapy to determine how well this

is tolerated in patients and evaluate the
added therapeutic benefit of the combined
treatment. Immunotherapy may provide a
treatment option for patients with cancers
that did not respond to currently approved
therapies. Additionally, immunotherapy
may also allow for treatment of cancer at
an earlier stage, for example after surgical
removal of the tumor, but prior to detection of any new cancer growth. Cancer
metastasis is often difficult to detect until later stages and the metastatic lesions
are often what results in the death of the
patient. The ability to utilize immunotherapy to kill these metastatic cancer cells
provides an additional treatment option
for these very aggressive cancers, and
due to its specificity, may be better tolerated than current treatments. Advances in
medical imaging technology are providing new insights into how the interaction
of cancerous cells occurs with other cells
at the molecular level [10]. State-of-theart optical microscopy technology has
been applied by scientists to investigate
fluorescently labelled immune and tumor
cells in living animals. Results from such
research have shown that in-vitro experiments that are considered an important
component of immunotherapies may not
be accurate. The reason is that the action
of cells in culture is significantly different
compared to when immunotherapies are
applied to the human body. In this regard,
fluorescent labels have been developed

to track cells and monitor their migration

and the duration of their interaction with
other cells [10]. It has been found that the
interaction of immune cells with one another along with the cancer cells is much
stronger inside the body than outside the
body. The work done by Philippe Bousso
has shown that it takes several hours for
a T-cell to kill a single cancer cell in the
body [11].
Concluding Remarks: Out of all four
methods of immunotherapy mentioned
above, the T-cell immunotherapy has
been the most successful method of cancer treatment to date. Although the ACT
method is still in the experimental stage
and requires FDA approval, it offers great
promise in the cancer immunotherapy
field. Biotech companies are coming together to move the science from the laboratory to the market place. More research
is ongoing to deal with the side effects
of immunotherapy and investigations are
continuing to better understand why all
patients do not benefit from this treatment.
Undoubtedly, the FDA approved drug ipilimumab or yervoy either by itself or in
combination with other antibodies like
anti-PD1 has opened new doors to potentially lifesaving treatment options and has
given hope to many cancer patients.
References for further reading
1. 
Chemotherapy activates cancer-associated fibroblasts to maintain colorectal
cancer-initiating cells by IL-17A. Lotti F,
Jarrar AM, Pai RK, Hitomi M, Lathia J,
Mace A, Gantt Jr. GA, Sukhdeo K, DeVecchio J, Vasanji A, Leahy P, Hjelmeland
AB, Kalady MF and Rich JN. The Journal
of Experimental Medicine (2013) 210,
2851-2872.
2. 
Calling cells to arms. Elert E. Nature
(2013)504, S2-S3.
3. Enhancement of antitumor immunity by
CTLA-4 blockade. Leach TR, Krummel
MF and Allison JP. Science (1996) 271,
1734-1736.
4. 
Cancer Immunotherapy. Couzin-Frankel
J. Science (2013). 342, 1432-1433.
5. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. Robert
C, Thomas L, Bondarenko I, O'Day S,
M D JW, Garbe C, Lebbe C, Baurain JF,
Testori A, Grob JJ, Davidson N, Richards
J, Maio M, Hauschild A, Miller WH Jr,

Vacuum Technology & Coating March 2014 www.vactechmag.com or www.vtcmag.com 

Gascon P, Lotem M, Harmankaya K, Ibrahim R, Francis S, Chen TT, Humphrey R,

Hoos A, Wolchok JD. New England Journal of medicine. (2011) 364, 2517-2526.
6. Ipilimumab: in the treatment of metastatic
melanoma: A summary of recent studies.
Ascierto PA. Tumeri (2013) 99, e302-305.
7. Lebbe C et al. 2008 ESMO oral presentation. Abstract 7690.
8. Adoptive immunotherapy for cancer: harnessing the T-cell response. Restifo NP,
Dudley ME and Rosenburg SA. Nature
Immunology (2012). 12, 269-281.
9. Chimeric Antigen receptor (CAR)- Engineered Lymphocytes for Cancer Therapy.
Ramos CA, Dotti G. Expert Opinion in
Biological Therapy (2011) 11, 855-873.
10. 
Medical imaging: Removing the blindfold. Bourzac K. Nature (2013) 504,
S10-S12.
11. 
Two photon imaging of intratumoral
CD8+ T-cells cytotoxic activity during
adoptive T-cell therapy in mice. Breart B,
Lemaitre F, Celli S, Bousso P. Journal of
clinical Investigation (2008) 118, 13901397.

Contributing Editors
Dr. Shyamasri Biswas received her Ph.D.
in Biotechnology jointly from Banaras
Hindu University, India and the University
of Potsdam in Germany in 2003. She was
awarded the prestigious German Academic Exchange Service (DAAD) sandwich
model international scholarship and carried out her Ph.D. thesis work in the Department of Physical Biochemistry at the
University of Potsdam, Germany. She also
received the Council for Scientific and
Industrial Research fellowship in India.
Dr. Biswas has held research positions in
protein biochemistry, structural biology,
biotechnology and molecular biology at
top-tier US institutions. Her most recent
affiliation has been with the University of
Florida where she has worked as a postdoctoral scientist in the Department of
Biochemistry and Molecular Biology. Dr.
Biswas has published over twenty peer-reviewed research papers in prestigious
international journals in the field of biotechnology that include Nature Structural
Biology, Journal of Biological Chemistry,
Structure and Biochemistry. She has also
given several talks at national and international meetings and has been an invited reviewer for a number of international
journals. Dr. Biswas can be contacted at
shyabiswas@gmail.com.
Dr. Megha Agrawal received her Ph.D.
in Biotechnology in 2010 from the Indian Institute of Technology Roorkee,
which is one of the premier institutions
in India with an outstanding reputation
across the globe. She was awarded the
prestigious National Fellowship of Council of Scientific and Industrial Research
to carry out her Ph.D. work. Her research
interests lie predominantly in the area of
Neuroscience with emphasis on neurodegenerative disorders. Her initial research
involved determining the role of various
herbal compounds and trace elements in
neuroprotection within rodent models of
neurodegeneration. During an active research career of more than eight years,
Dr. Agrawal has made significant contributions to develop a rapid, cost effective
and more sensitive mechanism based

vtc@vtcmag.com 

in-vitro model of ischemic stroke as first

tier of screening of neuroprotective drugs
for their anti-stroke potential. To further
achieve these objectives, and to broaden
and solidify her experience and knowledge of stroke, she joined an internationally recognized expert in stroke research
as a postdoctoral fellow at the University
of Florida. Currently, she is working on
how placental derived hormones affect
neurodevelopment at the Childrens National Medical Center, Washington DC.
Dr. Agrawals research has benefited a
number of her colleagues with whom she
has worked and her research has resulted in numerous publications in worlds
prestigious scientific journals in the field
of biotechnology. She has also given several talks at national and international
meetings. Besides research, she has considerable experience in writing research
grant proposals and has been an invited
reviewer for a number of international
journals. Dr. Agrawal can be contacted at
meghaagra@gmail.com.
Dr. Kim Van Vliet received her Ph.D. in
Biomedical Sciences from the University
of Florida, College of Medicine in 2007.
She carried out her Ph.D. thesis work in
the Department of Molecular Genetics
and Microbiology with a primary research focus in the area of virology, with
specific emphasis on protein biochemistry
and gene therapy applications. She has
published nearly a dozen peer-reviewed
research papers in highly regarded journals in the field of biotechnology and
biomedical science, including Journal of
Clinical Investigation, Molecular Therapy, and Journal of Virology. She has held
research positions in molecular biology,
protein biochemistry, structural biology
and biotechnology. Her most recent affiliation has been at the University of Florida where she has worked as an Assistant
Scientist. She has presented her work at
national and international meetings and
has been an invited reviewer for journals
in her field. Dr. Van Vliet can be contacted
at kimvanvliet2@gmail.com.

March 2014 Vacuum Technology & Coating