Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene therapy technology can help introduce new functional DNA to replace mutated genes. The idea first arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed that the same procedure could be done on humans to correct genetic defects by introducing therapeutic DNA. Currently, genetic modification of T lymphocytes has been the major area of research for treating malignant tumors. This technique seeks to create chimeric antigen receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the tumor that it is targeting.

1. Chimeric Antigen Receptor
In Gene Therapy
Duy-Khiem Chanh Pham
Kevin Hugins
Molecular Biology
March 13, 2014

2. 2
Introduction
Gene therapy was first conceptualized to alter debilitating fates of genetic diseases. Gene
therapy technology can help introduce new functional DNA to replace mutated genes. The idea first
arose in 1972 when Friedmann and Roblin authored a paper, “Gene therapy for human genetic
disease?”, demonstrating that exogenous DNA can be taken up by mammalian cells (1). They proposed
that the same procedure could be done on humans to correct genetic defects by introducing therapeutic
DNA. However, the first FDA-approved trial on humans was not until 1990 on two young females with
ADA-SCID, an immune deficiency disorder that leaves them defenseless against infections. Under Dr.
William French Anderson and a team of colleagues, the first successful human gene therapy using
reconstituted T lymphocytes to treat patients with ADA deficiency produced great results (2). Since then
clinical trials involving genetic engineering also show promising results in treating eyesight diseases,
Parkinson’s disease, and cancer. Currently, genetic modification of T lymphocytes has been the major
area of research for treating malignant tumors. This technique seeks to create chimeric antigen
receptor (CAR) in T cells by genetically modifying them in vitro and reintroduce them back into blood
circulation. The T cells are unique to every patient and the chimeric antigen receptors are unique to the
tumor that it is targeting.
Traditionally the battle with cancer has always been fought with chemotherapy, radiation
therapy, and even invasive surgeries. These treatments have made an astounding impact as far as being
able to destroy some cancerous cells. However, the biggest downfall to chemotherapy or radiation
therapy is the non-selective targeting of cells both cancerous and normal host cells. A more innovative
approach has now gone through successful clinical trials to combating cancer, specifically leukemias
such as chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In fact, Dr. Carl
June and his team from a Penn medicine was nationally recognized for being able to treat leukemia
patients using their own immune cells. The findings were published in an article, late of 2013, has been
feature “in more than 20 top newspapers across the nation” (3). Dr. June and his team started the trial
in summer of 2010 with three CLL patients whom were the very first to this procedure. Two of the three
patients are now still in remission. The procedure expanded to treat ALL patients and also showed
amazing results. Findings from three different trials with adult patients with CLL, pediatric patients with
ALL, and adult patients with ALL collectively produced remarkable responses. In adult patients with CLL,
15 out of 32 responded very well to treatment including 7 of them going into complete remission. In 22
pediatric patients with ALL, 19 of them went into complete remission and all 5 patients with ALL also
showed complete remission (4).
Furthermore, cancer treatments have never been cheap but one of the major contributors to
this cost is multiple treatments that extend over a period of time along with transplants when
treatments fail. This is why research is an ongoing process for a more effective one-time approach.
With CAR immunotherapy, it seeks to accomplish this and may be the tip of the iceberg for the future.
Reported on NBC news, an article by Marilyn Marchionne claims, “lab costs are now $25,000, without a
profit margin” for gene therapy. The Leukemia and Lymphoma Society has also donated 15 million
dollars to research groups testing out specifically the CAR approach and expanding it (5). This paper will
aim to explain the details of CAR immunotherapy, the structure of the receptors, the mechanism in
which it works, the vector used for delivery, and the possible side effects.

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Receptor Structure and Function
With the introduction and advancement of cancer therapies using T-cells modified with chimeric
antigen receptors (CAR), researchers are having success in overcoming two inherent problems that the
immune system has when responding to cancer cells. The cells of the immune system are continually on
the hunt for cells that are presenting non-self antigen on the cell surface. Cancer cells are essentially
self cells in which cell division has gone awry. When cells of the immune system come into contact with
these cancer cells they recognize the antigens on the cell surface as being self and the leukocyte
continues on searching for other invaders.
The body has a second major process in recognizing infections using major histocompatibility
complex or MHC. One of the functions of MHC’s is to alert immune system cells that a self-cell has been
infected internally. The infected cell upregulates the processes involved with MHC. It digests some of
the foreign protein, attaches it to an MHC and expresses the antigen protein and MHC on the cell
surface. When T Cells encounter these MHC molecules they are activated and an immune response is
initiated. (11) However, many types of cancer have the ability to prevent the upregulation of MHC so the
immune system does not recognize the pathogenic cells. Again, the immune system is prevented from
recognizing a problem with the proliferating cancer cells and is unable to mount a response.
The first publication regarding CAR was in 1989 by Gross et al. The process involved placing a
modified receptor and signaling complex on a T cell. The goal of this process was to modify a patient’s
own T-Cells so they could recognize and attack cancer cells while undergoing normal expansion in vivo in
order to provide an expanding and persistent immune response. (8)
A native T cell has 2 primary components, the T cell receptor (TCR), which is designed to interact
with MHC, and the signaling portion (CD3) which activates the T cell after an antigen is bound. In the
first attempts at modifying T cells to attack cancer cells researchers tried to modify the antigen
recognition site on the T cell. It was discovered that while this worked well in vitro there was poor
response in vivo. This was because as the modified T cells circulated through the lymphatic system,
when they reached the thymus they were selected out because the antigen recognition portion was
very similar to self-antigens. Gross got past this obstacle by synthesizing an entirely new receptor that
could very specifically target malignant cells while avoiding negative selection processes in the thymus.
(7)
The first step utilizes antibodies which are produced in B-Cells.
As is shown in the figure to the right, an antibody is made up of four
basic units, two heavy chains and two light chains. At the top of the “Y”
the heavy and light chains both contain what is known as the variable
region. It is there that the antigen binding sites for the antibody is
located. Antigen binding sites have very high specificity for specific
antigens. Researchers are able to engineer this binding site specifically
for antigens presented on cancer cells in an individual patient. These
antibodies can then be clonally expanded in a lab either in mice or
isolated tumor cells. After sufficient quantities of the antibodies are
made, the variable portions of the heavy and light chains are removed
from the constant subunits of the antibody. This product is termed a
single chain variable fragment, or scFv. These scFv’s will eventually be
incorporated into viable T-Cells.
Figure 1
http://www.biology.arizona.edu/immunolog
y/tutorials/antibody/graphics/antibody.gif

4. 4
Like antibodies, T-Cells also have receptors containing antigen binding sites. But, the binding
site simply recognizing and attaching to an antigen is just the first part of T-Cell activation. In close
proximity to the receptor is a signal transduction complex that is
responsible for providing the T-Cell an intracellular signal signifying that it
has found something that is a threat that needs to be responded to. This
signaling complex is referred to as CD3. CD refers to “cluster of
differentiation”. The T-Cell CD3 has two subunits that have been given the
designation of the Greek letter zeta. The body of a ζ unit penetrates deep
into the T-Cell and signals it that the pathogen it has attached to needs to
be destroyed. These ζ subunits are a critical component of the chimeric
antigen receptor. An scFv alone would bind to the cancer cell but without a
signal transduction unit no response would occur.
After obtaining CD3 ζ subunits the CAR can be assembled. The light
and heavy variable chains obtained from the antibodies are attached to
each other using a flexible linker.
Flexibility is critical so that antigen
binding sites are able to conform to
targeted antigens on the surface of the
cancer cells. A ζ subunit is then
attached to the scFv with a spacer that must contain a hydrophobic
region that can be inserted into the membrane of the T-Cell which is
being modified.
Typically a viral vector is used to introduce the engineered
receptor into T-Cells that have been harvested from the patient. The
T-Cell population is then expanded before being introduced to the
patient. Currently this process takes approximately two weeks from
start until the cells are ready to be infused into the patient.
After expansion the chimeric T-Cells are introduced into the patient. The modified T-Cells with
the chimeric antigen receptors function like native T-Cells. When they locate and bind to the target
antigen on cancer cells the T-Cells release perforins and granzymes which lyse the cell membrane and
break down the cancerous cell bringing about its death. Additionally, since the modified T-Cells circulate
through the body in the blood and lymph, if the cancer has metastasized, the CAR T-Cells can locate and
destroy the renegade cells in other parts of the patient’s body long before their physician has discovered
its proliferation. Because the modification of the T-Cells was
accomplished through a virus, researchers hypothesize that
persistent immunity will occur as a result of periodic
reactivation of the virus. (9)
The engineered structure of chimeric antigen receptors
has evolved and improved over time with in vitro and in vivo
studies. As shown in the figure, signaling enhancements have
evolved as researchers seek to create a more robust response
from the CAR T-Cells. These changes have helped increase the
effectiveness of the T-Cells. With better signaling mechanisms
the ability to kill a cancer cell has increased significantly and
Figure 2
http://course1.winona.edu/kbates/Im
munology/images/figure_05_06.jpg
Figure 3
http://upload.wikimedia.org/wikipedia/co
mmons/4/47/CAR_cartoon.png
Figure 3
http://www.hindawi.com/journals/bmri/2010/956304/fig3/

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proliferation, resistance, and persistence of the T-Cells have all been enhanced. As additional in vivo
trials on humans are approved this mechanism will continue to be fine-tuned. (10)
The Vector System
There are several ways to introduce therapeutic DNA to cells but the most common and still
widely use is through viral vectors. Viruses can be rendered non-pathogenic but still be effective in
incorporating DNA into host’s cells for protein expression. A few considerations should be taken when
choosing a vector for delivery such as dividing or non-dividing target cells, and short or long-term
expression. Some of viruses that have been used as vectors include lentivirus, adenovirus, herpes virus,
and retrovirus. Although all of these viruses have their advantages and drawbacks, the adenovirus and
retrovirus demonstrate to be most useful.
The adenovirus is a non-envelope virus that utilizes double-stranded DNA. It allows up to 5 kb
of exogenous DNA to be inserted using recombinant DNA technology and the transmission of these
genes to host nucleus without disturbing the chromosome. Viral DNA is taken within the nucleus and
exists as “extra” DNA that will be replicated and expressed into proteins. Therefore, it will not likely
disrupt important cell functions. Another feature of the adenoviral-vector is the viral DNA will vanish
making it less effective for chronic diseases but a more potent system to stimulate an immune response
against cancer (6). Incorporating adenovirus into a host cell is first accomplished through certain
interactions on the outside membranes of the cell and the virus. The structure of the adenovirus (Figure
1) has a fiber region that binds to the cell receptor. A second stimulatory interaction is through a motif
found on the virus and an integrin molecule on the host cell. Both of these interactions are required for
the entry of the virus following the endocytic pathway by forming a vesicle surrounded by a clathrin coat
(Figure 2).
Figure 4 Figure 5
http://www.genetherapynet.com/viral-vectors/adenoviruses.html http://www.genetherapynet.com/viral-vectors/adenoviruses.html
Adenovirus used as vectors have shown to be advantageous because they can infect various
human cells with proper receptor, there are at lease 43 different serotypes in humans, and they can
cause mild symptoms that usually accompanies a common cold. Furthermore, adenoviruses can be
easily employed with therapeutic DNA using recombinant DNA. Once the necessary interactions are
establish with host lymphocytes and the virus gains entry it has high gene transfer efficiency with low
chance of mutations.
Another viral vector used in modification of immune cells are retroviruses. These viruses are
enveloped and use single-stranded RNA in the form of mRNA. Retrovirus infects host cells and replicate
using reverse transcription, a process where RNA is transcribe to DNA before going through

6. 6
transcription then translation and then expression of genes. This is opposite to conventional processes
where DNA is transcribed to RNA and RNA is translated into proteins.
Figure 6 Figure 7
http://www.ohsu.edu/xd/about/services/integrity/upload/IBC_Presentation-Choosing-a-Viral-Vector-System.pdf http://www.ohsu.edu/xd/about/services/integrity/upload/IBC_Presentation-Choosing-a-Viral-Vector-System.pdf
The structure of the retrovirus (figure 3) shows the viral RNA genome surrounded by structural
proteins (gag) and further encase within an envelope with surface glycoproteins. Retroviruses possess
its own enzymes such as integrase, reverse transcriptase, and protease to carry out its unique reverse
transcription process in immune cells. A basic mechanism depicting retrovirus introduction (Figure 4)
into host immune cells for gene expression shows a process for long-term through integration.
Retroviruses can only infect dividing cells, a slight disadvantage compared to adenoviruses.
Both viruses ultimately can achieve the same endpoint by introducing therapeutic genes to host
for expression. Choosing a proper vector system to use is not easy and is heavily dependent on the
circumstances. Adenoviruses utilize a more transient gene expression whereas retroviruses offer long-
term expression. Adenoviruses also can infect dividing and non-dividing cells, trigger a high immune
response in target cells with great transduction efficiency. Retroviruses is more effective targeting non-
dividing cells, can produce a moderate response in target cells with moderate transduction efficiency.
Although these vectors are most commonly used, clinical trials are not limited to these two but are
continuously trying different vectors.
Conclusion
The recent breakthroughs in the use of chimeric antigen receptor modified T-cells in the
treatment of cancer in which all traditional treatments have failed shows great promise for the future.
A National Cancer Institute article declared that some Doctors and Researchers see immunotherapy as
“the fifth pillar” of cancer treatment. (10) There is still research and expanded human trials needed
before modified T-cell treatment for cancer can begin to be used on a wider basis. Most of the success
in this arena has been with various types of leukemia. Researchers are having less success with solid
tumors such as is seen in lung or brain cancer. With current techniques there is still a risk of serious side
effects including death. The most common deleterious side effect experienced by patients is cytokine
release syndrome, a condition in which the inflammatory response is excessive and has a significant
systemic effect on the patient’s whole body. However, recent progress seems to be developing quickly
and researchers are optimistic that soon targeted immunotherapy techniques will dramatically alter the
way cancer is treated.

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