1. Luke Brennan
4/21/2015
Review of Adoptive T-Cell Immunotherapy
INTRODUCTION
One of the ‘hallmarks’ of cancer is its ability to evade detection and destruction by the body’s
immune system by essentially hiding in plain sight1
. Cancer is also inherently derived from the body’s
own tissue, and harbors many of the signals that identify it as ‘self,’ which contributes to its
camoflauge1
. These two factors have made cancer so difficult to treat, because the immune system is
insufficient and man-made chemicals and radiation therapy used to attack cancer inevitably targets
unintended healthy tissue.
These issues and more prompted the development of immunotherapy for cancer treatment.
Much like how a vaccination conditions the adaptive immune system to respond to a certain pathogen,
immunotherapy would allow the immune system to sense and destroy cancer, though it uses different
means. This is often referred to as the cancer immunosurveillance hypothesis, and it gained significant
support after the verification of human tumor-associated antigens (TAAs) which are identifiers unique to
cancer, and can be exploited to discern cancer from healthy tissue2
.
Although immunotherapy is used to treat cancer in many ways, the scope of this paper will be
limited to Adoptive T-Cell Therapy (ACT). T-cells are lymphocytes, or white blood cells, which are present
throughout the body and attack pathogens and cells marked for apoptosis. T cells usually destroy cells
that would become cancerous before they even become a problem, when these mutated cells aren’t
destroyed and proliferate in an uncontrolled way they become cancers. ACT is the process by which T-
cells (originally extracted from the body, in most cases and for the purposes of this paper) with cancer-
recognizing properties are introduced into the patient to combat cancer.
Harnessing the power of these cancer sensitive T cells for use against cancer has great potential
for many reasons:
“1) T cell responses are specific, and can thus potentially distinguish between healthy
and cancerous tissue;
2) T cells responses are robust, undergoing up to 1,000-fold clonal expansion after activation;
3) T cell response can traffic to the site of antigen, suggesting a mechanism for eradication of
distant metastases;
4) T cell responses have memory, maintaining therapeutic effect for many years after initial
treatment.”2
As elegant a solution as immunotherapy may seem for the above reasons, many challenges
must be overcome before this becomes a viable treatment for mainstream cancer patients, as discussed
later in this review. Regardless, the following sections describe two main types of ACT based on the anti-
tumor activity of the lymphocytes collected from the patient.
ACT USING TUMOR-INFILTRATING LYMPHOCYTES
This type of ACT is dependent on the collection of lymphocytes with anti-tumor cytotoxic
activity, called tumor-infiltrating lymphocytes (TILs), against the cancer in question2
. Fortunately, such T-
cells have been identified in the tumor samples of up to 80% of melanoma patients, with admittedly
lower frequency in other forms of cancer5
. In fact, though not universal, lymphocyte infiltration of tumor
tissue has in fact become a hallmark of cancer1
. In the cases where such cells are detected and can be
extracted, these TILs can be cultured and grown ex vivo.

2. This process begins with the TILs from a “micro culture derived from a single tumor fragment or
106
viable cells derived from a single-cell enzymatic digestion [of a resected tumor specimen]”29
. These
cells are grown and cultures split as needed for two weeks with high dose interleukin 2, after which
point each culture is kept at 0.8-1.6x106
mL-1
in flasks and generates roughly 5x107
cells (the required
minimum being 3x107
cells) from each culture after 3-5 weeks29
. The cultures are then tested for activity
and specificity by an immunosorbent assay after stimulation with tumor cells, and cultures deemed
active undergo a rapid expansion phase for 2 weeks with donor feeder cells, anti-CD3 OKT-3 monoclonal
antibody, and high dose interleukin 2 to promote expansion 29
. In total, the entire manufacture process
takes roughly 6-8 weeks31
. The expanded colonies of TILs are then reintroduced to the body to attack
the cancer with greater numbers than before. This approach capitalizes on the ‘strength in numbers’
strategy, but with the added benefit of more specific TILs from the testing and selection processes
performed ex-vivo29
. This type of ACT would be considered the most feasible, but it sacrifices efficacy.
ACTs using TILs are limited in a few ways. First, this treatment is simply not an option for any
patient in whom TILs cannot be identified or extracted. Even for those patients from whom TILs can be
extracted, there is growing concern that these TILs may have little to no effect on tumor progression
despite their presence in areas of tumor inhibition2
. Further, to avoid unintended autoimmune attack,
these TILs generally have low affinity to the self-antigens and germline antigens that are usually
overexpressed by cancerous tissues (but are shared by healthy body tissues)6
. While this is still the most
effective method of ACT to date, these limitations have encouraged development of the second type of
ACT.
ACT USING ENGINEERED T CELLS
This method of ACT makes use of the T-cells’ time ex vivo (as described in the methods section)
to enhance T-cell function by changing receptors to give the cells improved affinity to self-antigens or
novel specificity, and/or by improving their proliferative capacity through alterations in signaling
functions7
. Because the cells are being engineered, the T-cells do not necessarily need to be TILs or even
from the tumor region, and can be collected from peripheral blood samples which are much easier to
collect, and are present in every patient8
. This higher affinity and novel specificity is largely achieved
through expression of heterodimeric T-cell receptors (TCRs) in T-cells which can allow tighter binding to
the self-antigens overexpressed on cancer, or neo-antigens, which are epitopes that are only present in
cells with cancerous mutations9
. To achieve this, a gene corresponding to the engineered TCR is
introduced into the T cells by a retroviral vector32
. The TCR then “combines TCR-alpha and TCR-beta
genes” and undergoes MHC restriction, a process that ensures it only recognizes peptide antigens when
they are presented on the body’s MHC molecule10
. Another method to enhance affinity is to reduce N-
glycosylation on TCR chains11
. Surprisingly, self-antigens (specifically genes of the cancer testis family*for
more info look at 12
) rather than neoantigens, are more often the targets of such engineered TCRs12
.
Some of the first trials of this type have been for the NY ESO-1-specific and HLA-A2-restricted
TCR for use in melanoma, myeloma, and synovial cell sarcoma13
. These studies have been making great
progress and have not shown toxicity. Toxicity is a large concern in these trials, after all these cells are
being engineered to attack self-antigens that are present on healthy body tissue. In fact in another study
a TCR engineered for enhanced MAGE 3 affinity and HLA-A1-restriction had off target recognition of the
muscle protein Titin, resulting in lethal cardiac toxicity for both patients14,15
. This will be discussed
further along in the paper. Another problem of ACT with genetically engineered T-cells is that, due to a
multitude of factors known and unknown, it has been less successful than ACT with TILs16,17
. This being
said, ACT remains relevant because of how much it can improve with better protocols, and because it

3. greatly expands the range of ACT to cancers like neuroblastoma, synovial cell carcinoma, and colorectal
carcinoma (among others) where TILs cannot be extracted18,19, 20
.
The synthesis of these genetically engineered T-cells begins with T-cells from ficoll-purified
PMBCs, which are activated with OKT-3 antibodies29
. They are then transduced with a retroviral vector
that encodes the desired antigen-specific TCR, cultured for 2 weeks, and transduced and expanded
under cGMP 32,33
. These activated T-cells are then transduced again with retroviral vectors in
RetroNectin-coated cell bags, inoculated in a WAVE bioreactor for two days, and expanded with a
continuous perfusion regime29
. After this, the beads (from cGMP treatment) are removed and the cells
are formulated for infusion29
.
This total process takes about 2 weeks and is of a large enough scale to support multiple clinical
trials29
.
CAR T-Cells
One other noteworthy category of genetically engineered T-cells for use in ACT is the CAR-
modified T-cell. This modification exploits the fact that “the cytoplasmic tail of the TCR CD3zeta chain
[can] activate T-cells without the rest of the receptor complex”21
. Despite disappointing results from first
generation CARs22
, second and third generation CARs have shown antitumor effects and have remained
in patients for up to a decade (but also as little as a month)*for more information about the modifications of second and third
CARs go to 23
. What is promising is that response rates are generally encouraging, over 50% of patients
achieving some kind of remission in studies at the University of Pennsylvania, and that this response is
correlated with CAR T-cell proliferation, indicating that the remission is related to the presence or
activities of the CAR T-cells10
.
CAR modification represent a valuable alternative to TCR modification because only CAR-
modified T-cells can recognize a cancer cell that has lost MHC expression, a condition which applies to
many cancers24,25
. Also, CAR-modified T-cells are sometimes able to remain in the body and fight cancer
much longer than TCR-modified T-cells, though the longevity of each varies substantally26,27
. This being
said, TCR-modification has its place, as it is “able to sense the entire intracellular proteome that is
presented by MHC molecules” and accordingly can attack a wider range of molecules in the cancer
cells10
. It also requires about 10x less target antigen expression (sometimes less than 10 ligands) to
induce cytolosys28
.
DISCUSSION
ACT involving any type of T-cell is not yet ready for the standard treatment of cancers on a large
scale. This is because of several factors both biological and practical.
T-cell Inhibition
First, both the cancer and the immune system itself have several methods of inhibiting an
immune response to cancer tissue. As T-cells are maturing in the thymus, they are tested for recognition
to self-antigens, and those that are particularly reactive are deleted to avoid autoimmunity34
. These
especially active T-cells that the body stops from maturing are needed to recognize the over-expressed
self-antigens on the surface of cancer cells, so this limits the effectiveness of any TIL related ACT10
. T-
cells are also inhibited from attacking cancer in maturity by peripheral tolerance mechanisms, which
consists of 3 categories2
:
1. Inhibition from the T-cell itself or its nature, like low activity around inflammation*for more information
see 35

4. 2. Inhibition from the Tumor, like the expression of checkpoint molecules*for more information go to 36, this is
an area of relatively high development after trials in melanoma (37,38).
3. Inhibition from regulatory T-cells and myeloid derived suppressor cells*for more information see 39
.
The first two sources of inhibition are difficult to handle, as they are meant to stop autoimmune
attack on one’s own body tissues2
. Simply turning these inhibitory mechanisms off could cause severe
autoimmune disease.
Toxicity
Toxicity is another huge concern, with off-target autoimmunity having lethal ramifications in
both TCR-modified T-cell ACT studies (see above) and CAR-modified T-cells which can cause B-cell
aplasia, lysis syndrome, and cytokine release syndrome22,40,41,10
. To make matters works, the toxicity
from these ACTs can target the brain or heart, and cannot always be managed with corticosteroids10
.
The problem here is that T-cells are being engineered to have greater recognition of self-antigens, and
while this allows them to detect the overexpressed self-antigens on cancer cells, it is difficult to
eliminate all off target effects since almost every cell in the body has these self-antigens. It goes without
saying that work is being done to stop this off target autoimmunity, and one noteworthy idea is to add
inducible mechanisms for T-cell apoptosis when engineering the T-cells ex-vivo so that they could be
stopped quickly and easily if toxicity were to become an issue10
.
Longevity
The final biological issue with ACT is response and longevity, specifically for engineered T-cells.
As noted above, most TCR-modified T-cells can often last for only a few months, and CAR-modified T-
cells aren’t much better. This is only counting the patients who respond well to the treatment. For ACT
to become a mainstream treatment, researchers need to find ways to allow the engineered cells to have
antitumor effects in a larger proportion of patients, and to have longer lasting effects in each patient.
One way of prolonging the effects of T cells is by immunodepletion of the regulatory immune
system, which can be achieved by total body irradiation and lymphodepleting chemotherapy2
.
Lymphodepletion before ACT can allow better proliferation of the reactive T cells by lowering the
numbers of regulatory T cells and myeloid-derived suppressor cells, enhancing innate immunity, and by
increasing cytokines42
. This lymphodepletion has improved results in mouse models of B16 melanoma43
.
Also, for stage IV melanoma response rates were 49% with 1x lymphodepletion and 72% with 3x
lymphodepletion44
. Finally, checkpoint inhibitors are proving to be a great way to assist cancer
therapy*for more information see 37, 38
.
Culturing
The practical problems of ACT are the time and resources necessary to culture and test these T-
cells for the 6-8 weeks31
, and a minimum of 2 extra weeks to incorporate any genetically engineered
changes required for TCR or CAR modification29
. This must be done over for every single patient (the
epitome of individualized medicine) as each cancer is different, presents unique markers, and
accordingly requires T-cells with unique receptors2
. This delay of at least 6-10 weeks makes this therapy
somewhat sluggish, and the weeks of cell culture requires hours of work for a researcher. Using current
methods, it would be unfeasible to run these ACT protocols for the general population in all but very
rare cancers.
Cost

5. All the time and resources necessary to culture these T-cells inherently makes this a costly
process. The hospital stay required to have the cells injected is also costly, as it is essentially a form of
transplant2
. It is nearly impossible to even estimate a cost seeing as the protocols change rapidly as
development of this field proceeds, and as costs are not always publicly reported in clinical trials2
. For a
very rough estimate, sipuleucel-T, a cancer vaccine used to treat prostate cancer, requires the isolation
of immune cells from each patient, in-vitro culturing, and infusion back into the patient, all just like ACT,
and it costs $93,000 all told44
.
THE FUTURE FOR ACT
ACT is certainly still a treatment under development. Its simple premise inspires hope for
success in the future, but the long list of limitations admittedly casts doubt. What will likely determine if
ACT becomes a widely used therapy in the near future is how the cost-to-benefit ratio changes in
relation to other therapies like chemotherapy and radiation. Unless the process of T-cell culture and
engineering can be majorly streamlined and automated, it seems unlikely that ACT will be able to
compete with chemotherapy and radiation therapy for most cancers.
Of course, the cost-to-benefit ratio goes hand in hand with the biological effectiveness of ACT.
Many versions of this therapy pose a toxicity risk to the patient, which is often quite severe. While this
may be quite difficult or even impossible to eliminate completely given the need for enhanced affinity to
self-antigens, there needs to be a way of minimizing the potential harm to the patient. This could come
in the form of an inducible apoptosis mechanism for the modified T cells, or by focusing efforts on
neoantigens as receptor targets rather than self-antigens. These engineered T cells must also last longer
in the host in order to produce significant anti-tumor effects, so these cells need to proliferate better
and avoid degradation. Finally, better receptors need to be engineered for use in TCR and CARs, though
given the transformation between first and third generation CARs there seems to be little doubt that
these improvements will come with time.
To its credit, ACT could eventually enable patients to attack even aggressive, metastatic, and
heterogeneous cancers with very limited toxicity or side effects. Perhaps this dream will propel the
research needed to bring this form of therapy to fruition.
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