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Immuno-Oncology Profiling for

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Enabling Immunotherapy Discovery

W E L C O M E to the IntelliCyt eBook on Immuno-Oncology
I
mmuno-oncology discovery is key to understanding
the immune system and its processes in order to enable
the development of effective cancer therapies.
Immuno-oncology trains the body’s immune system to
target specific cancer cells and gives the immune system
the ability to combat cancer on a long-term basis.
IntelliCyt is the market leader in suspension-cell
analysis platforms for immunology and immuno-
oncology profiling for drug discovery and translational
research. The iQue Screener can rapidly and cost
effectively profile physiologically relevant model systems
for phenotypic endpoints using multiplexed cell and/
or bead-based assays.
Our easy-to-use instruments, software, and reagent kits
are optimized to work together and designed to conserve
precious samples, use less reagents, and minimize time-
to-answer. Our high-content solution offers fast and rich
per-cell information that delivers the deep insight that
complex biology demands.
In this eBook, we would like to share some articles that
provide useful information on the field of immuno-oncology.
Take a look at the webinars and papers and learn why
leaders in immuno-oncology have made the iQue Screener
platform an integral part of their discovery process.
The IntelliCyt Team
Contents Photos courtesy of: iStock / Dragonimages / AzmanJaka / GregorBister / gmutlu / nicolas_

21 Therapeutic
Antibodies—
5 Cancer in 10 Immunotherapy A Success Story
Cell Therapy’s Shows Promise Use of high-throughput
screening platform for
Crosshairs for Myeloma multiplexed assays significantly
The immunotherapy challenge Cellular immunotherapy helps aids drug discovery of
is to identify the right tumor treat myeloma patients. therapeutic antibodies.
targets.
31 A High-Throughput,
13 Immunotherapy 16 Immunotherapy Radioactivity-Free
Opens Two Fronts, Tightens the Siege Assay for
against Cancer, on Solid Tumors Cell-Medicated
Innate and Translational scientists
Cytotoxicity
gather to crank up
Adaptive immunological machinery, A high-throughput screening
Cancer treatment recruits let fly at cancer’s ramparts. assay that captures multiple
various immune cells to launch biological markers when
coordinated attacks that beat determining cell-mediated
back aggressive tumors. cytotoxicity.
Cancer in Cell
Therapy’s Crosshairs
A doptive immunotherapy has emerged as a
promising weapon in the therapeutic arsenal
being mobilized to fight cancer. In this approach,
T cells are isolated from a patient’s tumor,
genetically engineered using one of several
different approaches, and infused back into
the patient.
Of various strategies for adoptive immuno-
therapy, the use of chimeric antigen receptors
(CARs) has received considerable attention.
Typically, sequences that encode the variable
regions of antibodies are modified and then
5 | GENengnews.com
spliced into the T-cell receptor (TCR)
intracellular domains that activate T cells.
This chimeric construct is cloned into a retro-
Additional Content
APPLICATIONS IN ACTION
viral vector and used to generate a population of
T cells that express the CAR, with activity specific Cells and Beads—Better Together
for an antigen expressed by the patient’s tumor. To learn how to analyze mixtures of
These modified T cells, when infused into the cells and beads, download the pdf.
patient, specifically target tumor cells that express
the antigen, resulting in a rapid immune response. Download now!
t
Target discovery is a large part of cell therapy,
as are investigations into TCRs, CAR engineering,
and the use of tumor-infiltrating lymphocytes
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Cancer in Cell Therapy’s Crosshairs
(TILs) against tumor antigens.
Creating designer T cells that target specific
tumors involves a collaborative effort from scientists
in a variety of disciplines, in partnership with physi-
cians who deal with the realities of cancer therapy on
a daily basis. To this end, the Memorial Sloan Kettering
Cancer Center established the Center for Cell Engi-
neering. Michel Sadelain, M.D., Ph.D., is the center’s
director and heads a research group studying the
mechanisms governing transgene expression, stem
cell engineering, and other genetic strategies to bolster
the immune response in cancer patients.
A significant area of Dr. Sadelain’s research
focuses on the CD19 receptor. He explains, “We
chose CD19 (over 15 years ago) because of where
this molecule is expressed, as well as where it is not
expressed.” Since CD19 is found on the cell sur-
face in most leukemias and lymphomas, but no-
where else in the body except for B-lineage cells, it is
an attractive target for therapy. In addition, CD19
has been implicated in tumor survival, making it
more likely to be present on—and less likely to be
6 | GENengnews.com
lost from—all tumor cells.
Dr. Sadelain points out that the most
impressive results from CAR T-cell therapy have
been achieved in acute lymphoblastic leukemia
(ALL), first in adults and then in children.
However, he says, “One of the big questions is
whether this approach will work in solid tumors.”
In theory, CAR therapy should be applicable to
a wide range of cancers. The challenge facing
researchers is “to identify the right targets and to
appropriately adapt the T-cell engineering strategies
to the immune barriers opposed by different
tumor types.”
Dr. Sadelain’s group is pursuing targets,
such as prostate-specific membrane antigen
and mesothelin, for which there is substantial
information already available in the scientific
literature. His group is also searching for new
candidates based on studies from tumor samples.
“We have open protocols for ALL, chronic
lymphocytic leukemia (CLL), certain lymphomas,
and Waldenstrom disease,” he says. “We will
soon open a trial for patients with certain forms
of mesothelioma, lung adenocarcinoma, and
breast cancer.”
Hematologic Cancers
The first trials of CAR-based therapy addressed
hematologic cancers, largely due to the extensive
knowledge base surrounding surface antigens
expressed on hematologic cells. Researchers,
however, still face considerable challenges when
engineering CARs.
Marcela Maus, M.D., Ph.D., director of
translational medicine and early clinical develop-
ment at the University of Pennsylvania’s Abramson
Cancer Center, notes that “the process for manufac-
turing CARs is more complex than making a pill.”
She adds that different kinds of cancers require
different targets, and that finding good targets
can be difficult.
The tools offered by synthetic biology have
helped, in some cases, to facilitate the development
of CARs. “Swapping and testing various intracellular
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Cancer in Cell Therapy’s Crosshairs

signaling domains and CAR domains like Lego bricks—or ‘biobricks’—has

become a relatively routine set of experiments to do in the lab,” says Dr. Maus.
Ultimately, though, extensive clinical trials are required to determine the best
engineered domains and study their safety and efficacy.
Dr. Maus notes that CAR-directed approaches show the greatest promise
in B-cell malignancies such as B-cell acute lymphoblastic leukemia (B-ALL) and
non-Hodgkin’s lymphomas. Accordingly, the University of Pennsylvania has
teamed up with Novartis to offer CAR therapies on a global scale.
Other hematologic malignancies are attractive targets as well. According
to bone marrow transplant research, these types of tumors are easily accessible
by T cells. Dr. Maus adds, “CAR-modified T cells can form memory, which
means there is potential for a long-term remission.” Because they are generated
with the patient’s own immune system, Dr. Maus explains, “CAR T cells
combine the potential for cure that bone marrow transplants have, but they
don’t have the same risks.”

Solid Tumors

John Maher, Ph.D., senior lecturer, immunology, department of research

oncology, King’s College London, reiterates the promise shown by targeted
T cells in treating hematologic malignancies. “Much excitement surrounding

Electron micrograph of magnetic beads stimulating

T cells in preparation to make CAR T cells.
University of Pennsylvania Abramson Cancer Center

7 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Cancer in Cell Therapy’s Crosshairs
the use of gene-targeted T cells concerns their high
degree of effectiveness in the treatment of B-cell
malignancy, especially ALL,” says Dr. Maher.
He adds, however, that some approaches used
in B-ALL target both healthy and diseased B cells,
ultimately leading to hypogammaglobulinemia. This
toxicity can be treated by intravenous or subcutaneous
immunoglobulin replacement therapy. “Engineered
T cells that recognize B-lineage targets, such as CD19,
can consequently provide a highly effective
therapeutic approach,” explains Dr. Maher.
Dr. Maher’s research focuses on extending the
targeted T-cell approach to solid tumors—a field
of study fraught with obstacles. “Tumor-specific
targets are very few and far between,” says Dr.
Maher, “meaning that we are generally forced to
target molecules that are also expressed in healthy
tissues.” In addition, T-cell homing to metastatic
solid tumors is not as efficient as for hematological
malignancies. For those T cells that do reach the
target, it is difficult to maintain functionality and
viability.
8 | GENengnews.com
For these reasons, Dr. Maher’s group is
examining specific targets in high-profile cancers,
such as the ErbB family of receptor tyrosine kinases.
“It’s difficult to find a solid tumor type in which
dysregulation of the ErbB network is not a common
event,” notes Dr. Maher. He adds that ErbB also
makes an attractive target because its dysregulation
contributes directly to pathogenesis. Further, the
ErbB family is the most successful cancer-associated
target for monoclonal antibody therapy, for
example, in agents such as herceptin (anti-ErbB2)
and cetuximab (anti-ErbB1).
Dr. Maher’s research initially focused on ovarian
cancer, but his group plans clinical trials in head and
neck cancers later this year. His approach addresses
concerns about safe targeting—a universal challenge,
since ErbB receptors are widely expressed in healthy
tissues, although at low levels—by using regional
delivery systems. “We will inject the cells directly
into head and neck tumors,” he states. “In ovarian
cancer or mesothelioma, intracavitary delivery may
be used.”
Future directions for Dr. Maher’s research include
investigating additional tumor-selective targets, and
methods to improve T-cell trafficking to tumors.
At the same time, his group is examining ways to
mitigate on-target toxicities associated with CAR
T-cell therapy. “We have data suggesting that this
approach can be combined with conventional
therapies, such as chemotherapy, that further
sensitize tumor cells to CAR T-cells,” concludes
Dr. Maher.
Although viral vectors are still popular for
engineering T cells, researchers are examining other
methods that can improve efficiency and lower cost.
Laurence Cooper, M.D., Ph.D., professor in pediat-
rics at the M.D. Anderson Cancer Center, is using
one such approach—taking advantage of in vivo
transposition systems such as Sleeping Beauty (SB).
Dr. Cooper uses the SB system to engineer CAR
T cells to target tumor antigens. His research also
focuses on pediatric cancers including acute leukemia.
Dr. Cooper explains that CAR T cells have
already shown dramatic results in pediatric patients,
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Cancer in Cell Therapy’s Crosshairs
and these data will “provide the foundation for
next-generation clinical trials targeting childhood
cancers that are refractory to conventional
therapies.”
Dr. Cooper’s approach involves a “one for many”
strategy, unlike conventional CAR T cell therapy. In
his method, genetically modified CAR T cells are
sourced from healthy donors in advance of when they
are required for therapy. The modified cells can then
be infused into a patient when required for immuno-
therapy. The approach addresses key disadvantages
of conventional CAR T cell therapy—the time and
expense associated with generation of the CAR and
9 | GENengnews.com
propagation of the T cells.
“We are developing off-the-shelf T cells before
the patient arrives at a treatment facility,” asserts
Dr. Cooper. “This enables a biobank of T cells to
be pre-prepared, which could be a one-time cost.”
Additionally, Dr. Cooper’s group is working on
manufacturing patient-derived T cells in real time,
with the goal of eliminating the need for ex vivo
propagation after gene transfer. CAR T-cell therapy
holds the most promise in childhood leukemia and
lymphoma, but Dr. Cooper is optimistic that the
approach will see success in solid tumors, such as
neuroblastoma, as well. n
Immunotherapy Shows
Promise for Myeloma
S timulating a patients’ immune system to
combat diseases like cancer lies at the heart of
immunotherapy and in recent years it has emerged
as one of the most promising treatment options.
Now, a clinical study from University of Maryland
School of Medicine, the Perelman School of
Medicine at the University of Pennsylvania, and
Adaptimmune, a clinical-stage biopharmaceutical
company, has published data that shows significant
success using this technique against multiple
myeloma.
Typically, immunotherapies fall into one of
three categories: cytokine, antibody, and cellular.
10 | GENengnews.com
Cytokine treatments have been administered for
various cancers and attempt to modulate the im-
mune response toward the disease and have been
modestly successful. Antibody therapies have shown
great promise for a variety of cancers and have be-
come the focus of many biopharmaceutical compa-
nies of the past several years. These molecules bind
to the target antigen on the cell surface, marking it
for destruction by the immune system.
Cellular therapies have been slow to make
their way into the clinical realm as the method
has been wrought with an array of technical
difficulties. However, recent advances in
sequencing and genome editing tools have enabled
researchers to engineer a patients’ own immune
cells to target specific tumor subtypes—multiple
myeloma in the case of the current clinical trial.
“This study suggests that treatment with
engineered T cells is not only safe but of potential
clinical benefit to patients with certain types of
aggressive multiple myeloma,” explained lead
author Aaron Rapoport, M.D., professor of medical
oncology at the University of Maryland School of
Medicine. “Our findings provide a strong founda-
tion for further research in the field of cellular
immunotherapy for myeloma to help achieve even
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Shows Promise for Myeloma

better results for our patients.”

The findings from this study were published recently in Nature Medicine
through an article entitled “NY-ESO-1–specific TCR–engineered T cells
mediate sustained antigen-specific antitumor effects in myeloma.”
More than 24,000 new cases of multiple myeloma are diagnosed each
year, with long-term response rates being extremely low and median survival
rates of three to five years.
“The majority of patients who participated in this trial had a meaningful
degree of clinical benefit,” Dr. Rapoport noted. “Even patients who later
relapsed after achieving a complete response to treatment or didn’t have a
complete response had periods of disease control that I believe they would
not have otherwise experienced. Some patients are still in remission after
nearly three years.”
In this study, patients’ T cells were engineered to express an affinity
enhanced T cell receptor, specific for the tumor antigens known as
NY-ESO-1 and LAGE-1. Close to 60% of advanced myelomas have
been reported to express NY-ESO-1 and/or LAGE-1—correlating to
tumor proliferation and poorer outcomes.

In cellular immunotherapy, healthy T cells are

removed from patients, genetically engineered to target
their cancer, and injected back into the body. NIAID

11 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Shows Promise for Myeloma
In the current study, 20 patients were treated
using cell immunotherapy, of which 14 (70%) had
a near complete or complete response after three
months. Median progression-free survival was 19.1
months and overall survival was 32.1 months.
Additionally, the researchers found that the
engineered T-cells were migrating to their intended
target zone within the bone marrow and showed
persistence that correlated with clinical activity
against antigen-positive myeloma.
“Multiple myeloma is a treatable but largely
12 | GENengnews.com
incurable cancer. This study reveals the promise that
immunotherapy with genetically engineered T-cells
holds for boosting the body’s ability to attack the
cancer and provide patients with better treatments
and control of their disease,” stated E. Albert Reece,
M.D., Ph.D., M.B.A., dean of the University of
Maryland School of Medicine, who wasn’t directly
involved in the current study. “This trial is also an
excellent example of significant scientific advances
that result from collaborations between academic
medical institutions and private industry.” n
Immunotherapy
Opens Two Fronts
against Cancer,
Innate and Adaptive
S taging the immunological equivalent of a
two-front war, MIT scientists simultaneously
activated both arms of the immune system to halt
the growth of a very aggressive form of melanoma
in mice. The MIT scientists, like many other
researchers, decided to use an approach called
cancer immunotherapy. Ordinarily, this approach
involves either the activation of the innate immune
13 | GENengnews.com
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Multiplexed Detection of Mouse
system or the stimulation of T cells. The MIT
researchers, however, mustered both innate and
adaptive immune forces.
Cytokines Using QBeads and
the iQue Screener
To learn about miniaturized assays,
“An antitumor antibody can improve adoptive download the pdf.
T-cell therapy to a surprising extent,” said K. Dane
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t
Wittrup, Ph.D., a professor of chemical engineering
at MIT. “These two different parts of the immune
therapy are interdependent and synergistic.”
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Opens Two Fronts against Cancer, Innate and Adaptive
To date, immunotherapeutic campaigns
against cancer have proven difficult. The
immune system often fails to recognize the
appearance of tumors as a call to arms. And
it often seems a reluctant recruit, whether the
“I want you” is issued to raise antibodies or
enlist T cells. Dr. Wittrup and his colleagues,
however, made the discovery that they could
generate both types of immune responses
while they were experimenting with improving
antibody drug performance with a signaling
molecule called IL-2, which helps boost
immune responses.
The scientists described their work
earlier this year in the journal Cancer Cell,
in an article entitled, “Synergistic Innate
and Adaptive Immune Response to
Combination Immunotherapy with Anti-
Tumor Antigen Antibodies and Extended
Serum Half-Life IL-2.”
“We find that a combination of an anti-
tumor antigen antibody and an untargeted
IL-2 fusion protein with delayed systemic
14 | GENengnews.com
clearance induces significant tumor control
in aggressive isogenic tumor models via a
concerted innate and adaptive response
involving neutrophils, NK cells, macro-
phages, and CD8+ T cells,” they wrote.
“This combination therapy induces an
intratumoral ‘cytokine storm’ and extensive
lymphocyte infiltration.”
About a dozen IL-2-enhanced antibody
therapies have gone through Phase I clinical
trials. However, most of these efforts failed,
even though the antibody-IL-2 combination
usually works very well against cancer cells
grown in a lab dish.
The MIT team realized that this failure
might be caused by the timing of IL-2 delivery.
When delivered to cells in a dish, IL-2 sticks
around for a long time, amplifying the
response of natural killer cells against cancer
cells. However, when IL-2 is injected into a
patient’s bloodstream, the kidneys filter it A concerted innate and adaptive attack involving neutrophils, NK
out within an hour. cells, macrophages, and CD8+ T cells beat back cancer in isogenic
Dr. Wittrup and his colleagues overcame tumor models. kniazev_iv/Fotolia
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Opens Two Fronts against Cancer, Innate and Adaptive
this by fusing IL-2 to part of an antibody molecule,
which allows it to circulate in the bloodstream for
much longer. In tests in mice with a very aggressive
form of melanoma, the researchers found they could
stop tumor growth by delivering this engineered
form of IL-2, along with antibody
drugs, once a week.
To their surprise, the researchers found that
T cells were the most important component of the
anti-tumor response induced by the antibody-IL-2
combination. They believe that the synergy of
IL-2-induced cells and cytokines, and the antibody
treatment, creates an environment that lets T cells
attack more effectively.
“The antibody-driven innate response creates
an environment such that when the T cells come in,
they can kill the tumor. In its absence, the tumor cells
establish an environment where the T cells don’t
work very well,” Dr. Wittrup said.
15 | GENengnews.com
Cells called neutrophils, which are considered
the immune system’s first line of defense because
they react strongly to foreign invaders that enter
the skin through a cut or other injury, were also
surprisingly important.
The researchers also found that when they
delivered an antibody, IL-2, and T cells targeted to
the tumor, the adoptively transferred T cells killed
cancer cells much more successfully than when only
T cells were delivered. In 80–90% of the mice,
tumors disappeared completely; even when tumor
cells were reinjected into the mice months after the
original treatment, their immune systems destroyed
the cells, preventing new tumors from forming.
“Adoptive transfer of antitumor T cells together
with this combination therapy,” the authors of the
Cancer Cell article concluded, “leads to robust
cures of established tumors and development of
immunological memory.” n
Immunotherapy
Tightens the Siege
on Solid Tumors
I n anticancer campaigns, the immune system has
often shown too little fighting spirit. It can be too
civilized, too restrained—unless it is specially out-
fitted and guided. Measures that can drive the im-
mune system to exert itself more strenuously, more
aggressively, include monoclonal antibodies, cancer
vaccines, checkpoint inhibitors, and adaptive cell
therapy—all the tools and techniques of immuno-
oncology.
16 | GENengnews.com
The proliferation of immuno-oncology tools
and techniques was evident at the recent Translat-
ing Science into Survival conference. This event,
which was held in New York City was organized
by the Cancer Research Institute, the Association
for Cancer Immunotherapy, the European Academy
of Tumor Immunology, and the American Associa-
tion for Cancer Research. The organizers evidently
anticipated that this event, like previous immuno-
therapy events, would be fairly intimate. Yet it sold
out quickly and ultimately strained to accommodate
1,400 attendees.
The event’s popularity was probably at least
in partly due to recent immunotherapy successes
against blood cancers. For example, adaptive cell
therapy approaches have shown promise in small
trials, and work along these lines continues apace,
as several presentations demonstrated. Moreover,
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Tightens the Siege on Solid Tumors

lessons derived from this work may be applied more broadly, even to the treatment
of solid tumors.

CAR-Modified T Cells

For example, in a presentation entitled “Engineered T cells for cancer therapy,” the
University of Pennsylvania’s Carl June described his team’s progress in using chimeric
antigen receptor (CAR)-modified T cells to treat patients with chronic lymphocytic
leukemia (CLL). “We previously reported preliminary results on three patients with
refractory CLL,” Dr. June noted. “Here we report the mature results for our initial trial
using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL.”
The overall response rate in CLL patients was 8/14 (57%), with four complete
remissions and four partial remissions. All responding patients developed B cell
aplasia and experienced cytokine release syndrome, coincident with T cell
proliferation. Minimal residual disease was not detectable in patients who
achieved complete remission, which, Dr. June suggested, indicated that “disease
eradication may be possible in some patients with advanced CLL.”
Dr. June also summarized a separate investigation that asked whether the CAR
A group of killer T cells surrounding a cancer cell.
cells used against CLL would also be effective against multiple myeloma. At first Alex Ritter, Jennifer Lippincott Schwartz, and Gillian Griffiths, National Institutes of Health
glance, this may seem odd, since the CAR cells that were effective against CLL target
CD19, and CD19 expression is all but absent from myeloma cells. That is, myeloma
cells don’t traditionally express CD19 on their surface because they arise from the
most mature type of lymphocytes—plasma cells.
Dr. June’s team, however, proceeded on the chance that they would be able to

17 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Tightens the Siege on Solid Tumors
incorporate their anti-CD19 CAR T cells into a
Immunotherapy
therapy that would target early precursors of
successes against
myeloma cells. This therapy, which was
blood cancers
have been in the administered to a patient with refractory
news lately. multiple myeloma, involved an infusion of
iStock/twilightproductions
the patient’s own stem cells along with
lymphodepleting chemotherapy (melphalan)
as well as an infusion (two weeks later) of
anti-CD19 CAR T cells.
The patient experienced transplantation-
related side effects during the time prior to
receiving CTL019, including neutropenia and
thrombocytopenia, nausea, fever, and an infection.
After receiving the engineered cells, she
experienced no fevers or other signs of cytokine
release syndrome, a condition that has been
observed in other patients undergoing CTL019.
At last evaluation, 12 months after treatment,
the patient exhibited a complete response with no
evidence of progression. According to Dr. June,
“This response was achieved despite absence of
CD19 expression in 99.95% of this patient’s
neoplastic plasma cells.”
18 | GENengnews.com
While the results presented by Dr. June pertain
most directly to blood cancers—specifically, the
inducement of favorable patient responses to
treatment—they may also apply more broadly.
For example, they demonstrate that a “living
drug” may exert its effects indirectly. Also, they
emphasize the importance of managing toxicity
and ensuring the expansion of modified cells. They
also raise the issue of introducing cells that may
demonstrate longevity.
More generally, uncertainties surrounding the
differential expansion and persistence of distinct
cell populations over time can complicate dosing.
Similarly, with respect to toxicity, cytokine release
and the generation of tumor-shredding products
might be considered side effects or, really, evidence
that a therapy is working.
Yet engineered cells may also attack both
malignant and healthy cells directly. That is,
engineered cells may be sensitive to target proteins
that stud both cancer cells and also, if only to a
lesser degree, normal cells. In CAR T-cell therapies
against leukemia and lymphoma, side effects
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Tightens the Siege on Solid Tumors
related to direct attacks on normal cells has been
manageable. Such side effects, however, may be
more severe if adaptive cell therapies are directed
against solid tumors.
Leading up to the Translating Science into
Survival conference, Dr. June’s group published a
study that described an approach for managing
target-mediated toxicity. The approach, called
affinity tuning, involves generating CAR T cells that
are sufficiently insensitive to ignore normal cells,
which are relatively target-sparse, and yet sensitive
enough to latch onto cancer cells, which are
relatively target-rich.
In a paper (“Affinity-Tuned ErbB2 or EGFR
Chimeric Antigen Receptor T Cells Exhibit an
Increased Therapeutic Index against Tumors in
Mice”) that appeared September 1 in Cancer
Research, the case was made that “affinity-tuned
cells” could exhibit robust antitumor efficacy
similar to high-affinity cells, but spare normal cells
expressing physiologic target levels: “The use of
affinity-tuned scFvs offers a strategy to empower
wider use of CAR T cells against validated targets
19 | GENengnews.com
widely overexpressed on solid tumors, including
those considered undruggable by this approach.”
Tumor Microenvironment
At the Translating Science into Survival event,
the presentations concerning solid tumors
emphasized that adoptive cell therapy could
become more effective if obstacles in the tumor
microenvironment could be overcome. For example,
the University of Pennsylvania’s Ellen Puré, in a
presentation entitled, “Tumor stroma:
Immunomodulatory functions and a target of
immunotherapy,” explained that stroma can be
a barrier to T cells, including CAR T cells.
Stromal components such as fibroblasts and
the extracellular matrix can play myriad functions
in cancer. For example, Puré noted, reactive stroma
enriched in growth and angiogenic factors presents
chemoattractants that promote the recruitment
of bone marrow-derived cells and can modulate
inflammatory and immune cell function, all of
which can contribute to its tumor-permissive nature
relative to normal stroma.
“A significant portion of cancer-associated
fibroblasts in virtually all human carcinomas
express the cell surface protease fibroblast
activation protein (FAP),” Puré continued. “Our
studies indicate that FAP+ cells are required for
the generation and maintenance of desmoplastic
stroma and that depletion of FAP+ cells can inhibit
tumor growth through both immune-dependent
and immune-independent mechanisms.”
Another take on the tumor microenvironment
was presented by Wolf H. Fridman, Cordeliers
Research Centre, Paris. In a talk entitled, “Cancer
subtypes and their immune microenvironments,”
Dr. Fridman described how his group was elabo-
rating on the Immunoscore concept, which goes
back at least as far as 2006. Basically, Immunoscore
builds on the insight that in many patients, the
density of T cells near tumor cells could be a better
predictor of survival than traditional staging based
on a cancer’s size and spread.
In general, for a patient’s prognosis to be
favorable, immune cells need to infiltrate a solid
tumor. But recent work also suggests that more
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Immunotherapy Tightens the Siege on Solid Tumors
immune cells may not always be better. Apparently
some immune cells are less helpful than others.
Some may even be deleterious, depending on the
interactions that occur between a tumor’s
microenvironment and the immune system.
“Our team studied the immune infiltrates
of pulmonary metastases from colorectal cancer
(CRC) and renal cell carcinoma (RCC),” reported
Dr. Fridman. “As in primary tumors, a high
density of CD8+ T cells correlated with good
prognosis for CRC metastases, while it correlated
with a bad prognosis for RCC metastases.”
“In addition, in both cancer types, we identified
subgroups of poor-prognosis patients with high
tumoral lymphocyte infiltration, in the context of
high expression of genes related to inflammation,
immunosuppression, and angiogenesis,” he
continued. “These results suggested that the identity
20 | GENengnews.com
of the tumor cells, rather than the organ where they
grow, is critical for shaping the immune contexture
of a given tumor.”
A particular cancer, then, may have an ecology
of its own, one in which the overall disposition of
elements—tumor cells, immune cells, extracellular
matrix elements, and so on—matters, much as the
overall disposition of chess pieces matters in a game
of chess. Even though it might be advantageous to
occupy a particular space on the board, apparently
not any chess piece will do. Frustratingly, one’s own
pieces may be poorly positioned, so as to get in
each other’s way.
Nonetheless, as Dr. Fridman concluded,
the integration of molecular and immune
tumor phenotypes could guide the selection
of immunotherapies “appropriate to specific,
potentially responding groups of patients.” n
APPLICATION NOTE
Therapeutic Antibodies
A Success Story
A ntibody-based therapeutics are the fastest
growing and most successful therapeutic
modality for treating diseases such as cancer,
cardiovascular disease, autoimmune disorders and
infectious disease.1 It is predicted that by the year
2020 there will be 70 therapeutic antibodies on the
market, generating revenue of $125 billion.2
The success of this class of drugs stems from
innovative technical advances in antibody
engineering and development, including display
screening technologies, bispecific antibodies,
21 | GENengnews.com
Additional Content
and antibody drug conjugates. These technical
innovations, combined with significant scientific
advancements toward our understanding of the
WEBINAR
Identification of Antibodies
complex molecular mechanisms underlying human Against Challenging Targets
disease, present new opportunities for improving Using High Throughput
human health with antibody therapeutics. Multiplex-Screening Approaches
The continued advancement in this field
Watch now!
t
is dependent on the successful discovery and
development of novel therapeutic antibody
candidates. These efforts are being driven by
scientists in discovery labs from pharma, biotech
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story
and academics, who need powerful tools that
can deliver rapid, multifactorial results to fully
understand the ability of candidate antibodies to
interrupt the cellular and molecular processes
leading to disease states. In this white paper, we
describe how the iQue Screener, the fastest
suspension based high throughput screening system
available, is used to perform multiplexed screens
for antibody binding to either cell surface or to
circulating target antigens. We will also discuss the
use of the same platform to carry out high content
assays to evaluate the effects of lead candidates in
multiplexed cell based and secreted protein assays.
Antibody Screening—
Moving Beyond ELISA
Because therapeutic antibodies are large biologic
molecules, their molecular targets are usually
extracellular, either on the surface of cells or circulating
in blood or other tissues. Screening hybridomas and
other libraries for antibody binding to each of these
types of targets has historically involved the use of
22 | GENengnews.com
enzyme-linked immunosorbent assays (ELISAs).
ELISAs were developed over 40 years ago, and have
been a staple in antibody screening labs. ELISAs are
performed by coating a single target antigen onto
the wells of assay plates, followed by addition of
individual samples from an antibody library
(e.g., hybridoma, phage display). Wells containing
antibodies that bind to the immobilized antigen
are detected through a change in color due to an
enzyme/substrate reaction.
While robust and relatively straightforward to run,
ELISAs have a number of disadvantages that can
limit the success of modern antibody screening labs:
• Primary screens that test binding to a single
antigen require subsequent secondary and
sometimes tertiary screens with control antigens
to confirm specificity and/or cross-reactivity.
• ELISAs are not ideal for screening antibodies
that bind to cell surface antigens, because these
antigens are extracted from the cell membrane
and purified before adsorbing to the plastic
ELISA plate. This often leads to disruption of
conformational epitopes that can be important
targets for therapeutic antibodies.
• Finally, in order to minimize background signal,
ELISAs require multiple wash steps to remove
unbound antibodies and detection reagents,
resulting in long, labor-intensive screening
workflows.
Multiplex High-Throughput Screening
for Antibody Discovery with
the iQue Screener
Recent advances in assay technology are beginning
to transform the antibody discovery process. At the
center of these advances is the ability to perform
multiplexed, multiparameter assays at high
throughput screening speeds. By collecting more
information in primary screens, scientists are able
to simultaneously identify hits based on specificity
and cross-reactivity. Inclusion of more information
early in the screening process builds confidence in
potential hits, and increases the likelihood that
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story

candidate molecules will successfully proceed through downstream

steps in the discovery and development process.

The iQue Screener is a high throughput screening platform based

on flow cytometry that rapidly processes samples in suspension
(Figure 1). This unique capability makes the iQue Screener a powerful
tool for performing high content antibody screening campaigns.
It provides:
• Content: Multiplex assays to simultaneously test binding to
target and control antigens in primary screens
• Usability: Antibody binding assays on either intact cell surface
antigens or circulating antigens
• Speed: Mix and read high throughput screens

Figure 1. iQue Screener and technology

Encoding Technology—Screen against Multiple
Antigens in Every Well

The IntelliCyt iQue Screener was designed to perform high throughput

assays on cells and beads in suspension, which enables a powerful
multiplexing approach known as encoding technology.3 With
encoding technology, scientists can combine multiple populations
of cells or beads bearing different antigens of interest into the wells

23 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story

A B
C
Figure 2. Encoding technology for cell- and bead-based antibody screening
of assay plates, and screen against multiple target antigens in the same
experiment. This greatly increases the power and robustness of
antibody screening campaigns, and allows for an enhanced screening
workflow. Figure 2 illustrates encoding technology and its use in antibody
screening assays. For cell based antigens (Figure 2A), cell lines expressing
different target antigens on their surface are individually stained with fluorescent
encoding dyes at different concentrations. For soluble antigens (Figure 2B), beads
encoded with different fluorescent dyes are coated with different target antigens.
For screening, encoded cells or beads are mixed together, and then distributed
into wells of screening plates (Figure 2C). Antibodies from hybridoma, phage
display or other libraries are added to the plates and tested for binding to the
different antigens in the wells.
By incorporating high throughput encoding into their assays,
antibody screening scientists can:
• Simultaneously test antibodies for binding to target and control antigens.
This provides an internal reference control within each well, which improves
confidence in hits. Also, including control antigens in primary screens can
lessen the need for secondary counter screens, which streamlines the overall
antibody screening workflow.
• Screen for cross reactivity to related antigens. Encoding allows scientists to
include multiple antigens in their primary screens to search for cross-reactive
antibodies that bind to families of target antigens such as cytokines. This

24 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story

technique can also be used to screen for anti-

B C
bodies that bind to a target antigen from mul-
tiple animal species. This is important when
A
developing therapeutic candidates specific for
human target antigens that can also be used in
animal models of toxicity and efficacy.
• Combine multiple projects into a single screen-
ing campaign. Encoding can also be used to
screen a single library against multiple unrelat-
ed antigens. Assay plates can be set up contain-
ing encoded cells or beads presenting different
antigens of interest. Multiple antibody libraries
can then be tested in the same screen, greatly
improving the overall workflow.
Figure 3. ForeCyt analysis of multiplex, cell based antibody screen using encoding technology
High-Throughput Screens Using Encoding
Technology and the iQue Screener
encoding dye, and cells expressing negative control
Following are some examples of how the iQue Simultaneous screening against target antigens on their surface were left unstained. The
Screener is being used to perform more effective and control antigens on intact cells cells were mixed together and distributed into wells
antibody screens, increasing the chance of In this example, two cell lines were used, one of the assay plates. Test antibodies were added to
discovering lead candidates that will be expressing a control antigen and one expressing a the plates and following a 30 minute incubation,
successful in the clinic. target antigen. Cells expressing target antigens on anti-IgG antibodies labeled with a red fluorescent
their surface were stained with a green fluorescent dye were added to the wells to detect test antibodies

25 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story

bound to each cell type. After a one hour incubation, Figure 4. Workflow for a cell-based species
plates were read on the iQue Screener and data cross-reactivity experiment
were analyzed using ForeCyt software (Figure 3).
Figure 3A shows populations representing the two
cell types were easily identified in scatter plots by
different fluorescent intensities on the Y axis,
which represents the green channel (Cell Encoder
Fluorescence). Cells from each population with
antibody bound to their surfaces exhibited increased
fluorescence in the red channel (Antibody Binding
Fluorescence). Heat maps in ForeCyt easily identify
wells containing cells with antibody bound to their
surface (Figure 3B). The two heat maps represent
each population of cells from the same plate
(Figure 3C).

Screening for cross reactivity
using cell-based assays
This example demonstrates the use of multiplexed
cell based assays by scientists at Xoma Corporation
to identify antibodies that react with a cell surface
receptor from different animal species. In these
experiments, CHO cells were engineered to
individually express the same target receptor from
different species: human, monkey, rat and mouse
(Figure 4). The parent cell line, and lines expressing
the species-specific receptors were labeled separately
with different concentrations of the encoding dye.
The five different cell lines were combined into a
single mixture, which was then distributed into wells
of the assay plates. After sampling, the differentially
encoded cell lines within each well were easily
distinguished using ForeCyt software, which
enabled the identification of cross-reactive
antibodies. In these experiments, each 96-well
assay plate contained 12 antibodies, tested at
8 concentrations each. Each well contained five

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Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story

encoded cell lines. Plate read times of five minutes

or less, combined with encoding technology, enables
large scale experiments to be performed in short time
frames. Large data sets comparing the binding
characteristics of antibodies to the different
receptors can easily be generated (Figure 5).
These cross-reactivity experiments enable the rapid
discovery of antibodies that bind to both human
andanimal receptor analogues. Using this approach,
potential drug candidates that can be used in humans
and also be tested in preclinical models of efficacy
and toxicity. This has the potential of dramatically
improving the success of therapeutic antibody
discovery efforts.

Screening for cross reactivity using

bead-based assays
Affimers developed by Avacta are small engineered
binding molecules designed to be more robust than
antibodies, and can be generated very rapidly. This
Figure 5. Representative data from cross-reactivity experiments for 12 antibodies
example demonstrates the use of multiplex bead-
based assays by scientists at Avacta to screen affimer
libraries generated from phage display panning

27 | GENengnews.com
 Human PD-L1

Human PD-L2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
Mouse PD-L1 D
E
F
G
H
I
J
Mouse PD-L2 K
L
M
N
O
P

Fc Fragment Control

Figure 6. Avacta’s affimer screening workflow

28 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story
Figure 7. Data from the screen, demonstrating the identification of clones that bound to both
human PD-L1 (red line) and mouse PD-L1 (light blue bars), but not to human or mouse PD-L2 (green
and grey bars, respectively). No clones that bound to control beads coated with the Fc fragment
were identified (purple bars). Using multiplex bead based assays, Avacta is employing large scale
experiments on the iQue Screener platform to significantly improve their affimer screening workflow.
29 | GENengnews.com
outputs for cross-reactive binders to different immune checkpoint
molecules. Encoded beads were coated with four different check-
point modulating proteins: human PD-L1, human PD-L2, mouse
PD-L1, and mouse PD-L2. Since these proteins were expressed
as Fc fusions, a fifth encoded bead was coated with Fc fragment,
and served as a negative control (Figure 6). The five beads were
combined into a single mixture, which was then distributed into
wells of 384-well assay plates. A library consisting of 768
affimers selected by panning against human PD-L1 was screened
for binding to the different PD- L1 and PD-L2 antigens (Figure 7).
Because five binding assays were run in every well, the screen
required only two plates and 7 µg of each target protein. The
time to perform the entire screen was less than one hour.
Running this same screen using single-plex ELISA assays would
have required ten 384-well plates (or forty 96 well plates) and
several mg of each target protein.
Summary
Multiplexed high throughput screening is becoming an
integral component in the discovery of therapeutic antibodies
because it increases confidence in screening hits, improves
workflow and enables the identification of lead candidates
with increased functionality. With the iQue Screener platform,
Immuno-Oncology Profiling for Drug Discovery and Translational Research • Therapeutic Antibodies—A Success Story

scientists are using suspension assay formats and

encoding technology to develop powerful high
content screens for their antibody libraries. n

References
1. Chames, P., et al. (2009) “Therapeutic antibodies: successes,
limitations, and hopes for the future.” British Journal of
Pharmacology 157: 220-233.
2. Ecker, D.M., et al. (2015) “The therapeutic monoclonal
antibody market.” mAbs 7: 9-14.
3. Black, C.B., et al. (2011) “Cell-Based Screening Using
High-Throughput Flow Cytometry.” Assay and Drug
Development Technologies 9:13-20.

30 | GENengnews.com
APPLICATION NOTE
A High-Throughput,
Radioactivity-Free Assay
for Cell-Mediated Cytotoxicity
I mmunotherapy promises to be a powerful
approach for treating a variety of diseases—
most notably cancer.1–5 However, development of
novel immunotherapeutics has been limited by the
lack of high-throughput methods to screen for
effective molecular entities or, in the case of
adoptive cell therapy, genetically engineered cells.
Most current assays that measure cell-mediated
cytotoxicity (CMC), such as the chromium release
31 | GENengnews.com
Additional Content
WEBINAR
assay, are difficult to perform on large numbers
of samples, can only report on a single biological
readout like cell membrane integrity, and
Immunology Screening on
the iQue Screener:
Assays for Immune-Modulators
cannot differentiate between effector and target and Immunotherapy
cells. Methods based on flow cytometry, such as
Watch now!
t
the CFSE assay,6 can assess CMC at the level of an
individual cell and enable discrimination between
effector cells and target cells. Traditional flow
cytometry is slow, however, making it unsuitable
Immuno-Oncology Profiling for Drug Discovery and Translational Research • A High-Throughput, Radioactivity-Free Assay for Cell-Mediated Cytotoxicity
for use as a high throughput screening (HTS) assay.
Here we demonstrate a fast, efficient, radioactivity-
free CMC assay with low sample input requirements,
enabling miniaturization to 384-well plates. Using
the iQue Screener and differential labeling of effector
and target cells, this method can capture multiple
facets of biology—apoptosis markers, signal
transduction markers, cell permeability, proliferative
capability, and more—in a single well, providing a
rich and highly quantitative data set specific to each
cell type present. With this approach, researchers and
drug discovery teams can quickly screen through
compounds and conditions, building a detailed
understanding of the molecular events occurring in
each well and speeding insight into development of
immunotherapeutic approaches.
Materials and Methods
CELLS AND REAGENTS
Jurkat cells, clone E6.1 (TIB-152 from ATCC), were
used as target cells and NK-92 cells (CRL-2407
from ATCC) were used as effector cells. Jurkat cells
were maintained in log growth phase at 37°C with
32 | GENengnews.com
5% CO in 1640 RPMI medium supplemented with
10% fetal bovine serum (Seradigm). NK-92 cells
were cultured at 37°C and supplemented with 20
ng/ml rhIL2 (Sigma) and 1 µM hydrocortisone
(Sigma). All assays were conducted in Jurkat cell
medium. NK-92 cells were used in CMC assays
48 hours after passage into new medium. Signal
transduction inhibitors PP2, U73122, Sunitinib,
and SP600125 were obtained from Sigma. Each
inhibitor was solubilized in 100% DMSO (various
concentrations) and stored at -20°C until use.
CMC TITRATION ASSAYS
The iQue Screener CMC workflow is illustrated in
Figure 1. Prior to the start of the assay, Jurkat cells
(target) were encoded using the MultiCyt FL4 Cell
Proliferation and Encoding Kit (IntelliCyt) according
to the protocol. Briefly, the cells were prepared in
batch and excess dye removed by washing before
use in the assay. The encoding protocol takes
approximately 20 minutes. The following steps
were performed in a single 384-well plate with thirty
replicates of each condition and a total assay volume
of 20 µL/well. Unlabeled NK-92 cells were serially
diluted (1:2) over 12 steps from 60,000 cells/ well
down to 30 cells/well. At the start of the assay, each
dilution was mixed with labeled Jurkat cells (6,000
cells/ well) and incubated for four hours at 37°C.
The resulting effector:target ratios ranged from
10:1 down to 1:200.
To assess CMC, we monitored two different
readouts— apoptosis using the Caspase 3/7 reagent
from the MultiCyt Apoptosis Kit (IntelliCyt), and
cell viability using the MultiCyt FL3 Cell Membrane
Integrity reagent (IntelliCyt). Both reagents were
used as stated in the product protocols, and were
added to the reaction at the same time as the NK-92
cells. After four hours, the 384-well plate was placed
directly into the iQue Screener and analyzed.
CMC INHIBITION ASSAYS
The CMC inhibition assay was performed as
described for the CMC titration assay with the
following modifications. A 16-step, 1:2 dilution
series of each inhibitor was prepared with the
following as the highest concentrations: Sunitinib
Immuno-Oncology Profiling for Drug Discovery and Translational Research • A High-Throughput, Radioactivity-Free Assay for Cell-Mediated Cytotoxicity

at 25 ng/ml; PP2 at 100 mM; U73122 at 37.5 mM;

SP600125 at 150 mM. NK-92 cells (12,000 cells/
well) were preincubated at 37°C for 20 minutes with
each dilution of inhibitor, and then mixed with
Jurkat cells (6,000 cells/well). An effector:target
ratio of 2:1 was used for this study, with a total
reaction volume of 20 µL. Each condition was
performed in triplicate.

IQUE SCREENER AND FORECYT ANALYSIS

The iQue Screener provides high-throughput
measurements using a flow cytometry detection
engine and patented air-gap delimited sampling
technology, which can sample wells with zero
dead volume. Samples are delivered in a
continuous stream to the detector engine without
needing to pause between each sample. Individual
wells are separated by discrete air gaps ensuring
accurate discrimination of each sample (Figure 2).
ForeCyt Software was used to acquire data, set
Figure 1. Three-step iQue Screener cell mediated cytoxicity (CMC) assay workflow.
appropriate gates, quantify, and plot CMC titration
and inhibition data as described in the Results and
Discussion section.

33 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • A High-Throughput, Radioactivity-Free Assay for Cell-Mediated Cytotoxicity
Figure 2. iQue Screener Technology provides
multiparameter, high- content analysis with as
little as 1 µL of sample and zero dead volume.
34 | GENengnews.com
Results and Discussion
RCMC ASSAY OVERVIEW
The iQue Screener-based CMC assay is a simple,
three-step process that enables rapid screening of
multiple conditions (Figure 1). First, target cells are
labeled with an encoding dye, and then incubated
with effector cells and reporter dyes, all in the same
well of a multi-well plate. At the end of the
incubation, the plate is placed into the iQue
Screener for analysis. The resulting data is analyzed
using ForeCyt Software to set appropriate gates,
allowing discrimination of target cells from effector
cells and quantification of cell membrane integrity
and caspase 3/7 activation in individual cell types.
The advantages of this assay stem not only from
its easy implementation as a high-throughput screen
but also from the streamlined protocol, which
reduces hands-on time. By adding staining reagents
at the beginning of the assay, the labor involved in
downstream steps is minimized while still providing
robust results.
Data from an example assay using Jurkat target
cells and NK-92 effector cells is shown in Figure 3.
Figure 3A shows two distinct cell populations,
the background fluorescence of unlabeled NK-92
cells and a higher intensity signal generated by
fluorescence-encoded Jurkat cells.
Gating the Jurkat cells and analyzing just this
population in the FL1 channel for caspase 3/7
activation enables quantification of Jurkat
apoptosis. Because only apoptotic cells with activated
caspase 3/7 will display fluorescence in the FL1
detection channel, we can determine the percent of
apoptotic target cells in the well by comparing the
number of FL1-positive cells (Figure 3B, right peak)
to FL1-negative cells (Figure 3B, left peak).
Similarly, by analyzing the data for the Jurkat
target cells in the FL3 channel, we can measure
the relative amount of cell death mediated by the
effector cells (Figure 3C). The higher intensity
peak consists of FL3-positive cells with damaged
membranes (Figure 3C, right peak). A gate can be
drawn to quantify the number of target cells that
 Immuno-Oncology Profiling for Drug Discovery and Translational Research • A High-Throughput, Radioactivity-Free Assay for Cell-Mediated Cytotoxicity

are detected as FL3-positive, and therefore reports on

the number of non- viable cells, whereas the lower-
B C intensity “negative” peak is comprised of the viable
cells that have excluded the dye and are not fluorescent
A (Figure 3C, left peak).
Finally, the same analysis can be done on the
NK-92 cells to verify that they remain unaffected
by incubation with the target cells (Figure 3D, E).
In this case, the same gates used to assess activation
D E of caspase 3/7 (FL1) and cell viability (FL3) for Jurkat
cells were applied to the NK-92 cells for an unbiased
analysis.
Note that for this example we chose to detect
caspase 3/7 activation and cell membrane integrity,
but a number of other reporters can be simultaneously
detected and quantified for each cell type. Examples
of other MultiCyt reagents that have been validated in
Figure 3. Analysis of iQue Screener CMC assay. Target cells are encoded with a dye that fluoresces in the multiplex with either the caspase or the cell viability
FL4 channel, enabling differentiation from unstained effector cells (A). Both target and effector cells can reagents include Annexin V binding, mitochondrial
then be queried for viability (B, D) or caspase activation (C, E) separately, even though both are present membrane depolarization (MultiCyt Apoptosis Kits),
in the same well.
cell proliferation (MultiCyt Cell Proliferation and
Encoding Kit), and detection of secreted cytokines
(QBeads PlexScreen and DevScreen Kits).

35 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • A High-Throughput, Radioactivity-Free Assay for Cell-Mediated Cytotoxicity

ASSAY SENSITIVITY, CELL TYPE

A B
Figure 4. CMC Titration Assay. Cell viability (A) and Caspase 3/7 activation (B) assessment demonstrate that
Jurkat target cells are killed by NK92 effector cells in a dose-dependent manner. For each data point, n = 30
with the curves simply connecting each data point.
REPRODUCIBLE QUANTIFICATION
DEMONSTRATED BY EFFECTOR CELL TITRATION
To demonstrate the ability of the iQue Screener
CMC assay to provide highly quantitative informa-
tion, we assessed the dose-response relationship of
NK-92 effector cells on CMC of Jurkat target cells.
Keeping the number of Jurkat cells fixed at 6,000
cells/well, we added increasing amounts of NK-92
cells, changing the effector:target ratio from 1:200
to 10:1 (Figure 4). Each data point is the average of
measurements from 30 different wells, with error
bars as indicated.
Examination of the caspase 3/7 activation
and cell viability plots for the NK-92 cells show
background levels of caspase activation and cell
viability, with higher variability at the lower NK-92
concentrations likely a reflection of measurement
uncertainty at extremely low cell densities.
DISCRIMINATION DEMONSTRATED BY
CMC INHIBITOR DOSE-RESPONSE ASSAY
To demonstrate the sensitivity and specificity
of the iQue Screener CMC assay, we performed
the assay in the presence of increasing amounts of
known CMC inhibitors that target different signaling
pathways (Figure 5)—sunitinib (tyrosine kinase
inhibitor), PP2 (Src inihibitor), U73122 (PLC
inhibitor), or SP600125 (JNK inhibitor). Note that
the ability to observe the effects of pathway-specific
inhibitors on both effector cells and target cells
helped us to quickly uncover an experimental
artifact— general cytotoxicity rather than
specific CMC inhibition—that could have led to
misinterpretation of the results in the absence of
the effector cell data.
For this assay we selected a fixed 2:1 ratio of
NK-92 cells to Jurkat cells, which results in baseline
values of ~75% of Jurkat cells staining positive for
caspase 3/7 activation, and ~60% of Jurkat cells
staining positive for membrane permeability
(non-viability).

36 | GENengnews.com
 Immuno-Oncology Profiling for Drug Discovery and Translational Research
A B

The effect of pathway inhibitor compounds on CMC can be seen in Figure 5,

with increasing inhibitor concentration generally resulting in less cell membrane
permeability (Figure 5A, C, E, and G) and caspase 3/7 activation (Figure 5B, D, F,
and H) in Jurkat cells. There are two intriguing exceptions.
Figure 5A and B show the effects of sunitinib on both Jurkat target cells and
NK-92 effector cells. While this tyrosine kinase inhibitor appears to have no effect on C D
CMC based on the stable levels of membrane permeability and caspase 3/7 activation in
the Jurkat target cells (Figures 5A, B), it increases caspase 3/7 activation in NK-92 effector
cells (Figure 5B), while leaving cell membrane permeability in NK-92 cells unaffected.
This effect on NK-92 cells would not have been visible using a traditional Cr51 release
assay, and raises questions on the interpretation of the Jurkat caspase 3/7 data.
A second intriguing result comes from U73122. At the highest concentration of
U73122, Jurkat target cells show a reverse in the trend of decreased cell membrane E F
permeability and caspase 3/7 activation. Examination of these two readouts in NK-92
cells shows that at higher concentrations of U73122, NK-92 cells also display increased
cell membrane permeability and caspase 3/7 activation (Figure 5E, F), suggesting that

Figure 5. Pathway-specific inhibition of CMC. Four different compounds show

varying degrees of CMC inhibition on Jurkat target cells (blue curves) as assessed by cell
G H
membrane permeability (A, C, E, G) and caspase 3/7 activation (B, D, F, H). For each data
point n = 3, with the data fit using a four parameter logistic nonlinear regression
model. Notably, sunitinib and U73122 also have an effect on the NK-92 effector cells
(red curves), which would not have been detected using a traditional Cr51 release assay.

37 | GENengnews.com
Immuno-Oncology Profiling for Drug Discovery and Translational Research • A High-Throughput, Radioactivity-Free Assay for Cell-Mediated Cytotoxicity
U73122 might possess general cytotoxic activity
at these concentrations instead of specific CMC
inhibition. This is in contrast to other inhibitors,
such as PP2 and SP600125, which show no in-
creased cell membrane permeability or caspase 3/7
activation at any concentration in NK-92 cells.
Thus, the ability to measure endpoints in
both effector and target cells enabled a possible
explanation of the anomalous U73122 data at high
concentrations. Importantly, if only the highest
concentration of U73122 had been examined, its
effectiveness as an inhibitor of CMC would have
been underestimated. In the context of a HTS where
primary screening is typically performed using a
single concentration of the test compound, this type
of underestimation could lead to early elimination
of a potentially potent compound.
Conclusion
The iQue Screener CMC assay is a sensitive,
specific, and high-throughput assay that can provide
a rich data set of quantitative information in a cell
38 | GENengnews.com
type-specific manner. It overcomes the problems of
the traditional Cr51 release assay by removing the
need to work with a radiolabeled isotope, reducing
hands-on time, and increasing reproducibility
and measurement precision. Like flow-cytometry
approaches, the iQue Screener CMC assay enables
discrimination of signal generated by effector cells
versus target cells. However, the iQue Screener
CMC assay goes one step further and improves
on flow-cytometry-based approaches through its
HTS format. Further, the potential for additional
multiplexing with readouts such as quantification
of secreted cytokines provides even more biological
context. By simplifying and miniaturizing the CMC
assay, the iQue Screener enables researchers to get
results faster while conserving precious sample, thus
providing an opportunity to accelerate discovery and
development of new immunotherapeutics. n
References
1. Anguille S, Smits EL, Bryant C, et al. Dendritic Cells as
Pharmacological Tools for Cancer Immunotherapy. Pharmacol
Rev. 2015;67(4):731-753. doi:10.1124/ pr.114.009456.
2. Chester C, Marabelle A, Houot R, Kohrt HE. Dual antibody
therapy to harness the innate anti-tumor immune response
to enhance antibody targeting of tumors. Curr Opin Immunol.
2015;33:1-8. doi:10.1016/j. coi.2014.12.010.
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Rethink Possible
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