Multiplexing Phenotype and Function for More Biologically Relevant Insights - 36

Related Article from
Addressing Logistical Challenges
"CAR T-cell therapy has proven to be very effective within the context of liquid
tumors," said Sadik Kassim, Ph.D., vice president, process and analytical development, Mustang Bio. "But the major challenge in the field has been getting the clinical
response to a CAR T-cell therapy within a solid tumor setting."
In his CAR-T Congress presentation, Dr. Kassim drew on lessons from Mustang
Bio's collaboration with the City of Hope Beckman Research Institute and Medical
Center. Specifically, he referenced a recent study led by the City of Hope's Stephen J.
Forman, M.D., and Behnam Badie, M.D.
The study, which appeared in the New England Journal of Medicine, suggested that
while a leukemia or lymphoma may be impacted by a single systemic infusion of
tumor-specific CAR T cells, solid tumors may require multiple, regional infusions. To
flesh out this notion, City of Hope investigators described a novel administration
approach against multifocal glioblastoma, one of the most lethal cancers. Multifocal
glioblastoma has a five-year survival of about 5%-due in part to the brain's sequestration behind the blood-brain barrier.

Targeting Liquid and Solid Tumors
Finding (and hitting) the right target can be difficult. "The antigens that are present
in solid tumors are usually expressed in other tissues of the body, or there is great
heterogeneity," said Peggy Sotiropoulou, Ph.D., R&D manager, Celyad. "Consequently,
the antigens are not expressed in all the tumor cells." She added that tumors that do
express the antigens may reside in an immunosuppressive microenvironment, that
is, an environment that can suppress the activity of CAR T cells.
To tackle these issues, Celyad engineered T cells expressing a CAR composed of
the full-length human natural killer group 2D (NKG2D) receptor fused to the CD3ζ
cytoplasmic signaling domain. As its name suggests, the NKG2D receptor is derived
from natural killer cells. The CD3ζ chain, which is found in most antibody-based
CARs, provides the primary stimulatory signal within the T cell when the NKG2D
receptor binds to a ligand.
The NKG2D receptor targets eight different ligands that are normally expressed in
cells after infection and other types of stress. Most important, as Dr. Sotiropoulou
noted, these ligands are expressed by most types of cancer cells, but they are not
expressed at all (or above very low levels) by healthy cells

After tumor resection, CAR T cells specific for the interleukin-13 receptor α chain
variant 2 (IL13Rα2) were infused weekly directly into the cavity created by removal
of the primary tumor. Subsequently, the therapy was administered weekly into
the cerebral ventricles, which allowed systemic distribution within the nervous
system.

The NKG2D-based CAR serves as the basis of Celyad's lead CAR T-cell oncology
candidate, CYAD-01. (Celyad's NKG2D-CD3ζ CAR T cell was previously referred to
as CAR T NKR-2.) NKG2D attracts a costimulatory adapter protein, DAP10, which is
normally expressed in natural killer cells and T cells.

"Even though they administered at a certain location, they observed a regression of
the tumor at a distal site, in the spinal cord," noted Dr. Kassim. "The point is, within
the context of solid tumors, the trafficking of the CAR T cells is probably going to be
important. One aspect of that is the route of administration."

Besides recognizing the formation of DAP10-stabilized NKG2D constructs, Celyad
evaluated whether CYAD-01 would perform better if it incorporated an additional
cytoplasmic costimulatory domain. When Celyad tried adding CD28 or 4-1BB
costimulatory domains, which are known to confer interesting properties to classical
scFV-based CAR T cells, the company found that neither CD28 nor 4-1BB provided
any advantages to NKG2D-based CAR T cells in vitro.

If single-infusion protocols are inadequate against solid tumors, monotherapies may
be insufficient as well. Treatment may require a checkpoint antibody, for example,
or something to promote inflammation. Preconditioning regimens have yet to be
optimized. "What will it take for the CAR T cell, post infusion, to really take and start
expanding," asked Dr. Kassim, "and what would give the cells room? Those protocols
are still being investigated."
36

| January, 2019

Unlike CD28 and 4-1BB, DAP10 does offer distinct advantages. "The overexpression
of DAP10 allows T cells to express NKG2D-based CAR at the cell surface and tends to
increase the secretion of proinflammatory cytokines," a Celyad poster paper indicated. "The increased secretion of these proinflammatory cytokines is of particular
interest in the context of solid tumors."


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Multiplexing Phenotype and Function for More Biologically Relevant Insights

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