IsoPlexis eBook - 10

Polyfunctional Strength Offers a New Approach to Defining Quality in Cell Therapy

Most immunotherapy revolves around T cells. But immune
system monitoring is deceptively tricky. Current technologies are inadequate to assess critical quality, function and
variability attributes that drive efficacy, potency, and safety
especially as the mechanism for both clinical effects and
cytotoxicity can be heavily mediated by cytokines (functional proteins through which immune cells send and
receive signals; tracking them to the cells that secrete them
is not a simple endeavor).

A single cell secretes few copies of a protein. To address this
technical hurdle, the IsoPlexis microchip contains a parallelized series of microchambers that are encoded with a full
copy of a multiplexed antibody array. This allows single cells
to be captured and the small number of multiple protein
copies secreted to become sufficiently concentrated for
their automatic measurement with a standard immunofluorescence sandwich assay based on antibody capture and

Take cancer immunotherapy as an example: blood
contains many proteins and immune cell phenotypes, of
which anti-tumor immune cells make up a small subset.
Even important cell subsets, such as effector T cells, have
a large clonal diversity. The few clones that might have a
therapeutic impact are mixed along with all other cells,
illustrating the need for a single cell technology to locate
the cells of interest.

Using a straightforward workflow, the technology isolates
thousands of immune cells and identifies polyfunctional
cells (that secrete multiple cytokines). Polyfunctional cells are
recognized as key effector cells contributing to the development of potent and durable cellular immunity against viral
infection, cancer, and other diseases.

In 2017, Dr. Rong Fan from Yale University and an IsoPlexis
co-founder, along with scientists at IsoPlexis, tested the
index on samples from various CAR-T studies. They found a
correlation of the single-cell PSI with patient response and
non-response. To date, PSI has been repeated used to assess
the function of the immune response in multiple clinical
trials focusing on immunotherapy, autoimmune and inflammatory diseases, and vaccine development.

Data through Time

Democratizing PSI

Early single-cell proteomics data from melanoma patients
treated with engineered T cells that had MART-1 TCR specificity revealed high functional heterogeneity of MART-1
cytolytic T lymphocytes despite the cells being homogenous in phenotypes.1 Polyfunctionality was confined to a
small subgroup of cells.

Through its innovative technology, IsoPlexis has democratized PSI measurements and increased the cytokine panel
size to approximately 40, allowing for higher data multiplexing. Importantly, all data underlying the PSI calculation, the
protein secretion signature from each individual cell, are
retained and allow for deeper dives into how cells evolve
over time and a history of unique cytokine contribution.

The secreted cytokines reflect immune cell functions. In an
immunotherapy, a T cell is tasked to target and kill cancer
cells, recruit other immune cells of specific types, promote
or mediate inflammation, replicate, or induce other immune
cells to replicate-upwards of about 20 distinct functions.
The challenge is to monitor different T-cell populations along
with their functional and anti-tumor performance. Phenotyping, for example using flow cytometry, only provides for
population statistics, not functional performance.



The cytokine panel was expanded and melanoma patients
re-evaluated over the course of a clinical trial.2 After
deconstructing the secreted proteins into biological function buckets, this study showed that the secreted protein
response from tumor-antigen-specific T cells was highly

coordinated, and that 5-10% of the T cells were polyfunctional and dominated the anti-tumor immune response.
T cells can perform 20-30 separate functions; the most
effective ones perform all or most of the functions whereas
the least effective may not perform any functions. A simple
metric was developed to understand the data. This metric
has evolved to become the PSI.

In a 2018 publication in Blood, pre-infusion CD4+ and CD8+
CAR-T cell samples from 20 of 22 non-Hodgkin lymphoma
patients in a CAR-T clinical trial were profiled.3 This study
revealed that PSI could be used as a quality-control metric

IsoPlexis eBook

Table of Contents for the Digital Edition of IsoPlexis eBook

IsoPlexis eBook - 1
IsoPlexis eBook - 2
IsoPlexis eBook - Contents
IsoPlexis eBook - 4
IsoPlexis eBook - 5
IsoPlexis eBook - 6
IsoPlexis eBook - 7
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IsoPlexis eBook - 9
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