The Scientist November 2019

24

CREDIT LINE

I

n CAR-T cell therapy, clinicians obtain T

cell source material directly from a patient

(autologous), or from a donor (allogenic),

modify them, and re-introduce them to the

patient. Working with autologous cells can be

complicated by insufficient source material

quality or quantity, and expanding the cells to

the requisite number takes time that the patient

may not have. Allogenic cells can be prepared

in advance and mass produced, but they may

be rejected by the patient, and in the worst case,

begin attacking the patient’s own cells.

Vladimir Senyukov is the Director of

BioAnalytical Development at Precision

Biosciences, where he investigates the

development and manufacturing of allogenic

CAR-T cell products. We spoke to him about

working with allogenic CAR-T cells to unlock

their therapeutic potential.

Using single-cell functional proteomics

for CAR-T cell therapy development

Senyukov’s goal is to learn what actually

drives a cell to be polyfunctional – to produce

multiple types of cytokines instead of a single

cytokine – and to determine a way to apply

that knowledge to improve CAR-T cell therapy

in the future. Recent research found improved

patient outcomes where polyfunctional T cells

were present. For Senyukov, this indicates that

genetic factors drive CAR-T cell heterogeneity,

given that all the cells within a CAR-T product

are ostensibly generated from the same

starting material. But what is the relationship

between these genetic factors and cytokine

secretion profile? This is what Senyukov is

trying to figure out with the help of single-cell

functional proteomic approaches.

“My current project involves using IsoPlexis to

profile the cytokine signatures of polyfunctional

cells that we might have in a cell population,”

said Senyukov. “Then we take those cells

and employ a single cell RNA sequencing

approach where we can also try to identify

that polyfunctional cytokine signature in the

RNAseq data. From there, we can try to identify

what else—what other genes are associated

with that signature—and then try to find the

next genotype or other properties of a cell that

makes them unique or different or better.”

Testing and verifying edited and

engineered CAR-T cells

Whenever cells have been altered in the

laboratory—especially on a genetic level—

researchers need to ensure that the edits

function as intended. “To test CAR-T cells and

figure out if they function properly,” Senyukov

said, “you usually test them against their targets.”

Researchers use three common metrics to

evaluate T cell function and response: “When

T cells are activated, they produce cytokines

for the microenvironment and make it more

pro-inflammatory. They [recruit] other cells

and make the target cells more sensitive for

killing, and that’s one of the first responses

usually from T cells when they encounter

a target. The second way that a T cell

responds is by killing, so we can measure

how effectively CAR-T cells kill the target

cells. The third expected response is that T

cells will proliferate—when T cells recognize

an antigen, they will increase the population

of T cells that are responsive to that antigen.”

Single-cell functional proteomics is valuable

here as well, explained Senyukov. “When

optimizing, or when changing manufacturing

processes to make better cells, it can be used

to monitor cellular changes. It is especially

valuable in screening for polyfunctionality,

which is key, because based on the literature,

polyfunctionality is one of the most important

factors driving clinical efficiency.”

Unlocking the potential of allogenic

CAR-T cells

Ultimately, Senyukov would like to break

new ground for allogenic CAR-T cells,

given that most of the existing research on

polyfunctional T cells comes from cases

of autologous cell transfer. “There haven’t

been too many studies on the importance

of polyfunctionality for allogenic material,

and there’s a need to find specific signatures

that could give us information about

functional efficiency in a particular patient

group. It’s also about trying to find other

pathways of gene expression associated

with polyfunctionality that could allow future

optimization of manufacturing processes.”

Understanding T-cell polyfunctionality:

How single cell proteomics data drive CAR-T cell therapy

research and development–an interview with Vladimir Senyukov

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