Agilent - Breakthroughs in Immunotherapy- 2019 - 11

Breakthroughs in Immunotherapy

off-target activities 6. This novel approach resulted
in a dramatic reduction in off-target cleavage activities while maintaining high on-target performance
(Figure 2).
Once you have edited your cells how will you
assess and validate both the desired and undesired functional consequences? With the
emergence of quantitative, time-resolved cell
analysis platforms researchers can make more
sensitive live-cell measures of function and simultaneously scan the temporal components of
an ever-evolving immune response in terms of
efficacy and persistence. For example, in a single
experiment, one can now test gene edits for both
killing efficiencies and serial killing at very low
effector to target ratios. The results can then be
related to inhibitory effects such as exhaustion,
anergy, and other escape mechanisms due to
interactions with a constantly evolving tumor and
its microenvironment.
A great example of the value of combining functional sensitivity with time-resolution was recently
published using an xCELLigence system (ACEA
Biosciences now a part of Agilent). In this study,
a CAR T cell strategy to address the heterogeneity of antigen expression within a cancer cell
population was investigated7. A common obser11

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Figure 2

Figure 2. The HBB gene, which contains the mutation that causes sickle cell disease was used as an
important, but challenging test to improve CRISPR specificity. It turns out that the favorite guide RNA for
editing the sickle cell mutation in the HBB gene yields quite a high level of off-target activity at another
genomic location. Adding a single modification at either position 5 or 11 of the HBB guide RNA sequence
dramatically reduced the level of off-target activity as shown in the gold bars on this graph while
maintaining a high level of activity at the on-target site. Figure adapted from Ryan et al., Nucleic Acids
Res. 46(2):792-803 (2018)

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Agilent - Breakthroughs in Immunotherapy- 2019

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