Off-target profiling of whole CAR T cells: Strategies for nonclinical safety assessment in cancer immunotherapy


Modification of a patient’s own T cells to carry a chimeric antigen receptor (CAR) recognising a cancer cell surface antigen can promote a specific anti-tumour response and potentially confer long-term immunity to cancer. Minimising the risk of triggering an inappropriate immune response through unanticipated off-target binding of the engineered CAR T cells is essential in ensuring patient safety.

Screening using human cell microarrays – also known as plasma membrane protein arrays (PMPA) – has been successful in identifying key receptors for orphan ligands and for phenotypic molecules as well as detecting potential off-target receptor interactions that may impact on the safety profile of lead biotherapeutic compounds.

Here we describe a PMPA approach for specificity screening of whole engineered CAR T cells as a supportive tool in safety assessment of novel cancer immunotherapies.

This study was presented at SOT 2017 – click here to download the poster.


Materials and methods

T cells, isolated from healthy donors and engineered to carry an anti-CD19 CAR, were provided to Retrogenix by bluebird bio, along with donor-matched untransduced T cell controls. All T cells were labelled with a far-red fluorescent cell dye in order to detect interactions with plasma membrane binding partners using Retrogenix’s PMPA technology as described here.

Optimisation of the ratio of T cells to the underlying HEK293s, as well as careful control of washing stringencies, was required to develop the technology in order to reliably and reproducibly identify specific binding events.

Primary screens: 13 slides were spotted in duplicate with different expression vectors encoding over 4,500 full-length human plasma membrane proteins. Human HEK293 cells were overlaid and reverse-transfected. Fluorescently labelled T cells were added to all 13 slides (n=2 replicate slides per T cell line) and gain of binding was detected.

Confirmation screen: All positive ‘hits’ from the primary screen were respotted on fresh slides and retested to confirm the specificity and reproducibility of the interactions (n=2 replicate slides per T cell line).



Screening of untransduced T cells against 4,500+ proteins identified a core panel of human plasma membrane proteins which may mediate CAR-independent interactions between normal T cells and human cells, providing a baseline for identifying interactions that are specific to engineered CAR T cells (Figure 1).


Figure 1. Confirmation screen results for untransduced T cells. The GFP spotting pattern of all ‘primary’ PMPA hits is shown on the left. Specific, reproducible interactions with untransduced T cells are visible on the right.

A full list of proteins hits for untransduced T cells can be found in the poster.

A clear, additional interaction with the primary target, CD19, was observed for all engineered anti-CD19 CAR T cells screened (Figure 2). No additional receptor binding to engineered CAR T cells was observed in this study, confirming no off-target binding partners amongst this extensive library of 4,500 human plasma membrane proteins, and no false positives.



Figure 2. Confirmation screen of all ‘primary’ PMPA hits. GFP spotting pattern is shown on left hand panel. The right hand panel shows the specific interaction with CD19 (highlighted in green) in addition to the core binding proteins observed for untransduced T cells.



Specificity screening of whole CAR T cells using PMPA technology presents a novel strategy to help de-risk immunotherapy development programmes and provide supporting data for IND submissions.

To discuss a CAR T specificity screen with a member of our science team then please do get in touch.

To browse our other case studies please click here, or return to our CAR T screening applications page.

Download the poster