Bringing order to the Chaos

Chimeric Antigen Receptor based immune synapses are a disordered punctate structure that is highly inefficient. We sought to alter that chaotic interface with an intracellular anchor domain to increase efficacy against solid tumors.
Bringing order to the Chaos
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Chimeric antigen receptor(CAR) technologies have been widely studied in various malignancy settings; most successfully in the treatment of leukemias. More recently, CAR T cell products have garnered numerous Food and Drug Administration (FDA) approvals and have begun to reshape the immunotherapy landscape for cancer treatment. Significant efforts towards optimization have been employed to make stronger and more effective CARs. These areas include exchanging various costimulatory and activating domains, genetic deletion of negative regulators, and increasing the affinity or diversity of the recognition domains. All these areas of exploration have been performed within the confines of a disordered and inefficient synapse formation.

Prototypical synapse formation between a T cell receptor (TCR) and the cognate human leukocyte antigen (HLA) results in a highly ordered and efficient synapse interface. TCR:HLA synapses look akin to a bullseye with a concentrically organized central, peripheral, and distal supramolecular activation complexes. Conversely, a  CAR:Antigen synapse is disjointed and results in a punctate structure with microclustered signaling islands. The sensitivity disparity between these structures is ~4-5 logs with the CAR synapse requiring significantly more antigen for successful downstream signaling and activation.

Given the extensive research and past efforts in CAR optimization we elected to take an orthogonal approach to altering the disordered synapse. CAR immune cell recognition and killing of cancer cells require the effector cell to polarize and shuttle lytic granules to the synapse. This cell polarizing process is tightly controlled and requires an elegant orchestration of connecting scaffolding proteins. Naturally occurring synapses utilize external recognition but also a sophisticated internal ordering that can exclude inhibitory proteins and help arrange and align the signaling componentry for a highly efficient synapse to form.

These scaffolding proteins align and order via Post-synaptic density protein-95, Discs large, Zona occuldens-1 (PDZ) domains and cognate PDZ binding motifs (PDZbm). There are approximately a couple hundred PDZ domain containing proteins in humans, and these are tightly regulated and almost exclusively interact with the extreme end of the carboxylate C-terminus. We elected to use the PDZbm of CRTAM, a late phase polarity protein expressed in numerous immune cell types, most notably in natural killer (NK) and T cells. This specific motif on CRTAM binds the scaffolding protein Scribble. This results in a cascade of organization that enhances nearly all aspects of the immune synapse formation. We have dubbed this new domain an ‘anchor domain’.

Considering the previous studies detailing the role of CRTAM activity in immune cells we hypothesized that adding this specific anchoring domain would enhance nearly every function of CAR modified immune cells. Starting with calcium flux, to granule polarization, to cell-cell avidity, cytokine production, cytotoxicity, invasion, and anti-tumor activity our novel CARs outperformed previous designs. Furthermore, given the universal nature of cell polarity we also demonstrated efficacy in both NK and T cells.

We employed a variety of novel techniques to assess the ‘synapse-tuning’ of our CARs. Most influentially were the avidity measurements that directly interrogated the strength of the synapse. This was further corroborated by the live cell imaging detailing the smaller synapses with a greater calcium influx indicating a more efficient signal. Finally, one of the most striking findings was the single dose efficacy of CAR NK cells in our models. This has never been observed previously, especially with a rechallenge at 4 months post treatment.

Immediate future explorations will involve in vivo tracking of CAR immune cells and super-resolution microscopy of synapse formation to detail more precisely the organization of the synapse and related proteins. Additionally, given the increased efficiency, we posit that the sensitivity to antigen on cancer cells will be enhanced and reduce the chance for immune escape by antigen loss. We are hopeful for the successful translation to the clinic as the pre-clinical data is promising for solid tumor treatments.

Our work is a proof-of-concept study, and the next steps will be to test the novel anchoring domain in diverse CAR designs and tumor models. We believe that an organized synapse will allow for a more optimized CAR and potentially unlock previously shelved ideas. Given the universal nature of cell polarity and the various cell types being exploited for immunotherapeutic use there is broad application to this novel domain. Scribble complexes play pivotal roles in all major components of cellular immunity. Macrophages, Dendritic cells, T, B, and NK cells all utilize Scribble polarity to orchestrate exquisite responses in various scenarios.

We believe that the ever-expanding armamentarium of CAR modified effector cells in the fight against cancer will undoubtedly be enhanced by synapse augmentation via anchor domains.

To see a recorded webinar discussing the paper use this link: https://bit.ly/41y6iep

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