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Since first approved by the FDA in 2017, T cells with chimeric antigen receptors (CAR T) have revolutionized the treatment of blood cancers like leukemia and lymphoma. Since then, research has been focused on improving the CARs to treat a wide range of cancers including solid tumors – that have proved to be more notorious to combat than blood cancers. In a study recently published in Nature Biotechnology, researchers attempt to upgrade the inherent cancer-killing ability of natural killer (NK) cells by optimizing the signaling at the immunological synapse. “Synapse” is a term frequently used in neuroscience, but what is an immunological synapse?
Immune cells can interact with one another and other cells of the body (like cancer cells) through a synapse – the region where cell resources (signaling molecules, secretory vesicles, cytoskeleton, etc.) become concentrated to facilitate the interaction. Some examples involving immunological synapse include but are not limited to, antigen-presenting cell priming a T cell, T and B cell interactions, and NK and cancer cell interactions. When NK cells detect abnormal cells that need to be lysed, they tether to the cell through adhesion molecules which initiate signals to activate multiple pathways. This activation massively changes the cytoskeleton and attracts surface receptor molecules that together form the immunological synapse around the point of adherence. Lytic granules also accumulate, which will ultimately be released onto the abnormal cell to destroy it. This is an elaborate process that is strongly regulated and controlled to avoid damage to healthy cells.
One of the reasons for the failure of CAR therapeutics against solid tumors has been attributed to poor synapse formation in between the T and NK cells. The absence of proper immunological synapse weakens the rate of signaling, and in turn, dims the activation of cells.
The researchers in the aforementioned paper hypothesized that if immunological synapse formation is improved, the NK cells could be activated more effectively. To test this, they first selected CD28z as the chimeric antigen receptor. This CAR targets the tumor-associated antigen: B7-H3, found on several solid tumors. They then modified its intracellular terminus by adding a domain called PDZbm. This domain is found across 400 proteins and has been described to help with cell polarization and synapse formations in immune cells.
From the in vitro experiments on B7-H3 coated slides and cancer cell lines, they found that CAR with the PDZbm domain enhanced not only the NK cell polarity and signaling at the synapse, but also the cell avidity (the strength of interaction or binding). Surprisingly, both CAR-only and non-CAR controls had similar binding strengths that were much lower than CAR-PDZbm, which further emphasizes the need for an improved immunological synapse in CAR cells. The researchers also noted increased calcium flux in NK cells with CAR-PDZbm, pointing towards a much stronger cell activation than controls. The increased rate of activation was accompanied by higher production of IFNγ (a key pro-inflammatory cytokine) and perforin (pore-forming glycoprotein) by the CAR-PDZbm NK cells.
The researchers then tested the CAR-PDZbm on 2D and 3D cultures, followed by mice models with adenocarcinomas and osteosarcomas. Studying these cells in 3D cultures shed light on the increased migratory response of CAR-PDZbm NK cells and higher invasion into tumors as compared to the controls. Moreover, all models of mice showed generally higher survivability in mice compared to controls. Lastly, when mice treated with CAR-PDZbm NK cells were rechallenged with cancer cells four months later, the mice rejected tumors within an average of 14 days, proving the long-term survivability and effectiveness of these cells. In summary, enhancing the immunological synapse largely improved the activity and effector functionality of CAR-NK cells. Like NK cells, when CARs of T cells were modified with the PDZbm domain, a similar ameliorated phenotype across in vitro and in vivo experiments was observed against solid tumors.
Generating CAR NK cells that persist when added back to the patient after in vitro treatments has been a challenge in using them as therapeutics. NK cells used in experiments here were derived from peripheral blood, and sustained in culture through low doses of IL2, instead of dependence on heavy cytokine dosing. Although detailed studies of how PDZbm-modified NK & T cells would behave in vivo need to be performed, the authors showed increased polarization, activation, and migration – hence paving the way to improving CAR therapies through modulation of immunological synapses.
Chockley PJ, Ibanez-Vega J, Krenciute G, Talbot LJ, Gottschalk S. Synapse-tuned CARs enhance immune cell anti-tumor activity. Nat Biotechnol. 2023 Feb 2. doi: 10.1038/s41587-022-01650-2.
Article author: Kevin Merchant. Kevin is a PhD student at Helmholtz Munich, working at the intersection of computational biology and drug development against Idiopathic pulmonary fibrosis. He aims to simplify latest research so that it becomes accessible to all.
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