1:IDIBAPS; 2:Hospital Clinic de Barcelona

On-target off-tumor toxicity is a major limitation in the development of CAR-T cells for solid tumours. Here, we hypothesized that modifying T-cells with a low-affinity CAR may be a safe therapy for the treatment of patients with Her2-positive breast cancers. CARs that contain a scFv targeting HER2 with low affinity and differ in the transmembrane (TMD) and intracellular domains (ICD) were designed and effectively expressed on the T-cells. The combination of the CD8α TMD with the 41BB ICD conferred the best antitumor effect with long-term persistence. Modifying the TMD of the Her2BBz-CAR strikingly impaired anti-tumour activity, suggesting a key role for the TMD on CAR-T cell function. Because high-affinity Her2-CART cells have induced severe toxicity in patients with cancer, we next compared the low-affinity Her2-BBz with a high affinity CAR. We demonstrated that high-affinity CAR-T cells can react to physiological levels of HER2 when cocultured with a panel of primary cell lines. By contrast, low-affinity Her2-CAR-T cells exhibited no reactivity, as evidenced by low of levels of CD107a expression and the inability to release cytokines and induce cell killing. Finally, using mice implanted with a HER2-low tumour in one flank and a HER2-high tumour in the other flank, we show that, while high-affinity CAR-T cells were able to completely eliminate all tumours, low-affinity Her2-BBz CAR-T cells could eliminate HER2-high tumours while sparing HER2-low tumours.

We conclude that the use of low-affinity Her2BBz-CAR-T cells could be a safe therapeutic option for patients with HER2+ breast cancer tumours.