Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function

T cells engineered to express chimeric antigen receptors (CAR-T cells) have shown impressive clinical efficacy in the treatment of B cell malignancies. However, the development of CAR-T cell therapies for solid tumors is hampered by the lack of truly tumor-specific antigens and poor control over T cell activity. Here we present an avidity-controlled CAR (AvidCAR) platform with inducible and logic control functions. The key is the combination of (i) an improved CAR design which enables controlled CAR dimerization and (ii) a significant reduction of antigen-binding affinities to introduce dependence on bivalent interaction, i.e. avidity. The potential and versatility of the AvidCAR platform is exemplified by designing ON-switch CARs, which can be regulated with a clinically applied drug, and AND-gate CARs specifically recognizing combinations of two antigens. Thus, we expect that AvidCARs will be a highly valuable platform for the development of controllable CAR therapies with improved tumor specificity.


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No data were excluded from the analysis.
The replication number is indicated in the legend of corresponding figures where applicable. All attempts at replication were successful.
For the in vivo tumor model in Figure 6d, equal mean tumor burden was achieved by the administration of a 1:1:1:1 mixture of Nalm-6, hEGFRt-Nalm-6, hHER2t-Nalm-6 and hHER2t-hEGFRt-Nalm-6 cells. For the in vivo tumor models in Figure 5c, mice were randomized to ensure equal mean tumor burden before T cell transfer. For the in vivo tumor model in Figure 5d, no randomization was performed due to technical limitations.
No blinding was performed. Objective assessment of the well-being of mice was achieved by the usage of stringent score sheets and the evaluation by one trained person.

Human research participants
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All cell lines tested negative for mycoplasma.
No commonly misidentified cell line was used.
This study did not involve wild animals.
This study did not involve samples collected in the field. Buffy coats from anonymous healthy donors were purchased from the Red Cross, Vienna. The researchers were blind to any covariate characteristics.
There was no recruitment of participants. Buffy coats were purchased from the Red Cross, Vienna, as described.