Direct control of CAR T cells through small molecule-regulated antibodies

Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. No statistical methods were used to determine sample size. For calculating statistics in in vitro studies, a minimum of triplicates was chosen to allow for this. For animal studies, 5 mice were included for each treatment group based on other published studies for establishing proof of concept.
No data were excluded.
The replication number and the number of donors tested are indicated in the legend of corresponding figures, where applicable. All attempts at replication were successful. The in vivo study shown was performed once but every effort was made to include critical controls in the study (e.g. untransduced T cell cohort and vehicle treatment cohort).
Animals were randomly divided into experimental groups in such a way that prior to T cell injection, mice in each cohort carried similar average tumor burden. For in vitro experiments, sample were allocated to identical cell-culture wells and there is no reason to believe the spatial location of the sample/well influenced experimental results.
Investigators were not blinded. For animal studies, mice in control and test groups were handled in a consistent manner to prevent study bias, and only quantitative measures were used to make conclusions. Fully blinded experiments were not possible due to personnel availability to accommodate such situations.

Human research participants
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Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
All cell lines were tested for mycoplasma contamination and they were free of mycoplasma contamination.
None of the used cell lines is listed in ICLAC database.
In this study were used 6-8 week old female NSG mice (NOD.Cg-Prkdcscid IL-2R!tm1Wjl/SzJ) obtained from The Jackson Laboratory. The mice were housed in a pathogen-free BSL2 biohazard facility with unrestricted access to water and food. The ambient temperature was restricted to 65 to 75 degrees F with 40-60% humidity. Mice were exposed to a 12:12h light-dark cycle.

This study did not involve wild animals
This study did not involve field-collected samples