PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells.

In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.

Antibody buffering of a ligand in vivo.

Clearance is the practical limit on drug action. Here we propose a means of slowing clearance, thereby extending drug lifetime in vivo by "antibody buffering." In this process, a drug and an anti-drug antibody are coadministered. Most of the drug is bound to the antibody, preventing the drug from acting, but also preventing its elimination. A dynamic free drug pool is established by reversible dissociation from the antibody. The free drug is active and can be eliminated, but the free pool is constantly replenished by reequilibration from the antibody-drug complex, giving a long effective lifetime. Here we explore antibody buffering experimentally by using a model compound, 2-phenyloxazol-5-one-gamma-aminobutyrate (Ox), as a drug proxy. We show that antibody buffering can extend by an order of magnitude the plasma lifetime of Ox in rats, and that the steady-state Ox level depends on the molecular properties of the antibody used to buffer the Ox. In addition, the anti-Ox antibody can be recharged with drug in vivo to extend Ox lifetime without additional antibody administration, making this technique even more suitable for possible clinical application.