4-1BB-Costimulated CAR-Mediated Non-Canonical NF-Kb Signaling Enhances CAR T Cell Survival and Suppresses Bim Expression

Chimeric Antigen Receptor (CAR) T cell therapy induces deep and durable responses in a large percentage of patients with B-cell malignancies. These responses often correlate with CAR T cell persistence in patients. The first two FDA-approved CAR-T cell therapies employ 2^nd generation CARs that use different costimulatory domains derived from either CD28 or 4-1BB. Data from pre-clinical studies as well as clinical trials suggest that 4-1BB CAR (BBz) T cells persist longer than CD28 CAR (28z) T cells.

One signal associated with cellular survival and activated by endogenous 4-1BB, but not CD28, is the non-canonical NF-kB (ncNF-kB) pathway. NcNF-kB is required for memory T cell persistence and enhances tumor cell survival by suppressing pro-apoptotic gene transcription. Therefore, we hypothesize that the BBz, but not the 28z CAR, activates ncNF-kB signaling, which promotes CAR T cell persistence by suppressing pro-apoptotic gene expression.

Using primary human T cells isolated from the apheresis products of anonymous healthy donors, we generated 28z or BBz T cells by lentiviral transduction following activation by CD3 and CD28. Induction of ncNF-kB signaling following CAR activation was assessed by western blot. NcNF-kB function was evaluated by concomitant expression of a dominant-negative mutant of NF-kB-inducing Kinase (dnNIK) to block the ncNF-kB pathway. In addition to pathway analysis by western blotting, T cell proliferation, immunophenotype and survival were assessed by bead-based counting via flow cytometry. To explore mechanisms affecting survival, pro- and anti-apoptotic gene expression was analyzed by qPCR and western blot over three weeks following restimulation through the CAR.

Following CAR activation, ncNF-kB signaling was detected in BBz but not 28z T cell lysates. Control mRFP BBz T cells expanded approximately 10 fold more than BBz T cells coexpressing dnNIK. DnNIK-expressing BBz T cells exhibited higher rates of cell death contributing to the observed differences apparent in ex vivo expansion. This cell death was associated with a 2 fold increase in the message and nearly 3 fold increase in the expression of the pro-apoptotic protein, Bim, in the dnNIK BBz T cells relative to control. In contrast, dnNIK 28z T cells had no appreciable difference in proliferation or survival compared with control.

Therefore, BBz, but not 28z signaling activates the ncNF-kB pathway, which protects BBz T cells from cell death likely by restricting the expression of the pro-apoptotic protein, Bim.