Understanding and targeting rare Ag–(CD19/CD20–) Follicular Lymphoma cells to prevent post-immunotherapy antigen escape

Follicular lymphoma (FL) is frequently and, perhaps wrongly, referred to as ‘incurable’; FL has been generally incurable with historical therapies.  Newer, T-cell mediated immunotherapies e.g. CAR-T cells and bispecific Abs (bsAbs) induce complete remission in a majority of FL patients suggesting that, if we can understand and address post-immunotherapy relapse, then current goal of FL therapy should be durable remission or cure.  

     Unfortunately, T cell therapies share a common limitation: tumoral antigen (Ag) escape; Ag– cells in heterogeneous tumors evade attack and lead to relapse. After anti-CD19 (aCD19) CAR-T therapy >40% of relapsing patients have CD19– tumors. Even more sobering, early data from trials of CD3xCD20 bsAbs have shown >60% of relapsing patients have CD20– tumors. One solution to treat Ag escape –re-targeting a distinct Ag (e.g. aCD22 CAR-T)– may be limited by i) concurrent loss of multiple tumor Ag and ii) lack of tumor-specific, homogeneously expressed Ag. 

     Instead of attempting to treat Ag escape, we recently proposed an innovative approach to prevent it, by enhancing geographically-localized T cell ‘Bystander Killing’. Specifically, we identified the critical role of a ‘Death Receptor’ gene called ‘Fas’ as mediating most Bystander Killing. We validated this finding in mouse and human models of CAR-T and bsAb therapies. We provided evidence from a large aCD19 CAR-T clinical trial that constitutive Bystander Killing may already be, partly, preventing Ag escape, by demonstrating that tumoral Fas-expression is an even greater predictor of survival than CD19-expression itself. Notably, we showed that inhibiting Fas regulators e.g. Bcl2, IAP, and HDAC family members, enhanced Bystander Killing. Thus, we hypothesize that:

1. rare Ag(CD19 or CD20)- FL cells exist prior to immunotherapy, cause Ag escape, and express some  proteins (‘regulators’) which prevent Fas-signaling, and
2. Ag- FL cells that avoid CAR-T/bsAb On-Target Killing are targetable by Fas-Mediated Bystander Killing which can be enhanced by inhibiting the above Fas-regulators. 

Aim 1: Characterize rare Ag- primary FL cells in preserved biopsies from prior trials, using a cutting edge technique called scRNAseq which analyzes the genes in each individual FL cell 

Aim 2: Demonstrate that bsAb-activated T cells can kill Ag- FL cells and that this Bystander Killing depends on the Fas gene

Aim 3: Determine whether inhibiting the regulators of Fas can increase Fas-Killing and Bystander Killing of Ag+ and Ag- FL cells

In the near future we also plan to characterize CD20- FL cells and intratumoral immune cells during CD3xCD20 bsAb therapy from serially obtained biopsies, in a new trial for FL patients (planned for Q3 2025) to assess enrichment of Ag– FL cells and of the Ag– FL cell-associated transcripts  defined in our pre-clinical studies herein to further validate the actionable targets.