Prevention of Antigen Escape By Modulation of Off-Target Tumor Killing in T Cells

CD19 antigen (Ag) escape after CAR-T therapy is common in B-ALL, accounting for > 40% of relapsing patients in early studies. Ag escape after CAR-T therapy is also well-described amongst patients with lymphoma as well as myeloma and glioblastoma. Following bsAb (BiTE) therapy, CD19 Ag escape occurs in >30% of relapsing B-ALL patients. CD20 Ag escape has been described even with the ‘gentle’ selective pressure of rituximab therapy in FL and other B-NHL. Taken that into account, it is not surprising that early data from trials of CD3xCD20 bsAb- a more effective therapy applying more potent selective pressure– have already revealed > 60% of relapsing patients with tumoral CD20 loss. Instead of attempting to treat Ag escape, we recently suggested an innovative approach to prevent it by utilizing geographically restricted Fas-dependent bystander killing. In our previous work, we have demonstrated the innate ability of T cells and CAR-T to target Ag^- negative cells through the process of bystander killing.

We hypothesized that Ag^-/lo (CD19^-/lo or CD20^-/lo) tumor cells which cause relapse, might be present at low levels prior to therapy, though these would be impossible to evaluate from paraffin-embedded biopsies. We used an exceptional resource- cryopreserved single-cells suspension biopsies to screen 20 biopsy samples and revealed the presence of rare (0.1% - 2.5%) CD19^-/lo and/or CD20^-/lo negative cells in all B-NHL biopsy samples prior to any therapy. These cells may be impervious to targeted therapy and drive relapse. When tested in a killing assay with autologous T cells and anti-CD19 (blinatumomab) or CD20 (epcoritamab) bsAbs, only Ag^-/lo target cells selectively evaded killing by the respective therapy. We then quantified the number of CD19 and CD20 molecules on target cells, revealing Ag thresholds for evasion of bsAbs-mediated killing. Interestingly, we also observed lesser magnitude, selective, non-targeted Ag loss e.g. CD19 loss with CD20 bsAbs but no CD20 loss with CD19 bsAb, apparently due to T-cell trogocytosis. Moreover, we managed to potentiate bystander killing of the Ag^-/lo targets, by utilizing a SMAC-mimetic cIAP1/xIAP inhibitor, revealed in our small molecule screen of Fas-induced apoptosis regulators.

Our next goal was to characterize rare Ag^-/lo (CD19^–/CD20^–) lymphoma cells from biopsies using scRNAseq with focus on targetable Ag (e.g. CD79b, CD22, CD74) and Fas-regulators (e.g. Bcl2, IAP, HDAC family members). To enrich these rare cells (cca. 0.1% of all B cells), we flow-sorted and remixed the Ag^-/lo and Ag⁺ lymphoma cells in near-to-equal ratios prior to downstream transcriptome analysis. Our analysis confirmed the existence of unique transcriptomic signatures of CD20^-/lo lymphoma cells and discrepancies between CD20 protein and its mRNA. Furthermore, despite heterogenous transcriptome signatures typical for tumors, CD19^-/lo and/or CD20^-/lo Ag cells respectively form distinct cell clusters. The lead targetable mRNA candidates found exclusively in Ag^-/lo cells in our preliminary screen are being functionally validated.

Lastly, we provide a comprehensive overview of pre-therapy hallmarks of Ag^-/lo tumor cells. These transcriptomic features may reveal the utility or futility of current combination strategies, e.g. if CD20^- FL cells exhibit concurrent loss of multiple B cell antigens as has been observed in B-ALL, then current strategies (e.g. mosunetuzumab plus polatuzumab vedotin) may be ineffective. Therefore, characterization of the ‘escaping’ tumor cells is critical for predicting optimal partner therapies.