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.

No relevant conflicts of interest to declare.