Englumafusp Alfa (CD19-4-1BBL) and Glofitamab Combination in Patients with Relapsed/Refractory Non-Hodgkin Lymphoma (R/R NHL): Biomarker Results from a Phase I Dose-Escalation Trial

Background: Englumafusp alfa is an antibody-like fusion protein that simultaneously targets CD19 on B cells and 4-1BB on T cells and other immune cells. In the presence of a T-cell receptor signal and strictly dependent on CD19 crosslinking, englumafusp alfa co-stimulates T cells via 4-1BB agonism that boosts T-cell effector functions and prevents T-cell anergy. An ongoing Phase I dose-escalation study (NCT04077723) is investigating the safety, efficacy, pharmacokinetics, and pharmacodynamics (PD) of intravenous administration of englumafusp alfa in combination with glofitamab in patients (pts) with R/R NHL. We recently presented preliminary clinical data demonstrating promising clinical activity and good tolerability in R/R NHL (Hutchings et al, ICML 2023). Here, we report preliminary peripheral blood (PB) and tissue biomarker analyses to demonstrate mechanisms of action (MoA), dose relationship, and baseline features associated with response.

Methods: Exploratory biomarker analyses included data from 84 pts with indolent or aggressive NHL (aNHL) dosed with 0.36–75mg englumafusp alfa, starting on Cycle 2 Day 8 (C2D8) and followed by combination with the fixed target dose of glofitamab (30mg), once every 3 weeks, from C3D1 for up to 12 cycles. Immune profiling of PB by flow cytometry and plasma cytokines analysis by ELLA were performed. Circulating tumor DNA (ctDNA) dynamics were evaluated by the Avenio NHL CAPP-Seq assay (Stokowski et al, ASH 2022) at C3D1, C5D1, and the end of 12-cycle treatment (EoT) in patients with aNHL. Baseline tumor biopsies (n=65) were analyzed by immunohistochemistry/immunofluorescence assays. On-treatment (OT) PD changes during the first five cycles of treatment were evaluated and compared to historical glofitamab monotherapy PD data generated in a phase I/II study (NCT03075696).

Results: Englumafusp alfa in combination with glofitamab induced PD changes in PB at all tested doses. Specifically, in a dose-dependent manner, englumafusp alfa in combination with glofitamab limited the PB expansion of fully differentiated PD1+ CD8+ effector memory T cells re-expressing CD45RA (Temra), previously shown to expand dose-dependently following glofitamab monotherapy (Broeske et al, Blood Advances 2022). Additionally, the combination also resulted in a significant PB expansion of the activated (HLA-DR+) and effector memory CD8+ T (T_EM) cells, and the ratio of T_EM to central memory and naïve CD8+ T cells compared to glofitamab monotherapy. Furthermore, we detected no significantly added cytokine release with the combination to that previously observed with glofitamab monotherapy, in line with the clinically observed safety profile. Preliminary analysis of ctDNA dynamics showed that at C3D1 33% (18/55), at C5D1 40% (15/38), and at EoT 53% (9/17) of the pts had no detectable ctDNA. Notably, 61% of pts with aNHL who achieved complete metabolic response (CMR) had no detectable ctDNA at any OT visit. Our preliminary analysis also indicated a significant (p=0.03) association between the observed OT expansion of CD8+ T_EM cells and the extent of decrease in ctDNA levels (mean mutant molecules per milliliter) at EoT relative to baseline. Finally, we observed ~60% of pts with low tumor infiltrating CD8+ T cells (<500 cells/mm² CD8+) and a more aggressive disease (>5000 cells/mm² proliferative (Ki67+) tumor cells) at baseline, both poor outcome correlates of glofitamab monotherapy, achieved a CMR with higher doses of englumafusp alfa.

Conclusions: In this study, we demonstrated the MoA of englumafusp alfa in R/R NHL and key PD effects in combination versus glofitamab monotherapy that will support optimal biological dose finding. Furthermore, the observed association between expansion of T_EM cells in PB and changes in ctDNA dynamics at EoT, albeit preliminary, supports ctDNA as a marker for depth and durability of response. Overall, our PD and biomarker observations so far strengthen the rationale of combining glofitamab with englumafusp alfa to drive long-term responses in this heavily pre-treated NHL patient population.

Acknowledgments: This study was sponsored by F. Hoffmann-La Roche Ltd. The authors would like to thank the patients who participated in this trial, their families, and their caregivers. Special thanks also to Daria Rukina, Iva Lelios, Philip Knuckles, Kat Reyskens, Grigori Singovski, and Joanne Hayward for their contributions to this work.