Potent CD8+ T-Cell Proliferation and Effector Differentiation Following Subcutaneous (SC) Mosunetuzumab Administration in Patients with Untreated, High Tumor-Burden Follicular Lymphoma (FL)
Authors: Falchi Lorenzo , Lin Ya-Hui , Melendez Lauren , Ghione Paola , Owens Colette , Hamlin Paul A. , Lue Jennifer Kimberly , Epstein-Peterson Zachary D. , Kumar Anita , Palomba Maria Lia , Torka Pallawi , Okwali Michelle , Ferreira Lopes Alexandra , Martinova Anastasia , Wood Lauren , Grieve Clare , Ramos Amador Walter , Zelenetz Andrew D. , Salles Gilles , Vardhana Santosha A 
Published: December 2023

Introduction:
Bispecific antibodies (BsAb) have emerged as a potent therapeutic option for patients with B-cell non-Hodgkin lymphoma, including FL. These drugs leverage endogenous CD3+ T-cells to enable cytotoxic tumor cell killing. While their mode of action has been hypothesized in in vitro and animal models, the mechanisms by which these drugs promote both initial and long-term immune-dependent disease control remains unknown. We recently initiated a multicenter phase 2 trial of the CD20xCD3 BsAb mosunetuzumab (mosun) as first-line therapy for patients with high-burden FL (NCT05389293). As part of this study, we performed serial sampling of both blood and lymph node tissues in order to elucidate T-cell changes associated with response and outcome. Here we present the first analysis of peripheral blood immune profiling.
Methods:
Patients with untreated, high-burden stage 2-4 FL in need of treatment received SC mosun at the dose of 5 mg on D1 and 45 mg on D8 and D15 of C1, and 45 mg on D1 of each subsequent 21-day cycle. Peripheral blood samples were obtained on C1D1, C1D8, C1D15, C2D1, C3D1, C5D1, and end of treatment (EOT, 24w from C1D1). Peripheral blood mononuclear cells (PBMCs) and plasma were isolated and cryopreserved. Samples were later thawed in batches and processed as follows: CITE-Seq + single-cell TCR sequencing was performed on viable CD11b-/CD19-/CD56-/CD3+ T-cells using the 10x Genomics Single Cell Immune Profiling Platform. RNA was extracted from 1 x 10 6 viable PBMCs using TriZol, followed by phenol/chloroform-based extraction, followed by bulk TCR sequencing using an in-house platform developed by MSKCC's Integrated Genomics Operation. The remaining cells were stained with antibodies targeting CD45RA, CD8, CD45, CD45RO, CD38, CD27, CD28, CXCR3, LAG-3, CD3, CD4, HLA-DR, CXCR5, CCR6, CD56, CD11b, CD19, CD39, PD-1, OX40, Tim-3, CCR4, Granzyme B, T-bet, FoxP3, Ki-67, and CTLA-4 and analyzed using a Cytek Aurora Spectral Cytometer. Flow cytometry analysis was performed using OMIQ. Statistical analyses were performed using a paired t-test.
Results:
A total of 45 samples obtained from 8 patients were analyzed, including samples from C1D1 (8), C1D8 (6), C1D15 (3), C2D1 (7), C3D1 (8), C5D1 (6), and EOT (7). High dimensional clustering of spectral cytometry data using the FlowSOM algorithm revealed 9 clusters of CD8+ T-cells corresponding to known CD8+ T-cell subsets. Among these subsets, we observed two distinct temporal patterns of CD8+ T-cells within the blood. On C1D8 there was a profound drop in circulating effector CD8+ T-cells as well as terminally differentiated CD8+ Temra cells (Figure 1), with a corresponding enrichment of naïve CD8+ T-cells (Figure). We also observed significant proliferation of newly activated T-cells on C1D8, followed by expansion of CD8+ T-cells expressing several activation and exhaustion markers including PD-1, Tim-3, and CD39, peaking on C2D1 (Figure 2). CD4+ T-cell responses were more modest with a small early increase in CD28+OX40+PD-1+ CD4+ T-cells on C1D8 and a drop in circulating GzmB+OX40+ CD4+ T-cells on C1D15.
Conclusion:
SC mosun therapy for newly diagnosed FL was associated with a unique peripheral blood immune profile, with early mobilization of pre-differentiated CD8+ T-cell effector cells and subsequent activation and expansion of newly primed CD8+ T-cells. Further immune phenotyping, including scRNA-seq and TCR clonal dynamics in the peripheral blood over time, as well as TCR clonality of distinct CD8+ and CD4+ T-cell populations are currently being analyzed on these and additional samples, and will be presented in detail.