Abstract 1815: Rapid serial killing of target cells by Vγ9Vδ2 T Cells in cynomolgus macaques and humanized mice treated with a CD20-directed heterodimeric butyrophilin 2A1/3A1 fusion protein
Authors: Lai Anne Y. , Gonzalez Louis E. , Murr Noah , Lenz Karen , Franklin Derek , Campbell Kristen , Brewer Faraha , Patel Arpita , Evans Kinsley , Hussein Mahmud , Wilson Keith , Fromm George , Schreiber Taylor H. , de Silva Suresh 
Published: April 2023

Abstract
CD3-directed T cell engagers provide clinical benefit in a variety of hematologic cancers, but it remains unclear whether γδ T cells (GDT) are present at sufficient numbers to be similarly harnessed because they represent only 1-5% of the total T cell pool. The Vγ9Vδ2 T cell receptor is activated by a butyrophilin 2A1 and 3A1 (2A1/3A1) heterodimer as “signal 1”, together with costimulatory signaling through CD28 or NKG2D as “signal 2”. Here we evaluated the ability of a GDT engager (GADLEN) comprising the extracellular domains of 2A1/3A1 adjoined via an Fc to an antibody fragment targeting the CD20 antigen, to mediate target-cell depletion in vivo at physiologically relevant frequencies of GDT. A humanized mouse model was developed wherein human peripheral blood mononuclear cells (PBMC) were engrafted to NSG-IL15 mice and a high degree of human chimerism persisted for over 3 weeks, including a circulating pool of human GDT comprising 0.5 to 3% of total T cells. Across different human donors, the GDT effector to target B cell ratio varied from 1:1 to 1:46. Following treatment with a B cell-targeted GADLEN, rapid and dose-dependent depletion of over 95% of human B cells was achieved in the peripheral blood and spleen, even at E:T ratios of 1:46. Based on these data, a repeat-dose toxicology study was conducted in cynomolgus macaques across a dose range of 0.1 to 25 mg/kg with the CD20-targeted GADLEN. This study also demonstrated dose-dependent B cell depletion within 2 hours of infusion. Durable receptor occupancy was observed on CD20+ cells for over 7 days, whereas transient occupancy was observed on circulating cynomolgus Vγ9Vδ2 T cells. GDT represented approximately 1-3% of total T cells in cynomolgus macaques, and E:T ratios ranged from 1:7 to 1:49. All treatments were well tolerated, and no evidence of cytokine release syndrome nor any other toxicities were observed. Interestingly, in both humanized mice and non-human primates, the proliferation of GDT was not required to achieve B cell depletion. In fact, over several weeks, the circulating frequencies of cynomolgus Vγ9Vδ2 T cells remained stable, and no evidence of either proliferation or migration out of the blood was observed. These observations, together with the speed at which B cell depletion occurred, suggest that the Vγ9Vδ2 T cells eliminated the B cell pool by serial killing following treatment with the GADLEN. Despite a GDT frequency of less than 5% of total T cells in all these studies, Vγ9Vδ2 T cells achieved rapid depletion of target cells following treatment with the GADLEN, without evidence of toxicity, even at high doses. Collectively, these results indicate that low frequencies of GDT can be harnessed to achieve similar target-cell killing potential as the broader CD3+ T cell pool but with potentially less toxicity.
Citation Format: Anne Y. Lai, Louis E. Gonzalez, Noah Murr, Karen Lenz, Derek Franklin, Kristen Campbell, Faraha Brewer, Arpita Patel, Kinsley Evans, Mahmud Hussein, Keith Wilson, George Fromm, Taylor H. Schreiber, Suresh de Silva. Rapid serial killing of target cells by Vγ9Vδ2 T Cells in cynomolgus macaques and humanized mice treated with a CD20-directed heterodimeric butyrophilin 2A1/3A1 fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1815.