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1203 Antigen targeted butyrophilin heterodimer-based bispecific engagers induce Vγ9δ2+ T cell-mediated anti-tumor activity
  1. Derek Franklin,
  2. Anne Lai,
  3. Faraha Brewer,
  4. Arpita Patel,
  5. Kinsley Evans,
  6. Mahmud Hussain,
  7. Louis Gonzalez,
  8. Keith Wilson,
  9. George Fromm,
  10. Taylor Schreiber and
  11. Suresh De Silva
  1. Shattuck Labs, Durham, NC, USA


Background Decreased antigen expression and antigen presentation via major histocompatibility complexes (MHCs) evades αβ T cell recognition. γδ T cells recognize stressed cells in an MHC-independent manner, and consequently, may be exploited to overcome immunotherapy resistance. The butyrophilin (BTN) 2A1/3A1 heterodimer specifically activates Vγ9δ2+ T cells, the predominant subtype in peripheral blood. BTN2A1 directly binds to the Vγ9 chain of the γδ T cell receptor (TCR), but only activates the γδ T cell if phosphoantigen-sensing BTN3A1 forms a heterodimer complex with BTN2A1. To mimic BTN-mediated activation of γδ T cells, we generated bispecific γδ T-cell engagers (GADLEN) containing heterodimeric BTN2A1 and BTN3A1 extracellular domains fused via inert Fc linkers to scFv domains targeting a tumor antigen. We previously reported that in the presence of costimulatory signals from either a cytotoxicity receptor (NKG2D) or T-cell co-stimulatory receptor (CD28), GADLEN compounds activated Vγ9δ2+ T cells to facilitate antigen-dependent tumor cell killing. The specificity of γδ TCR/BTN interactions and dependence upon a secondary co-stimulatory signal suggests that GADLENs could be used to redirect Vγ9δ2+ T cells against hematologic and solid tumors, with a lower risk of off-target activation common with other bispecific engagers. Here, we report the functional characterization of CD20- and B7H3-targeting GADLEN compounds for targeting heme malignancies and solid tumors, respectively.

Methods Specificity of CD20- and B7H3-targeting GADLENs were evaluated using ELISA and cell-based assays by flow cytometry. The functionality of the compounds to activate Vγ9δ2+ T cells and mediate killing of tumor cells was assessed in vitro in tumor co-cultures using flow cytometry and live cell imaging, as well as in vivo in murine xenograft models.

Results CD20- and B7H3-targeting GADLENs bound to human cells expressing CD20 or B7H3 and to Vg9d2+ T cells with low nanomolar affinity. GADLEN compounds activated Vγ9δ2+ T cells in in vitro co-culture assays resulting in degranulation and apoptosis of CD20+ or B7H3+ tumor cells, respectively. Importantly, GADLEN treatments induced the secretion of pro-inflammatory cytokines suggesting the potential of both direct and indirect tumor killing mechanisms via additional immune cell subset activation and recruitment. Introduction of CD20-GADLEN into NSG-hIL15 mice engrafted with human PBMCs efficiently depleted human CD20+ B cells in the blood and spleen. Similarly, coadministration of GADLEN with Vγ9δ2+ T cells reduced tumor growth in tumor xenografts.

Conclusions These results provide proof of concept for in vivo manipulation of γδ T cells using antigen targeted GADLENs for the treatment of hematologic and solid tumor malignancies.

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