Alcantara, M. | Search bioRxiv

bioRxiv2026

Specific killing of Ewing sarcoma by TCR-T cells targeting public neogene-encoded antigens

EWSR1::FLI1, the oncogenic chimeric transcription factor driving Ewing sarcoma (EwS)induces expression of exquisitely EwS-specific neogenes (Ew_NGs) through neomorphic binding and transcription activation at GGAA microsatellites in genomic regions that are silent in normal tissues. We show that peptides encoded by Ew_NGs are presented on HLA-I complexes on EwS cells. The cytokine secretion of CD8+ T cells specific for Ew_NG-encoded HLA-I-bound peptides is activated by all HLA-I-matched EwS cells but not by non-EwS cells. These T cells kill EwS cells in an HLA-I restricted manner. This cytotoxicity is dependent on the expression of EWSR1::FLI1 and of the corresponding Ew_NG. It can be reproduced by transduction of the TCR into donor T cells (TCR-T) which kill EwS cells in vivo. Moreover, we show that neither off target nor allogeneic activation are observed with TCR-T thus paving the way for cell therapy in relapsed/resistant EwS patients for which therapeutic options are very limited. Statement of significanceThe chimeric transcription factor EWSR1::FLI1 generates tumor-specific neogenes encoding neoantigen presented by the HLA-I molecules of Ewing cells. Neoantigen-specific CD8+ T-cell clones and engineered TCR-T cells can selectively recognize and kill EwS tumor cells in vitro and in vivo.

immunology↗

bioRxiv2023

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3

Decoy-oligodeoxynucleotides (D-ODNs) can target undruggable transcription factors, such as STAT3. However, challenges in D-ODN delivery and potency hampered their translation. To overcome these limitations, we conjugated STAT3-specific D-ODN to thalidomide (Tha), a known ligand to cereblon (CRBN, a component of E3 ubiquitin ligase) to generate a proteolysis-targeting chimera (STAT3DPROTAC). STAT3DPROTAC downregulated STAT3, but not STAT1 or STAT5, in target cells. Computational modeling of the STAT3DPROTAC ternary complex predicted two surface lysines on STAT3, K601 and K626 as potential ubiquitination sites for the PROTAC bound E3 ligase. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibitors, and CRBN deletion alleviated STAT3DPROTAC effect. Next, we conjugated STAT3DPROTAC to a CpG ligand targeting Toll-like receptor 9 (TLR9) to generate myeloid/B-cell-selective C-STAT3DPROTAC conjugate. Naked C-STAT3DPROTAC was spontaneously internalized by TLR9+ myeloid cells, B cells as well as human Ly18 and mouse A20 lymphoma cells, but not by T cells. C-STAT3DPROTAC decreased STAT3 levels to 50% at 250 nM and over 85% at 2 {micro}M dosing in myeloid cells. We also observed significantly improved downregulation of STAT3 target genes involved in lymphoma cell proliferation and/or survival (BCL2L1, CCND2, MYC). Finally, we assessed the antitumor efficacy of C-STAT3DPROTAC compared to C-STAT3D or scrambled control (C-SCR) against human lymphoma xenotransplants. Local C-STAT3DPROTAC administration triggered lymphoma regression while control treatments had limited effects. Our results underscore feasibility of using PROTAC strategy for cell-selective, decoy oligonucleotide-based targeting of STAT3 and potentially other tumorigenic transcription factors for cancer therapy.

cancer biology↗