Abstract 5589: <b> A biparatopic DLL3-targeting trispecific T-cell engager delivered by mRNA-LNP drives potent anti-tumor activity <i>in vitro</i> and <i>in vivo</i> </b>

Abstract Background: T-cell engagers (TCEs) targeting DLL3 show promising clinical activity in small cell lung cancer (SCLC), yet response rates, durability, and toxicities such as cytokine release syndrome (CRS) remain challenges. Because DLL3 is a low-abundance antigen, increased avidity can improve tumor cell killing. We developed an mRNA-lipid nanoparticle (mRNA-LNP) platform enabling sustained in vivo expression of a DLL3-targeting TCE, using the pharmacokinetic profile of mRNA to reduce CRS risk. To enhance penetration and avidity, we incorporated compact camelid VHH domains and a biparatopic design that engages two distinct DLL3 epitopes. Methods: A trispecific T-cell engager was engineered by fusing two DLL3-binding VHHs, each recognizing non-overlapping epitopes, with a CD3 agonist domain via flexible peptide linkers. The mRNA expression was optimized using codon engineering to balance Codon Adaptation Index (CAI) and Minimal Free Energy (MFE) across the full transcript, including untranslated regions (UTRs) and the coding sequence (CDS). The optimized mRNA was formulated into a novel LNP delivery system to generate the therapeutic candidate MTS108. Cytotoxic activity was assessed using T cell-dependent cellular cytotoxicity (TDCC) assays with human PBMCs co-cultured with DLL3-expressing SHP-77 or MC38-DLL3 target cells. In vivo efficacy was evaluated in both subcutaneous and orthotopic lung tumor models, including a humanized CD3ε transgenic syngeneic model and a human PBMC-reconstituted cell-derived xenograft (CDX) model. Results: In TDCC assays, MTS108 (mRNA-LNP) demonstrated potent tumor-killing activity against SHP-77 (low DLL3) and MC38-DLL3 (high DLL3) target cells. Against SHP-77 cells, MTS108 exhibited an EC50 more than 10-fold lower than that of the approved therapeutic Xaluritamig. It also showed significantly enhanced cytotoxicity against MC38-DLL3 cells relative to Xaluritamig. In vivo, MTS108 consistently and significantly outperformed the benchmark agent across both subcutaneous and orthotopic tumor models. Optimized mRNA design enabled robust expression in vitro and in mice, and both cell-culture supernatants and mouse serum containing mRNA-expressed TCEs displayed tumor-killing potency comparable to recombinant protein. In the subcutaneous MC38-DLL3 syngeneic model and the orthotopic SHP-77 lung cancer model, MTS108 achieved superior anti-tumor efficacy with an improved safety profile. Conclusion: These data demonstrate that an mRNA-LNP platform can effectively deliver a biparatopic DLL3-targeting trispecific TCE with potent and superior anti-tumor activity and a favorable safety profile. MTS108 represents a promising therapeutic candidate and supports advancement of mRNA-encoded TCEs toward clinical evaluation. Citation Format: Xue Qiao, Qiang Zhang, Lushuai Jin, Yao Lv, Shaoli Liu, Xiaoju Zhang, Xiaoyun Ma, Hongya Han, Wei Xu. A biparatopic DLL3-targeting trispecific T-cell engager delivered by mRNA-LNP drives potent anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5589.