Background: Systemic lupus erythematosus (SLE) and other B cell mediated autoimmune diseases are primarily characterized by dysregulated B-cell tolerance and pathogenic autoantibody productions, leading to progressive multi-organ damage. Conventional B-cell depleting agents, such as Rituximab, often fail to induce deep and durable remission. The widespread application of T-cell engagers (TCE) in autoimmune diseases is constrained by their narrow therapeutic index, driven by safety risks like cytokine release syndrome (CRS), as well as short half-lives that necessitate frequent administration. Although CD19-targeted CAR-T cell therapies can achieve deep B-cell depletion in organs, their broad clinical application is hindered by complex logistics, prohibitive costs and safety concerns. To address these unmet therapeutic needs, we developed CircMab, a novel platform levering Therorna’s proprietary circular RNA (circRNA) technology. By delivering LNP-encapsulated circRNA encoding CD19xCD3 bispecifics, this approach enables sustained, off-the-shelf in vivo production of TCEs. The unique closed-loop structure of circRNA confers resistance to exonucleases, thus allowing for robust and sustained antibody production compared to the linear mRNA. Furthermore, the continuous in-body production maintains steady therapeutic levels, avoiding the high initial peak concentration associated with traditional TCE dosing that often triggers CRS. This innovative circRNA-based TCE represents a promising alternative for the treatment of B cell driven disorders.

Objectives: To evaluate in vitro potency, in vivo efficacy, pharmacokinetic (PK) and safety profile of the CD19xCD3 circMab.

Methods: We leveraged a proprietary AI algorithm to design circRNA constructs. Following LNP encapsulation and transfection into HEK 293 T cells, the in vitro functionality of the secreted circMab was assessed using culture supernatant. We thoroughly characterized the functional profile of the secreted circMab, including its binding affinity, capacity for T-cell activation and proliferation, and cytotoxic potency against aberrant B cells. The in vivo pharmacokinetic (PK) profile was evaluated in the immunocompetent mice via administration of LNP-circRNA directly. Subsequently, efficacy against B-cell malignancies was assessed in humanized NOG-mice bearing Nalm-6-luc tumors. Finally, we utilized a non-human primate model to evaluate the PK, therapeutic efficacy and safety profile in a clinically relevant setting.

Results: in vitro profiling of circMab confirmed the picomolar binding affinity to both CD3 on T cells and CD19 on B cells, mediating robust T-cell activation, proliferation, and potent cytotoxicity. Notably, circMab demonstrated picomolar EC ₅₀ against autologous B cells in PMBCs derived from both healthy donors and patients with SLE. In a murine model, circMab demonstrated a superior PK profile compared with Blinatumomab, characterized by a higher AUC and a lower C _max . In the humanized tumor model, a single sub-microgram dose of LNP-circRNA achieved complete and sustained eradication of Nalm-6 tumors. In contrast, an equivalent dose of Blinatumomab only marginally delayed tumor progress compared to the PBS control. Following a single-dose administration of LNP-circRNA in cynomolgus monkey, circMab was rapidly expressed in serum within six hours. It reached C _max at 24 hours, then gradually declined, with sustained presence for over one month. Consistently, peripheral B cells were depleted to undetectable levels within 24 hours and sustained for one month, followed by recovery evidenced by the B cell subset repopulation. Importantly, no significant elevation in liver enzymes (ALT/AST) was observed. Cytokine release (IL-2, IL-6 and MCP-1) was transient and minimal, peaking at 6 hours and resolving by 24 hours without clinical signs of CRS or neurotoxicity.

Conclusions: Our study validated the translational potential of circMab, demonstrating distinct PK and efficacy profile compared with conventional TCEs. A single dose of LNP-circRNA injection resulted in sustained circMab production and profound B cell depletion in both murine and NHP models. Collectively, the results support further clinical development of circMab as a transformative therapeutic for both B cell malignancies and autoimmune diseases.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.