Background: B cell depletion therapy (BCDT), such as monoclonal antibodies, bispecific T cell engagers and CAR-T, has an extensive history of effectively treating B cell malignancies. Certain BCDT monoclonal antibodies, e.g. rituximab, are also approved to treat several autoimmune diseases [1], however demonstrate poor B cells depletion in deep tissue which may limit the long-term disease control or remission in patients. Recently, anti-CD19 CAR-T therapies, through thorough depletion of pathogenic B cells and “resetting” of normal B cell populations, have achieved compelling preliminary clinical efficacy including multi-year drug-free remission in multiple autoimmune diseases [2, 3], however CAR-T modality continues to pose challenges regarding safety, cost, and logistical complexity. T Cell Engagers (TCEs) represent an attractive alternate option to CAR-T, with approved CD19-targeting bispecific TCE Blincyto demonstrating strong potential in RA [4]. However, existing bispecifics have displayed key limitations such as T cell exhaustion, poor T cell proliferation, and insufficient durability of response. Here, we developed the tri-specific TCE CC312, targeting CD3 and the key co-stimulatory receptor CD28 on T cells and CD19 on B cells. By adding critical CD28 “two signal” co-stimulation, CC312 showed efficacy consistent with CAR-T therapies, achieving greater CD4/CD8 T cell activation, proliferation, T cell survival and B cell depletion versus Blincyto in a prior study in B cell malignancies, indicating strong potential to treat autoimmune disease.

Objectives: In the current study, the potential of CC312 in treating autoimmune disease was explored in translational models, including ex vivo studies with SLE/RA donor PBMCs, an in vivo efficacy study in SLE patient derived-PBMC NCG mouse models, etc.

Methods: B cell killing of CC312 was assessed in ex vivo cytotoxicity assays using human peripheral blood mononuclear cells (PBMCs) isolated from patients with autoimmune disease. T cell activation, proliferation and memory cell differentiation were also evaluated in response to patient-derived PBMCs. Moreover, to evaluate CC312’s durability of response compared with Blincyto, SLE/RA patient derived-T cells were repeat rechallenged with B cells from the same donor. The in vivo efficacy for SLE was investigated in an animal model in which NCG mice were engrafted with SLE patient-derived PBMCs. B cell depletion in blood was analyzed by flow cytometry and in kidney tissues by immunohistochemistry.

Results: Ex vivo , CC312 induced a dose-dependent potent depletion of B cells in PBMCs derived from donors with autoimmune diseases, with EC ₅₀ in pM range across donors and disease types. Compared with Blincyto, CC312 significantly enhanced T cell activation and proliferation due to CD28 co-stimulation. In addition, CC312 also exhibited augmented memory T cell differentiation, with higher proportion of central memory T cells (T _cm ) and effector memory T cells (T _em ). Moreover, CC312 displayed sustained T cell fitness, proliferation and killing effect in repeat rechallenge assay with SLE donor PBMCs, showing more durable response versus Blincyto. In vivo , CC312 thoroughly depleted blood B cells and plasmablasts as well as eliminated IgG and anti-dsDNA in peripheral blood in an SLE mouse model. At the end of this study, neither renal IgG deposits nor CD20 ⁺ B cell infiltrates were observed within kidney tissues.

Conclusion: CC312’s capacity to induce more complete T cell activation and longer T cell durability of response as well as more potently and thoroughly eliminate pathogenic B cells versus leading bispecific TCE Blincyto has been demonstrated in both ex vivo and in vivo studies. This highlights the importance of CD28 co-stimulation and indicates strong potential benefit of CC312 in achieving the depth of response needed to effectively treat B cell-related autoimmune diseases in the clinical setting.

REFERENCES: [1] M J Leandro, et al. Rheumatology (Oxford ). 2005 Dec;44(12):1542-5.

[2] Fabian Müller, et al. N Engl J Med. 2024 Feb 22;390(8):687-700.

[3] Carlo Tur, et al. Ann Rheum Dis. 2024 Sep 11:ard-2024-226142.

[4] Laura Bucci, et al. Nat Med. 2024 Jun;30(6):1593-1601.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.