DNA Hypomethylating Agents Preserve T-cell Stemness and Potentiate the Efficacy of CD3-Bispecific Antibodies

Bispecific antibodies targeting tumor-associated antigens and CD3 are promising therapeutic agents for both solid and hematologic cancers. CD3-bispecifics induce T-cell activation and cytotoxicity; however, prolonged T-cell receptor (TCR) stimulation can lead to chromatin rewiring and T-cell dysfunction, thereby limiting their full therapeutic potential. In this study, we investigate the combination of CD3-bispecifics with the DNA hypomethylating agent decitabine (DAC) and observe enhanced tumor growth inhibition in various preclinical models. Utilizing a prostate-specific membrane antigen (PSMA)×CD3 bispecific antibody for the treatment of prostate carcinoma and in vivo humanized mouse disease models, we catalog, at the single-cell level, the dynamics of T-cell epigenetic states during bispecific therapy and in combination with DAC. Importantly, this combination strategy preserves a T-cell factor 1 (TCF-1)–positive T-cell population and delays acquisition of a dysfunctional state at both chromatin and protein levels. At the DNA methylation level, TCR stimulation in the presence of DAC maintains a naïve-like pattern in gene loci associated with T-cell stemness. This study provides a resource for understanding the evolution of T-cell states during immunotherapy and mechanistic support for combining epigenetic modifiers with CD3-bispecifics in the clinic.