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 TCR stimulation can lead to chromatin rewiring and T cell dysfunction, thereby limiting their full therapeutic potential. Here, we investigate the combination of CD3-bispecifics with the DNA hypomethylating agent decitabine and observe enhanced synergistic tumor growth inhibition in various preclinical models. Utilizing a PSMAxCD3 bispecific antibody for 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 decitabine. Importantly, this combination strategy preserves a TCF-1+ 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 decitabine maintains a naive-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.