Efficient in vivo assembly of DNA encoded polyvalent BTEs for dual antigen targeting for broadening therapeutic impact in ovarian cancer.

The clinical potential of bispecific T cell engagers (BTEs) is limited by their short serum half-life and the complexity and cost of recombinant protein manufacturing. Currently, BTEs rely on external production and repeat dosing, limiting scalability and patient access. Here, we present a synDNA platform that enables in vivo assembly and long-term secretion of bispecific antibodies directly from host muscle. The system includes complementary "knob" and "hole" Fc chains on separate plasmids that pair only when co-expressed, ensuring controlled heterodimer formation and reducing homodimer assembly. Using ovarian cancer (OC) as a model of antigen heterogeneity, we generated dKBTEs targeting follicle stimulating hormone receptor (dK_FSHRxCD3) and Her2 (dK_Her2xCD3). Artificial intelligence simulations supported successful knob-hole pairing and presented significant challenges for homodimer formation. Co-transfected Expi293F cells secreted fully assembled bispecifics that bound antigen and CD3, activated primary human T cells, and induced potent antigen-specific cytotoxicity. OC patient peripheral blood mononuclear cells responded strongly to dKBTEs in vitro. In mice, dKBTEs showed superior pharmacokinetics and tumor control over first-generation BTEs. Combination therapy with both dKBTEs further improved tumor control. synDNA delivery allows simultaneous in vivo production of two dKBTEs without compromising expression. These findings establish synDNA delivery as a programmable approach for sustained delivery of complex biologics.