Background Her2 and Trop2, two tumor-associated antigens (TAAs), are well-established cancer targets validated by the approval of several anti-Her2 ADCs and an anti-Trop2 ADC. Her2 and Trop2 are expressed separately or co-expressed in cancer cells within tumors, reflecting the heterogeneity of tumor TAA expression. Currently, there is no approved bispecific ADC that targets both Her2 and Trop2. Dual TAA-targeting may reduce the probability of cancer escape, commonly seen in the treatment of single TAA-targeting ADCs, due to the heterogeneity of TAA expression.

Methods We developed a novel bispecific anti-Her2/anti-Trop2 ADC, BB-201. BB-201 was engineered using humanized anti-Trop2 and anti-Her2 VHH fragments, fused with a human IgG1 Fc, and conjugated with vc-MMAE. Whole cell binding assays were conducted to determine the binding affinity of BB-201 to TAAs. In vitro cytotoxicity assays were used to evaluate the cytotoxic effects of BB-201 on various cancer cell lines. The in vivo efficacy of BB-201 was tested in A431 and OE19 xenograft mouse models.

Results BB-201 maintained the high binding affinities of its parental VHH-Fc antibodies to Her2 and Trop2, with the affinity of the anti-Her2 arm 6-fold higher than that of the anti-Trop2 arm. BB-201 exhibited remarkable cytotoxic effect on cancer cells that express either Her2 or Trop2, or both. The potency of its cytotoxic effect was comparable to Trastuzumab-MMAE (T-MMAE) in high Her2-expressing cancer cells and to hRS7-MMAE in high Trop2-expressing cancer cells.

The in vivo efficacy of BB-201 was evaluated in an A431 model. A431 cells express high levels of Trop2 and low levels of Her2. In this model, both BB-201 and hRS7-MMAE treatments significantly inhibited tumor growth, achieving 61% and 67% of tumor growth inhibition (TGI), respectively. In contrast, treatments with T-MMAE and the unconjugated bispecific VHH antibody had no impact on A431 tumor growth.

Furthermore, BB-201 was tested in an OE19 model. OE19 cells express high levels of Her2 and medium levels of Trop2. Treatment with either BB-201 or T-MMAE significantly shrank the tumors. Four weeks after a single dose treatment, most mice in these two groups had no detectable tumors. While hRS7-MMAE treatment was able to shrink the tumors initially, the tumors gradually regrew in the fourth week post-treatment. No obvious side effects were observed in both in vivo studies.

Conclusions The novel anti-Her2/anti-Trop2 bispecific ADC BB-201 has superior anti-tumor activity in preclinical studies. BB-201 has a promising therapeutic potential as a first-in-class ADC drug candidate.