A novel CD28 x Nectin-4 costimulatory bispecific antibody for advanced bladder cancer.
Authors: Thomas Manley, Starlynn Clarke, Manpreet Kaur, Sebastian Moreno Arteaga, Soumili Chattopadhyay, Elaine Chen, Ruth Chu, Jacqueline Morgan, Cynthia Nguyen, Udaya Rangaswamy, Mathias Rickert, Katja Rueger, Imani Smith, Gizelle Taleno, Laura Davison, Katherine E Harris, Shelley Force Aldred, Nathan D. Trinklein
Journal: Journal of Clinical Oncology (2025)

810 Background: Effective T-cell activation is driven by multiple signals. CD3 bispecific antibodies (bsAbs) stimulate T-cell activation through the TCR. CD3 bsAbs demonstrate durable responses in hematological malignancies, but multiple clinical trials suggest that signaling through CD3 alone is insufficient to treat solid tumors. Recent results suggest that a robust T-cell response in solid tumors can be achieved by tumor-targeted costimulatory bsAbs that promote sustained T-cell activity. Bladder cancer is the tenth most prevalent cancer worldwide with suboptimal progression free survival in the locally advanced and metastatic setting. Efficacy of checkpoint inhibitors (CPI) in metastatic urothelial cancer (mUC) patients indicates the presence of tumor antigen-specific T-cell activity, and thus, treatment with a costimulatory bsAb as a single agent may elicit a durable and robust anti-tumor response. Here we describe RNDO-564, a novel CD28 x Nectin-4 bispecific molecule. RNDO-564 was developed preclinically with optimal potency to provide robust anti-tumor activity and safety by localizing co-stimulatory activity to the site of the tumor and avoiding super-agonist activity. Methods: High throughput antibody discovery was completed on CD28 and Nectin-4. A panel of agonistic bispecific CD28 antibodies was generated by combining high affinity Nectin-4 binders with CD28 binders of varying potencies using knobs-in-hole technology. Bispecific antibodies were tested for their ability to enhance T-cell mediated cytotoxicity and cytokine release on tumor cell lines expressing varying levels of Nectin-4 in the presence of suboptimal signal 1. Lead candidates were then tested for efficacy using in vivo syngeneic mouse models. Results: Affinity tuning of CD28 agonistic activity was performed by testing a diverse panel of CD28 effector arms to identify optimal costimulatory activity that lacked super agonist activity. One of these CD28 binders was paired with a high affinity Nectin-4 binder to generate RNDO-564. With an affinity tuned CD28 arm and a high affinity Nectin-4 arm driving localization to tumor cells, RNDO-564 exhibited potent and specific cytotoxicity and cytokine release to Nectin-4 expressing tumor cells in vitro. In vivo , RNDO-564 alone and in combination with an anti-PD-1 mAb resulted in dose-dependent tumor growth inhibition in signal 1-proficient syngeneic models, indicating clinical potential for both monotherapy and combination activity. In addition, RNDO-564 was well-tolerated in non-human primates and had normal pharmacokinetics after a single dose infusion. Conclusions: RNDO-564 has a high affinity Nectin-4 binding arm and an affinity-tuned CD28 arm optimized for activity in metastatic bladder cancer. A phase 1 study is planned to evaluate the safety, tolerability, PK/PD, and clinical activity of RNDO-564 alone and in combination with CPI in mUC.