A versatile KY-HC-mouse™nanobody platform enabling tumor-anchored, conditionally active 4-1BB bispecific antibodies

4-1BB (CD137) is an attractive co-stimulatory receptor for boosting anti-tumor T-cell responses, but first-generation systemic 4-1BB agonist antibodies have been limited by dose-limiting toxicity, highlighting the need for tumor-anchored, conditionally active agonists with an improved benefit-risk profile. We established a proprietary heavy-chain-only mouse (KY-HC-mouse™) platform to generate relatively low-affinity anti-4-1BB nanobodies that block 4-1BB/4-1BBL binding and require cross-linking for strong signaling, thereby conferring built-in conditional agonism. These nanobodies were formatted into Fc-silenced IgG (LALA mutation) bispecifics targeting PSMA (PSMA×4-1BB, KA-2722), CD19 (CD19×4-1BB, KA-6859) and CDH17 (CDH17×4-1BB, KA-A3438), as well as a trispecific antibody targeting CDH17×CLDN18.2×4-1BB (KA-A3601). CDH17×CLDN18.2×4-1BB (KA-A3601) was designed based on the frequent co-expression of CDH17 and CLDN18.2 in gastrointestinal tumors, enabling dual-antigen anchoring of 4-1BB co-stimulation. All molecules show relatively reduced 4-1BB binding compared with benchmark agonists but retain strong, high-avidity binding to their respective tumor antigens and drive potent, antigen-dependent activation of 4-1BB reporter cells. CD19×4-1BB (KA-6859) further demonstrates robust in vivo efficacy in hu-PBMC lymphoma models when combined with a CD20×CD3 T-cell engager Glofitamab, with clear tumor growth inhibition at 3 mg/kg KA-6859 plus 0.15mg/kg CD20×CD3 (QW*3), and exhibits favorable pharmacokinetics with sustained serum exposure in mice. CDH17×4-1BB (KA-A3438) and CDH17×CLDN18.2×4-1BB (KA-A3601) show strong tumor growth inhibition in MC38-CDH17 and MC38-CDH17-CLDN18.2 tumors established in B-h4-1BB C57BL/6 humanized 4-1BB (h4-1BB) mice, respectively, without ALT/AST elevation, indicating absence of detectable hepatotoxicity. CD19×4-1BB (KA-6859) exhibits a transient ExpiCHO-S expression yield of ~393 mg/L and a favorable pharmacokinetic profile in C57BL/6 mice, with terminal half-lives of approximately 16-21 days after i.v. dosing and ~10 days after s.c. dosing, and an s.c. bioavailability of ~82%. KA-6859 also displays high thermal stability and good overall stability, indicating favorable developability.Together, these data position the KY-HC-mouse™ 4-1BB nanobody platform as a versatile foundation for next-generation, tumor-anchored 4-1BB bispecifics and multispecifics, and for future combinations with checkpoint inhibitors in solid and hematologic malignancies.