Development of a novel humanized monoclonal antibody and efficacy profiling of site-directed antibody-drug conjugates for precision targeted therapy of CLDN18.2-positive cancers.

CLDN18.2, a tight junction protein aberrantly overexpressed in gastric, pancreatic, and other cancers but minimally expressed in normal tissues, is a promising target for antibody-drug conjugate (ADC)-based therapies. This study describes the development and preclinical characterization of h1D6-ADC-5, a novel ADC composed of a proprietary humanized anti-CLDN18.2 monoclonal antibody (h1D6) and the cytotoxic payload DUO-5, conjugated via site-specific technology. h1D6 was generated by immunizing CLDN18.2 knockout mice followed by humanization; it exhibited high specificity for CLDN18.2 (no cross-reactivity with CLDN18.1) and improved binding affinity and in vivo efficacy vs. existing antibodies (e.g., IMAB362). Using C-Lock site-directed conjugation, h1D6 was linked to DUO-5 (a tubulin polymerization inhibitor) to yield h1D6-ADC-5 with a controlled average drug-to-antibody ratio of 4.3. In vitro, h1D6-ADC-5 selectively bound CLDN18.2-positive cells (AGS-CLDN18.2, NUGC4), were efficiently internalized, and induced apoptosis by downregulating anti-apoptotic proteins and upregulating pro-apoptotic markers. It also exerted a "bystander effect," killing adjacent CLDN18.2-negative cells via released DUO-5. In vivo, 89Zr-labeled h1D6-ADC-5 showed prolonged tumor accumulation in CLDN18.2-positive xenograft, with significant tumor regression (nearly complete regression at 10 mg/kg) while no overt toxicity was observed based on body weight and histology. These preclinical data demonstrate that h1D6-ADC-5 is a promising candidate for treating CLDN18.2-positive cancers.