Preclinical characterization of LY4336619, a novel ALPPL2xCD3 bispecific antibody, for the treatment of ovarian and endometrial cancer

Development of CD3-bispecific antibodies has been challenging in solid tumors due to the difficulty identifying tumor antigens with high tumoral expression but with minimal or no expression in normal tissues. Placental-like alkaline phosphatase 2 (ALPPL2) is overexpressed in various solid tumors, including ovarian and endometrial carcinomas, while its expression in normal tissues is largely restricted to the placenta. This expression profile makes ALPPL2 a promising target for CD3-bispecific antibody therapies. We have developed a fully human common light chain-based ALPPL2×CD3 bispecific antibody, LY4336619, on an Fc-silenced IgG1 backbone designed to engage ALPPL2 on tumor cells and CD3 on T cells, facilitating targeted T cell-mediated cytotoxicity against ALPPL2-expressing tumor cells. LY4336619 was generated by pairing a novel anti-ALPPL2 monoclonal antibody, identified through a murine immunization strategy, with an anti-CD3 antibody. The binding, target selectivity, and cytotoxic activity of LY4336619 were evaluated using multiple ALPPL2-expressing cell lines. In vivo efficacy was evaluated using immunocompromised murine models engrafted with human tumor xenografts and human peripheral blood mononuclear cells (PBMCs). Additionally, a comprehensive proteomics analysis was performed to characterize ALPPL2 expression across tumor tissue specimens. LY4336619 specifically bound to ALPPL2-expressing cell lines but spared ALPI and ALPL, the other more widely expressed alkaline phosphatase family members; it also bound to the highly homologous ALPP family member, a protein with similar limited normal expression and association with similar tumor types. LY4336619 engaged with both CD3 and ALPPL2, and induced concentration-dependent T-cell activation and T cell-mediated cytotoxicity in co-culture assays with T cells and ALPPL2-expressing tumor cells. Furthermore, it demonstrated antitumor activity and relatively low cytokine release as a single agent in human PBMC-engrafted mice with human tumor xenografts representing a range of ALPPL2 expression levels. Additional preliminary studies revealed that the combination treatment with LY4170156, a clinical-stage anti-FRalpha antibody-drug conjugate (ADC), resulted in more sustained and pronounced tumor growth inhibition compared to either monotherapy alone. Our ALPPL2×CD3 bispecific antibody exhibited IgG-like pharmacokinetics and low immunogenic potential. These findings demonstrate that LY4336619 has target specificity, favorable functional activity, and a promising developability profile. LY4336619 has the potential as a first-in-class medicine to treat ALPPL2-positive solid tumors, including ovarian and endometrial cancers. An IND submission is anticipated for 2026.