Abstract 5318: SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells

Abstract Background: T cells require both signal 1 TCR engagement and signal 2 co-stimulatory signal to achieve complete activation and tumor recognition. CD28 is a signal 2 co-stimulatory receptor and activating CD28 increases T cell sensitivity, cytokine production and T cell survival. PD-1/PD-L1 signaling attenuates the T cells functional activity by inhibiting CD28 co-stimulation in the presence of TCR stimulation. Therefore, we developed SM2275, a tetraspecific VHH antibody against EGFR, PD-L1, CD28 and HSA, for the treatment of EGFR+/PD-L1+ solid tumors. Preclinical study demonstrated promising efficacy and favorable safety profile via a unique mechanism of action. Methods: SM2275 was developed using StarMab’s proprietary Quadbody multifunctional VHH antibody platform. It features two monovalent, low-affinity binding domains for EGFR and PD-L1, along with nanomolar low affinity CD28 binding domains arranged in a tandem format, followed by a half-life extending HSA binding domain. Differential binding affinities of SM2275 to EGFR/PD-L1 single-expressing versus double-expressing cell lines were analyzed by flow cytometry. Avidity-enhanced blockade of PD-1 on EGFR+PD-L1+ double-positive compared to PD-L1+ single-positive cells was analyzed via competitive binding assay and Jurkat PD1 NFAT Luciferase reporter assay. EGFR/PD-L1 dual-target-dependent CD28 activation was confirmed using a primary human T cell IL2 & IFN-γ release assay in the presence of TCR stimulation. In vivo efficacy against EGFR+/PD-L1+ double-positive human tumors were evaluated using a syngeneic tumor transplantation model of MC38-hEGFR/hPDL1 cell line into hPD-L1/hCD28 humanized mice. Results: SM2275 displays dramatically increased affinity for cancer cells co-expressing EGFR/PD-L1 compared to single-expressing cells. This leads to an avidity-enhanced PD-L1 blockade specifically on the surface of EGFR+PD-L1+ double-positive cancer cells, enabling localized reactivation of tumor-infiltrating T cells without systemic immune toxicity. Simultaneously blocking the PD-L1 signaling unleashed the inhibition of CD28 co-stimulation for the full T cell activation. In the presence of SM2275, EGFR/PD-L1 double-positive cancer cells induce significantly enhanced CD28 activation and cytokine secretion compared to single-antigen-expressing cells. In a syngeneic tumor transplantation model, SM2275 effectively inhibits the growth of MC38-hEGFR/hPDL1 tumors in hPD-L1-hCD28 humanized mice, with no apparent cytokine release and immune toxicity. Conclusions: SM2275 is a novel dual-EGFR & PD-L1-targeting, conditional CD28 agonistic antibody. It demonstrated promising efficacy and favorable safety profile in preclinical studies via a unique mechanism of action. Citation Format: Shihao Lu, Xiaodan Liu, Sheila Zhou, Wenjing Song, Huiqin Geng, Simin Yang, Hong Zhou, Siyao Xie, Yi Wei, Xing Zhang, Yanbin Liang. SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5318.