Abstract Non-small cell lung cancer (NSCLC) represents ∼85% of all lung cancer cases and remains one of the leading causes of cancer-related mortality in the United States. Approximately 3-7% of NSCLC patients harbor ALK rearrangements, most commonly the EML4-ALK fusion. Although ALK+ tumors initially respond well to ALK tyrosine kinase inhibitors (TKIs), patients still develop resistance. Lorlatinib, a third-generation ALK inhibitor, achieves ∼76% response rates in treatment-naïve patients; however, a substantial proportion experience disease progression within a few years, highlighting the urgency for new therapeutic strategies. The tumor immune microenvironment is increasingly recognized as a key contributor to resistance. Tumor-associated macrophages (TAMs) are the dominant immune population infiltrating NSCLC and can promote tumor growth, survival, and immune evasion. Here, we aimed to identify novel therapeutic strategies to stimulate macrophages to eliminate ALK+ lung cancer cells.Using in vitro co-culture assays, we found that macrophages markedly protect ALK+ lung cancer cells from lorlatinib, making the cancer cells significantly less responsive compared to cells cultured alone. We found that blocking macrophage immune checkpoints, such as the CD47/SIRPa axis, could restore macrophage activation and promote phagocytosis of the ALK+ lung cancer cells. Since systemic administration of anti-CD47 antibodies can result in dose-limiting hematotoxicity, we therefore developed a high-throughput bispecific antibody (bsAb) engineering platform to leverage the benefits of targeting CD47 while increasing specificity to the tumor microenvironment. We generated a library of bsAbs targeting CD47 with a second arm binding well-known tumor antigens including EGFR, EpCAM, HER2, TROP2, FOLR1, Nectin-1, Nectin-4, PD-L1, and CD71. Using live-cell imaging, we evaluated macrophage-mediated cytotoxicity for each bsAb across three ALK+ NSCLC cell lines. Among all constructs tested, a bsAb incorporating a low-affinity CD47-binding domain and a TROP2-targeting domain showed the strongest anti-tumor activity, significantly reducing tumor growth over time. This bsAb enhanced macrophage phagocytosis and decreased tumor area more effectively than lorlatinib alone. Importantly, combining the CD47×TROP2 bsAb with lorlatinib produced synergistic anti-tumor effects, offering a promising strategy to overcome or delay resistance. Overall, our study has identified novel bispecific antibodies that may be highly active for ALK+ lung cancer and our findings could be translated to the clinic to benefit patients in the future. Citation Format: Carlota Pages-Geli, Thomas Wienclaw, Juliano Ribeiro, Matheus Silva, Kipp Weiskopf. Unbiased discovery of novel macrophage-activating immunotherapies for ALK+ non-small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5548.
Pagès-Geli et al. (Fri,) studied this question.