INTRODUCTION:Macrophages are attractive targets for novel cancer immunotherapy approaches since they can infiltrate into the tumor microenvironment and have the capacity to engulf and destroy cancer cells by phagocytosis. The CD47/SIRPα axis is a key immune checkpoint that regulates macrophages' ability to attack cancer cells and has been the subject of intense preclinical and clinical investigation. AREAS COVERED:We review the scientific rationale for developing CD47/SIRPα-targeting therapies, and we summarize results of recent clinical trials that tested anti-CD47 antibodies, SIRPα-Fc fusion proteins, or anti-SIRPα antibodies in patients with lymphoma. We review signs of efficacy, opportunities for combination strategies, and challenges such as on-target hematologic toxicity and an 'antigen sink' that exists due to CD47 expression on blood cells. EXPERT OPINION:Multiple CD47/SIRPα-targeting therapeutics and multiple clinical trials have demonstrated encouraging results in patients with non-Hodgkin lymphoma, where objective responses have been observed in combination with rituximab and other anti-cancer agents. Next-generation approaches, such as bispecific antibodies and engineering efforts to reduce blood cell binding, are now under clinical development and may be successful strategies to unlock the extraordinary potential of the CD47/SIRPα immune checkpoint.

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.

Abstract Macrophages are critical effectors of antibody therapies for lymphoma, but the best targets to engage their function remain unknown. Here, we develop a ‘tactical surfaceome profiling’ strategy to define a comprehensive repertoire of surface antigens on B-cell lymphoma that can be targeted with antibodies to provoke macrophage attack. Across both mouse and human systems, we identify multiple unappreciated targets of opsonization as well as putative immune checkpoints. We incorporate this information into a high-throughput engineering strategy, creating 156 bispecific antibodies and identifying dozens that stimulate macrophage-mediated cytotoxicity. A heterodimeric scFv-Fc format enables our approach; it is superior to conventional bispecific antibody design and offers enhanced activity while reducing developmental complexity. Among the therapeutics we create, a bispecific comprising a SIRPα decoy domain and a CD38-targeting arm (WTa2d1xCD38) exhibits maximal efficacy with reduced risk of toxicity. This bispecific stimulates robust anti-tumor responses in xenograft models of aggressive B-cell lymphoma and shows benefits over anti-CD20 antibody rituximab. Our approach can be applied to other cancers to leverage anti-tumor responses by macrophages or other immune cells.

Understanding how tissues remodel in response to perturbations requires computational tools that can untangle condition-specific changes from the conserved tissue architecture. We present Haruka, a spatially aware contrastive learning framework that identifies salient (condition-specific) and background (shared) spatial domains across tissue slices and experimental conditions. Haruka integrates contrastive variational inference with an auxiliary microenvironment reconstruction task, enabling the model to learn spatial-context-informed embeddings that capture both perturbation effects and local neighborhood context. Through benchmarking on simulated and real datasets, Haruka outperforms state-of-the-art methods in detecting spatially heterogeneous responses. Applied to diverse spatial omics platforms, Haruka distinguished immunotherapy responders in melanoma, traced fibrosis progression in human lung tissue, and mapped treatment-resistant microenvironments in KRAS G12D -mutated lung cancer. Thus, Haruka provides a generalizable framework for spatial contrastive analysis, enabling systematic dissection of tissue organization, cellular plasticity, and microenvironmental remodeling across disease, development, and therapeutic response.

The awakening of dormant disseminated cancer cells appears to be responsible for the clinical relapses of patients whose primary tumors have been successfully cured months and even years earlier. In the present study, we demonstrate that dormant breast cancer cells lodged in the lungs reside in a highly mesenchymal, nonproliferative phenotypic state. The awakening of these cells is not triggered by a cancer cell-autonomous process. Instead, lung inflammation induced by the chemotherapeutic agent bleomycin effectively awakens dormant cancer cells, providing useful models for studying metastatic awakening. Mechanistically, the awakened cells shift from a highly mesenchymal to a quasi-mesenchymal phenotypic state in which they acquire tumorigenicity and proliferative ability. Once awakened, these cells can stably reside in this quasi-mesenchymal state and maintain their tumor-initiating ability, doing so without ongoing heterotypic signaling from the lung microenvironment. Epidermal growth factor receptor ligands released by the cells of the injured tissue microenvironment, including notably M2 type macrophages, promote dormant cancer cells to move toward this quasi-mesenchymal state, a transition that is critical for the awakening process. An understanding of the mechanisms of metastatic awakening may lead in the future to treatment strategies designed to prevent such awakening and resulting metastatic relapse.

Abstract Monoclonal antibodies are among the most effective treatments for patients with blood cancers, and they primarily function by marking cells for destruction by the innate immune system. Macrophages are critical innate effectors of antibody therapies for lymphoma, but their anti-tumor capacity is limited by the CD47/SIRPa interaction. CD47 acts a “don’t eat me” signal that is highly expressed on the surface of many lymphomas, and it prevents phagocytosis by binding to the inhibitory receptor SIRPa expressed on macrophages. In multiple clinical trials, CD47-blocking therapies have demonstrated encouraging signs of efficacy for B-cell lymphomas, particularly when used in combination with rituximab, an opsonizing anti-CD20 antibody. However, the best antibodies and combination strategies to activate macrophages remains unknown. Here, we sought to define the repertoire of cell surface antigens that can be targeted to stimulate macrophage-mediated destruction of B-cell lymphomas. To achieve this goal, we developed a high-throughput functional screening platform to measure the ability of primary macrophages to attack B-cell lymphoma cells. We successfully applied this system to screen monoclonal antibody libraries targeting hundreds of distinct cell surface antigens across both mouse and human systems. We conducted screens using each antibody as a single agent, in combination with anti-CD20, or in combination with anti-CD47. From these efforts, we identified CD24, CD38, CXCR4, CD71, and multiple other novel and unique antigens that could be targeted alone or in combination to exert maximal macrophage-mediated destruction of B-cell lymphoma. Of note, some of the identified targets are predominantly expressed by the lymphoma cells, whereas others are expressed by the macrophages and act as unappreciated immune checkpoints. In validation studies, we defined a multitude of new antibody combinations that robustly stimulate macrophages to attack and eliminate lymphoma cells. Since some anti-CD47-antibodies have been limited by on-target hematologic toxicity, we also used the identified targets to engineer a collection of novel bispecific antibodies that induce macrophage phagocytosis without causing hematologic toxicity. We generated 156 bispecific antibodies, produced them recombinantly, and demonstrated they can maximize macrophage-mediated cytotoxicity of human B-cell lymphoma cells while minimizing binding to healthy blood cells. In mouse xenograft models, these bispecific antibodies exhibited significant single-agent activity in a model of aggressive B-cell lymphoma. Thus, our study has led to the development of a multitude of novel therapeutic candidates and combination strategies that can be developed further to maximize anti-tumor function and benefit patients with lymphoma. Furthermore, our approach can be rapidly applied to other hematologic malignancies to create innovative bispecific agents that maximize anti-tumor responses by macrophages or other innate immune cells. Citation Format: Juliano Ribeiro, Carlota Pages Geli, José Velarde, Anna Meglan, Jasmine Blandin, Kyle Vaccaro, Marta Crespo, Kipp Weiskopf. Unbiased discovery of novel antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-038.

Macrophages hold tremendous promise as effectors of cancer immunotherapy, but the best strategies to provoke these cells to attack tumors remain unknown. Here, we evaluated the therapeutic potential of targeting two distinct macrophage immune checkpoints: CD47 and CD24. We found that antibodies targeting these antigens could elicit maximal levels of phagocytosis when combined together in vitro. However, to our surprise, via unbiased genome-wide CRISPR screens, we found that CD24 primarily acts as a target of opsonization rather than an immune checkpoint. In a series of in vitro and in vivo genetic validation studies, we found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, anti-CD24 antibodies exhibit robust Fc-dependent activity, and as a consequence, they cause significant on-target hematologic toxicity in mice. To overcome these challenges and leverage our findings for therapeutic purposes, we engineered a collection of 77 novel bispecific antibodies that bind to a tumor antigen with one arm and engage macrophages with the second arm. We discovered multiple novel bispecifics that maximally activate macrophage-mediated cytotoxicity and reduce binding to healthy blood cells, including bispecifics targeting macrophage immune checkpoint molecules in combination with EGFR, TROP2, and CD71. Overall, our findings indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer, and that the novel bispecifics we created may be optimal immunotherapies to direct myeloid cells to eradicate solid tumors.

Macrophages are critical effectors of antibody therapies for lymphoma, but the best targets for this purpose remain unknown. Here, we sought to define a comprehensive repertoire of cell surface antigens that can be targeted to stimulate macrophage-mediated destruction of B-cell lymphoma. We developed a high-throughput assay to screen hundreds of antibodies for their ability to provoke macrophages to attack B-cell lymphoma cells. Across both mouse and human systems, we identified multiple unappreciated targets of opsonization as well as putative immune checkpoints. We used this information to engineer a compendium of 156 bispecific antibodies, and we identified dozens of bispecifics that dramatically stimulate macrophage-mediated cytotoxicity of lymphoma cells. Among these, a bispecific comprising a SIRPα decoy domain and a CD38-targeting arm (WTa2d1xCD38) exhibited maximal efficacy while minimizing the risk of hematologic toxicity. This bispecific stimulated robust anti-tumor responses in multiple xenograft models of aggressive B-cell lymphoma. Our approach can be directly applied to other cancers to rapidly discover bispecific antibodies that leverage anti-tumor responses by macrophages or other innate immune cells.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed an unbiased, high-throughput screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we found that therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found that the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized antitumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations - including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of β2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

Abstract Macrophages hold tremendous promise as effectors of cancer immunotherapy, but the best strategies to provoke these cells to attack tumors remain unknown. Here, we evaluated the therapeutic potential of targeting two distinct macrophage immune checkpoints: CD47 and CD24. We found that antibodies targeting these antigens could elicit maximal levels of phagocytosis when combined together in vitro. However, to our surprise, via unbiased genome-wide CRISPR screens, we found that CD24 primarily acts as a target of opsonization rather than an immune checkpoint. In a series of in vitro and in vivo genetic validation studies, we found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, anti-CD24 antibodies exhibit robust Fc-dependent activity, and as a consequence, they cause significant on-target hematologic toxicity that was life-threatening in syngeneic mice. To overcome these challenges and leverage our findings for therapeutic purposes, we engineered a collection of 77 novel bispecific antibodies that bind to a tumor antigen with one arm and engage macrophages with the second arm. We discovered multiple novel bispecifics that maximally activate macrophage-mediated cytotoxicity and reduce binding to healthy blood cells, including bispecifics targeting macrophage immune checkpoint molecules in combination with EGFR, TROP2, and CD71. Overall, our findings indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer, and that the novel bispecifics we created may be optimal immunotherapies to direct myeloid cells to eradicate solid tumors. Citation Format: Anna Meglan, Juliet Allen, Kyle Vaccaro, José Velarde, Victor Chen, Juliano Ribeiro, Jasmine Blandin, Ranjan Mishra, Raymond Ho, Jennifer Love, Ferenc Reinhardt, George W Bell, Jin Chen, Robert Weinberg, Dian Yang, Jonathan Weissman, Kipp Weiskopf. CD47 predominates over CD24 as a macrophage immune checkpoint in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A055.

CD47 expression on canine cancer samples.

Recombinant production and evaluation of canine SIRPalpha protein.

Supplementary Table S1. Similarity between CD47 variants across species. Supplementary Table S2. Similarity between SIRPα variants across species. Supplementary Table S3. Summary of tumor necrosis scores following treatment with the indicated therapies. Supplementary Figure Legends. Supplementary Materials and Methods.

Supplementary Figure S1: SIRPabodies bind to CD20 and CD47 with weak affinity for CD47

Combination therapy with CD47-blockade and anti-CD20 antibodies produces in vivo efficacy with no overt toxicity.

1E4 antibody binds canine CD20 extracellular domain 2.

CV1 does not inhibit proliferation of CLBL-1 cells in vitro.

Ferroptosis is a form of regulated cell death with roles in degenerative diseases and cancer. Excessive iron -catalyzed peroxidation of membrane phospholipids, especially those containing the polyunsaturated fatty acid arachidonic acid (AA), is central in driving ferroptosis. Here, we reveal that an understudied Golgi-resi-dent scaffold protein, MMD, promotes susceptibility to ferroptosis in ovarian and renal carcinoma cells in an ACSL4-and MBOAT7-dependent manner. Mechanistically, MMD physically interacts with both ACSL4 and MBOAT7, two enzymes that catalyze sequential steps to incorporate AA in phosphatidylinositol (PI) lipids. Thus, MMD increases the flux of AA into PI, resulting in heightened cellular levels of AA-PI and other AA -con-taining phospholipid species. This molecular mechanism points to a pro-ferroptotic role for MBOAT7 and AA -PI, with potential therapeutic implications, and reveals that MMD is an important regulator of cellular lipid metabolism.

Disrupting the CD47–SIRPα checkpoint in tumour macrophages and delivering a tumour-opsonizing monoclonal antibody maximizes the macrophages’ cooperative phagocytic potency.