Spatially enforced engagement of PD-1⁺ T cells with AXL⁺ cells reprograms the tumor microenvironment and overcomes anti-PD-1 resistance

Background: AXL overexpression promotes tumor-cell plasticity, local immune suppression, and resistance to PD-1 blockade. Conventional anti-AXL and anti-PD-1 antibodies combinations act independently and do not ensure physical proximity between PD-1+ T cells and AXL+ cells within the immunosuppressive tumor microenvironment. Methods: To address this limitation, we developed a non-tandem, tetravalent IgG bispecific antibody designed to simultaneously engage PD-1 and AXL (BiTE-AXL/PD-1). Its antitumor activity was evaluated using in vitro functional assays and humanized and immunocompetent head and neck squamous cell carcinoma (HNSCC) models and compared with the combination of monospecific anti-AXL and anti-PD-1 antibodies. Spatial multiplex imaging was used to characterize treatment-associated changes in the tumor microenvironment of an anti-PD-1-resistant murine model. Results: BiTE-AXL/PD-1 enhanced T-cell activation, promoted AXL-dependent tumor-T-cell conjugation, and increased tumor-cell killing in vitro, outperforming the combination of the two monospecific antibodies. In humanized and immunocompetent HNSCC models, BiTE treatment significantly delayed tumor growth compared with the antibody combination and was well tolerated, with no overt toxicity. Spatial multiplex imaging revealed a shift from a fibroblast-dominated microenvironment characterized by exhausted CD8+ T cells toward dendritic-cell- and CD8+ T-cell-enriched niches associated with antitumor immune activity. Conclusion: BiTE-AXL/PD-1 promotes physical proximity between PD-1+ T cells and AXL+ cells, enhances antitumor immune responses, and remodels the immunosuppressive tumor microenvironment. These findings support PD-1/AXL bispecific targeting as a promising therapeutic strategy to overcome resistance to PD-1 blockade in HNSCC.

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Publication Details

Journal
bioRxiv (Cold Spring Harbor Laboratory)
Published
2026-10-28
DOI
https://doi.org/10.64898/2026.10.26.761755
Primary Topic
Cancer Immunotherapy and Biomarkers
Type
preprint

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preprint

Spatially enforced engagement of PD-1⁺ T cells with AXL⁺ cells reprograms the tumor microenvironment and overcomes anti-PD-1 resistance

Abraham Nissani, Tamar Shami, Monica Wainer, Leeat Keren et al.
2 links
bioRxiv (Cold Spring Harbor Laboratory)
Cancer Immunotherapy and Biomarkers
preprint

Spatially enforced engagement of PD-1⁺ T cells with AXL⁺ cells reprograms the tumor microenvironment and overcomes anti-PD-1 resistance

Abraham Nissani, Tamar Shami, Monica Wainer, Leeat Keren, Sankar Jagadeeshan, Uzi Hadad, Yaron Carmi, Moshe Elkabets, Raz Ben-Uri, Angel Porgador, Bar Kaufman, Kamran Waidha, Merav Cohen, Sarah Adi Eisdorfer, Ronnie Shapira‐Frommer, Amir Aharoni, Orit Itzhaki, Idan Milo, Yochai Wolf, Ksenia M. Yegodayev, Kong De, Ofir Cohen, Raghda Abu Shareb, Divyasree Marripati, Aseel Khateeb, Ekaterina Rudnitsky, Dinesh Babu Manikandan, Alice Raizman, Gregory Perelman, Oshrat Galibov-Levi, Hai Wang, Danialle Raviv, Gili Ezri, Jonatan Zorea, Noam Zuri, Ofra Novoplansky, Alona Veinblat
preprint en

Abstract

Background: AXL overexpression promotes tumor-cell plasticity, local immune suppression, and resistance to PD-1 blockade. Conventional anti-AXL and anti-PD-1 antibodies combinations act independently and do not ensure physical proximity between PD-1+ T cells and AXL+ cells within the immunosuppressive tumor microenvironment. Methods: To address this limitation, we developed a non-tandem, tetravalent IgG bispecific antibody designed to simultaneously engage PD-1 and AXL (BiTE-AXL/PD-1). Its antitumor activity was evaluated using in vitro functional assays and humanized and immunocompetent head and neck squamous cell carcinoma (HNSCC) models and compared with the combination of monospecific anti-AXL and anti-PD-1 antibodies. Spatial multiplex imaging was used to characterize treatment-associated changes in the tumor microenvironment of an anti-PD-1-resistant murine model. Results: BiTE-AXL/PD-1 enhanced T-cell activation, promoted AXL-dependent tumor-T-cell conjugation, and increased tumor-cell killing in vitro, outperforming the combination of the two monospecific antibodies. In humanized and immunocompetent HNSCC models, BiTE treatment significantly delayed tumor growth compared with the antibody combination and was well tolerated, with no overt toxicity. Spatial multiplex imaging revealed a shift from a fibroblast-dominated microenvironment characterized by exhausted CD8+ T cells toward dendritic-cell- and CD8+ T-cell-enriched niches associated with antitumor immune activity. Conclusion: BiTE-AXL/PD-1 promotes physical proximity between PD-1+ T cells and AXL+ cells, enhances antitumor immune responses, and remodels the immunosuppressive tumor microenvironment. These findings support PD-1/AXL bispecific targeting as a promising therapeutic strategy to overcome resistance to PD-1 blockade in HNSCC.

bioRxiv (Cold Spring Harbor Laboratory)
Ben-Gurion University of the Negev (IL), Tel Aviv University (IL), Sheba Medical Center (IL), University of Chinese Academy of Sciences (CN), Weizmann Institute of Science (IL), Tianjin Medical University (CN)
Israel Cancer Research Fund, United States-Israel Binational Science Foundation, National Natural Science Foundation of China, Israel Science Foundation
Good health and well-being
Cancer Immunotherapy and Biomarkers
DOIOpen Access PDFOpenAlex
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