{
  "abstract": "Background HER2-low tumors account for 60–70% of metastatic breast cancer (MBC) cases and, unlike HER2-overexpressing tumors, do not depend on HER2 signaling for survival. Trastuzumab-based treatments have failed to improve the outcomes of patients with HER2-low breast cancer. Despite promising results with trastuzumab-deruxtecan, significant systemic toxicity and disease progression were observed, highlighting the need to develop safer, more effective, and accessible therapies. 1 2 We evaluated the therapeutic potential of surface-engineered natural killer (SE-NK) cells conjugated with trastuzumab (Tz) for selective targeting of HER2-low breast cancer cells.Methods To establish a preclinical model, the triple-negative murine breast cancer cell line 4T1 was transduced with a lentiviral vector encoding human HER2. HER2 expression was validated using flow cytometry, confocal microscopy, and Western blot analysis. SE-NK cells were generated by embedding 25 µg of Tz onto the mouse-derived LNK cells 3 or human NK92 cells, using a hydrophobic linker. HER2-specific cytotoxicity was assessed in vitro by live cell imaging integrated with Caspase 3/7 assay against the 4T1-HER2-low cells and HER2-low MDA-MB-175 human breast cancer cells.Results Transduced 4T1-HER2-low cells expressed HER2 at levels comparable to those observed in human HER2-low breast tumors ( figure 1). In vitro cytotoxicity assays with human cells revealed that SE-NK92/Tz cells exhibited significantly enhanced tumor cell killing (91%, p<0.05) compared to unmodified NK cells. Similar trends were observed with SE-LNK/Tz cells against 4T1-HER2-low targets (figure 2).Conclusions We created, to our knowledge, the first murine cell line with human HER2-low status, validating their use as a preclinical model. This study establishes the feasibility of using SE-NK/Tz cells as a novel immunotherapeutic approach for HER2-low breast cancer. Our preliminary findings confirmed the targeted nature of this engineered platform, supporting its potential to overcome the limitations of current therapies. Future studies will explore mechanisms of immune activation and therapeutic efficacy in diverse in vivo HER2-low models. Acknowledgements We would like to thank, Dr. Young-Wook Won at U of North Texas for providing the linker reagent for the project and his assessment throughout the NK cell surface engineering process, The Illinois Cancer Health Equity Research (I-CHER) Center for funding this projectReferences Modi S, Jacot W, Yamashita T, Sohn J, Vidal M, Tokunaga E, Tsurutani J, Ueno NT, Prat A, Chae YS, Lee KS, Niikura N, Park YH, Xu B, Wang X, Gil-Gil M, Li W, Pierga JY, Im SA, Moore HCF, Rugo HS, Yerushalmi R, Zagouri F, Gombos A, Kim SB, Liu Q, Luo T, Saura C, Schmid P, Sun T, Gambhire D, Yung L, Wang Y, Singh J, Vitazka P, Meinhardt G, Harbeck N, Cameron DA; DESTINY-Breast04 Trial Investigators. Trastuzumab deruxtecan in previously treated HER2-low advanced breast cancer. N Engl J Med. 2022 Jul 7;387(1):9-20.Modi S, Saura C, Yamashita T, Park YH, Kim SB, Tamura K, Andre F, Iwata H, Ito Y, Tsurutani J, Sohn J, Denduluri N, Perrin C, Aogi K, Tokunaga E, Im SA, Lee KS, Hurvitz SA, Cortes J, Lee C, Chen S, Zhang L, Shahidi J, Yver A, Krop I; DESTINY-Breast01 Investigators. Trastuzumab deruxtecan in previously treated HER2-positive breast cancer. N Engl J Med. 2020 Feb 13;382(7):610-621.Tsutsui H, Nakanishi K, Matsui K, Higashino K, Okamura H, Miyazawa Y, Kaneda K. IFN-gamma-inducing factor up-regulates fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J Immunol. 1996 Nov 1;157(9):3967-73. PMID: 8892629.Abstract 325 Figure 1In Vitro and In Vivo Validation of a Murine HER2-Low Cell Line for Syngeneic Studies. (A) Flow cytometry analysis confirms low surface expression of human HER2 in transduced 4T1-HER2-low cells, comparable to theHER2-low human breast cancer cell line MDA-MB-175. Cells were gated on live, single-cell populations. (B) Confocal microscopy demonstrates membrane localization of HER2 in engineered 4T1-HER2-low cells. Cells were stained with anti-HER2 antibody (red) and DAPI (blue) to visualize nuclei, confirming HER2 surface expression. (C) Western blot analysis shows low HER2 protein expression in 4T1-HER2-low cells. Densitometric quantification reveals expression levels similar to those of MDA-MB-175 cells. (D) Orthotopic implantation of 4T1-HER2-low cells into BALB/c mice (n = 4) results in consistent tumor formation, validating their utility for syngeneic in vivo studiesAbstract 325 Figure 2Targeted Cytotoxicity of NK Cells in HER2-Low Tumor Co-cultures. Real-time imaging of tumor cell apoptosis over 48h using Incucyte Caspase-3/7 Green reagent. (A) The highest caspase activity was observed in MDA-MB-175 (HER2-low) cells co-cultured with SE-NK92 cells, which was statistically significant compared to naked NK92 cells, indicating targeting via HER2 expression. (B) 4T1 HER2-low cells co-cultured with SE-NK cells also show the highest caspase activity compared to naked NK cells",
  "authors": [
    {
      "affiliations": [
        "University of Illinois Chicago, Chicago, IL, USA"
      ],
      "name": "Maria J Godoy Calderon"
    },
    {
      "affiliations": [
        "University of Illinois Chicago, Chicago, IL, USA"
      ],
      "name": "Manali Patwardhan"
    },
    {
      "affiliations": [
        "University of Illinois Chicago, Chicago, IL, USA"
      ],
      "name": "Eric Gauchat"
    },
    {
      "affiliations": [
        "University of Illinois Chicago, Chicago, IL, USA"
      ],
      "name": "Vijayakrishna Gadi"
    }
  ],
  "title": "325 A next-generation immunotherapy targeting HER2-low breast cancer using surface-engineered NK/trastuzumab cells",
  "uid": "cc073cdf-6942-5f2a-a32a-11f7c2d7cb73"
}