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Herceptin-conjugated liposomes loaded with lipocalin-2 siRNA for the treatment of Inflammatory Breast Cancer

ABSTRACT: Inflammatory breast cancer (IBC) is a rare and aggressive subtype of breast cancer (BC) that regularly affects women younger than 40 years and accounts for 1 to 5% of all types of BC. There is not an optimal treatment for women with IBC. Approximately 40% of IBC patients are HER2+, which can be treated with the monoclonal antibody-based therapy, Herceptin (trastuzumab). However, more than 25% of patients become resistant to trastuzumab-based therapy in early stages and about 75% in late stages patients and effective second line targeted therapies are currently unavailable for IBC patients. Lipocalin-2 (LCN2) is a is a secreted protein involved in iron homeostasis, immune responses, transportation of siderophores for degradation, and epithelial cell differentiation. LCN-2 is highly abundant in certain subtypes of aggressive cancers including IBC. Reports have shown that LCN-2 is a potential target therapy for IBC patients. Here, we developed a double targeted therapy of liposomal-Herceptin formulation loaded with LCN2-targeted small interference RNA (siRNA). Trastuzumab DSPE-PEG(2000)-maleimide conjugates were synthesized for liposomal formulation, resulting in sizes of 18.3 nm and 73.7 nm for free DSPE-PEG(2000)-trastuzumab and trastuzumab-liposome, respectively. This formulation was efficiently internalized into HER2+ IBC cells, and reduced cell proliferation and tumor emboli formation in IBC cells. To further understand the molecular mechanism downstream LCN-2, we performed RNA-sequencing (RNA-seq), and we identified 139 differentially expressed genes upon siRNA-mediated LCN-2 knockdown in IBC cells. Ingenuity Pathway Analysis grouped the RNA-seq data into 25 canonical pathways altered downstream LCN-2. Together, this information suggests that LCN2-target siRNA within Herceptin-conjugated liposomes is a potential therapeutic strategy against IBC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277628 | GEO | 2025/07/30

REPOSITORIES: GEO

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Project description:Post expression from the host cell, a bio-therapeutic will be subjected to several downstream processing steps prior to final formulation. Analysis of the product under various buffer conditions at each stage is desirable to show that the integrity of the sample is maintained and to monitor any potential product loss. In this study we examine how varying buffer conditions during downstream processing impact the structural integrity of a biopharmaceutical product, with a specific focus on the model IgG1 antibody, mAb4, provided by FUJIFILM Diosynth Biotechnologies. Flexibility, stability, aggregation propensity, and bulk properties were evaluated across the buffers of four sample points, including perfusion media, purification stages, and formulation buffer. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible buffer. Although mAb4 and Herceptin present with similar charge state distributions (CSD) in native mass spectrometry (MS) experiments, they exhibit distinct unfolding patterns during the activated ion mobility-mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). The greater structural stability and aggregation onset temperature (Tagg) observed in Herceptin, are primarily attributed to the heavier glycosylation and kappa-class light chains present in Herceptin compared to the lambda-class light chains in mAb4. Hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that while mAb4 experiences substantial structural changes during purification, characterized by high flexibility, a low melting temperature (Tm), and prevalent repulsive protein-protein interactions, it eventually transitions to a highly compact and stable structure in buffers with higher salt concentrations and during formulation. Notably, in the formulated environment, the third constant domain (CH3) of the heavy chain retains flexibility and is identified as a region of interest for aggregation formation. Here we showcase the integration of MS and orthogonal techniques to attain comprehensive information which can be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

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Herceptin-conjugated liposomes loaded with lipocalin-2 siRNA for the treatment of Inflammatory Breast Cancer

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