BACKGROUND: The tumoricidal complex alpha1-oleate targets bladder cancer cells, triggering rapid, apoptosis-like tumor cell death. Clinical effects of alpha1-oleate were recently observed in patients with non-muscle invasive bladder cancer (NMIBC), using a randomized, placebo-controlled study protocol. AIMS: To investigate if there are dose-dependent effects of alpha1-oleate. MATERIALS AND METHODS: Here, patients with NMIBC were treated by intravesical instillation of increasing concentrations of alpha1-oleate (1.7, 8.5, or 17 mM) and the treatment response was defined relative to a placebo group. RESULTS: Strong, dose-dependent anti-tumor effects were detected in alpha1-oleate treated patients for a combination of molecular and clinical indicators; a complete or partial response was detected in 88% of tumors treated with 8.5 mM compared to 47% of tumors treated with 1.7 mM of alpha1-oleate. Uptake of alpha1-oleate by the tumor triggered rapid shedding of tumor cells into the urine and cell death by an apoptosis-like mechanism. RNA sequencing of tissue biopsies confirmed the activation of apoptotic cell death and strong inhibition of cancer gene networks, including bladder cancer related genes. Drug-related side effects were not recorded, except for local irritation at the site of instillation. DISCUSSION AND CONCLUSIONS: These dose-dependent anti-tumor effects of alpha1-oleate are promising and support the potential of alpha1-oleate treatment in patients with NMIBC.

• Keywords
• alpha1‐oleate complex, cell shedding, cellular uptake, gene expression, non‐muscle invasive bladder cancer,
• MeSH
• Administration, Intravesical MeSH
• Apoptosis drug effects   MeSH
• Oleic Acid * therapeutic use   MeSH
• Middle Aged MeSH
• Humans MeSH
• Urinary Bladder Neoplasms * drug therapy   pathology   genetics   MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Treatment Outcome MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Clinical Trial, Phase I MeSH
• Clinical Trial, Phase II MeSH
• Randomized Controlled Trial MeSH
• Names of Substances
• Oleic Acid * MeSH
• Antineoplastic Agents MeSH

BACKGROUND: The molecular content of urine is defined by filtration in the kidneys and by local release from tissues lining the urinary tract. Pathological processes and different therapies change the molecular composition of urine and a variety of markers have been analyzed in patients with bladder cancer. The response to BCG immunotherapy and chemotherapy has been extensively studied and elevated urine concentrations of IL-1RA, IFN-α, IFN-γ TNF-α, and IL-17 have been associated with improved outcome. METHODS: In this study, the host response to intravesical alpha 1-oleate treatment was characterized in patients with non-muscle invasive bladder cancer by proteomic and transcriptomic analysis. RESULTS: Proteomic profiling detected a significant increase in multiple cytokines in the treatment group compared to placebo. The innate immune response was strongly activated, including IL-1RA and pro-inflammatory cytokines in the IL-1 family (IL-1α, IL-1β, IL-33), chemokines (MIP-1α, IL-8), and interferons (IFN-α2, IFN-γ). Adaptive immune mediators included IL-12, Granzyme B, CD40, PD-L1, and IL-17D, suggesting broad effects of alpha 1-oleate treatment on the tumor tissues. CONCLUSIONS: The cytokine response profile in alpha 1-oleate treated patients was similar to that reported in BCG treated patients, suggesting a significant overlap. A reduction in protein levels at the end of treatment coincided with inhibition of cancer-related gene expression in tissue biopsies, consistent with a positive treatment effect. Thus, in addition to killing tumor cells and inducing cell detachment, alpha 1-oleate is shown to activate a broad immune response with a protective potential.

• Keywords
• BCG, alpha1‐oleate, bladder cancer, immune response, proteomic analysis,
• MeSH
• Interleukin 1 Receptor Antagonist Protein therapeutic use   MeSH
• BCG Vaccine * therapeutic use   MeSH
• Cytokines MeSH
• Immunity MeSH
• Interferon-alpha pharmacology   therapeutic use   MeSH
• Oleic Acid MeSH
• Humans MeSH
• Urinary Bladder Neoplasms * pathology   MeSH
• Proteomics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Interleukin 1 Receptor Antagonist Protein MeSH
• BCG Vaccine * MeSH
• Cytokines MeSH
• Interferon-alpha MeSH
• Oleic Acid MeSH

The protein-lipid complex alpha1-oleate, derived from HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), is identified as a molecular entity with significant therapeutic potential. Structural characterization of the complex and results of a successful placebo-controlled clinical trial are presented.

• Keywords
• Novel cancer therapy, bladder cancer, low toxicity, peptide-oleate complex, protein folding,
• Publication type
• Journal Article MeSH

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.

• MeSH
• Apoptosis drug effects   MeSH
• Endocytosis drug effects   MeSH
• Protein Conformation MeSH
• Oleic Acids chemistry   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Urinary Bladder Neoplasms drug therapy   genetics   pathology   MeSH
• Peptides chemistry   pharmacology   therapeutic use   MeSH
• Placebos MeSH
• Proton Magnetic Resonance Spectroscopy MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Amino Acid Sequence MeSH
• Endpoint Determination MeSH
• Thermodynamics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Clinical Trial, Phase I MeSH
• Clinical Trial, Phase II MeSH
• Research Support, Non-U.S. Gov't MeSH
• Randomized Controlled Trial MeSH
• Names of Substances
• Oleic Acids MeSH
• Peptides MeSH
• Placebos MeSH