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Mild hyperthermia influence on Herceptin(®) properties.

Escoffre JM, Deckers R, Sasaki N, Bos C, Moonen C - Radiol Oncol (2015)

Bottom Line: Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged.The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT.Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

View Article: PubMed Central - PubMed

Affiliation: Imaging Division, UMC Utrecht, Utrecht, the Netherlands.

ABSTRACT

Background: Mild hyperthermia (mHT) increases the tumor perfusion and vascular permeability, and reduces the interstitial fluid pressure, resulting in better intra-tumoral bioavailability of low molecular weight drugs. This approach is potentially also attractive for delivery of therapeutic macromolecules, such as antibodies. Here, we investigated the effects of mHT on the stability, immunological and pharmacological properties of Herceptin(®), a clinically approved antibody, targeting the human epidermal growth factor receptor 2 (HER-2) overexpressed in breast cancer.

Results: Herceptin(®) was heated to 37°C (control) and 42°C (mHT) for 1 hour. Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged. Immunological and pharmacological properties of Herceptin(®) were evaluated following mHT using HER-2 positive breast cancer cells (BT-474). Exposure of Herceptin(®) to mHT preserved recognition and binding affinity of Herceptin(®) to HER-2. Western-blot and cell proliferation assays on BT-474 cells showed that mHT left the inhibitory activities of Herceptin(®) unchanged.

Conclusions: The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT. Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

No MeSH data available.


Related in: MedlinePlus

Binding affinity of Herceptin® to HER-2. BT-474 cells were first incubated with unconjugated Herceptin® (5 × 10−6 to 5 × 10−2 mg/mL) and subsequently with FITC-Herceptin®. Fluorescence intensity on flow cytometry is plotted as a function of unlabeled Herceptin® concentration used for receptor saturation. Data expressed as mean ± SD calculated from three independent experiments and are fitted with Variable slope model (solid curve; confidence intervals, dotted curve) with a 95% confidence interval. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant).
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f3-rado-49-01-41: Binding affinity of Herceptin® to HER-2. BT-474 cells were first incubated with unconjugated Herceptin® (5 × 10−6 to 5 × 10−2 mg/mL) and subsequently with FITC-Herceptin®. Fluorescence intensity on flow cytometry is plotted as a function of unlabeled Herceptin® concentration used for receptor saturation. Data expressed as mean ± SD calculated from three independent experiments and are fitted with Variable slope model (solid curve; confidence intervals, dotted curve) with a 95% confidence interval. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant).

Mentions: To gain insight into the effect of mHT on the immunological properties of Herceptin®, the binding affinity of this antibody to HER-2 receptors was assessed by a competition assay on BT-474 cells using FITC-coupled Herceptin®. Figure 3 indicates that low concentrations of unlabeled and native Herceptin® (i.e., below 5×10−5 mg/mL) did not change the binding of FITC-conjugated Herceptin® to HER-2 receptors, while a concentration range from 1×10−4 mg/mL to 5×10−2 mg/mL of this antibody induced a significant inhibition of the binding of FITC-coupled Herceptin®. Heated Herceptin® led to a similar inhibition of FITC-conjugated Herceptin® as native Herceptin®. Moreover, native and heated Herceptin® show comparable IC50 values, i.e., 3.1×10−4 mg/mL and 3.6×10−4 mg/mL (p > 0.05), respectively. These results indicate that mHT does not modify the recognition and the binding affinity of Herceptin® to HER-2 receptors.


Mild hyperthermia influence on Herceptin(®) properties.

Escoffre JM, Deckers R, Sasaki N, Bos C, Moonen C - Radiol Oncol (2015)

Binding affinity of Herceptin® to HER-2. BT-474 cells were first incubated with unconjugated Herceptin® (5 × 10−6 to 5 × 10−2 mg/mL) and subsequently with FITC-Herceptin®. Fluorescence intensity on flow cytometry is plotted as a function of unlabeled Herceptin® concentration used for receptor saturation. Data expressed as mean ± SD calculated from three independent experiments and are fitted with Variable slope model (solid curve; confidence intervals, dotted curve) with a 95% confidence interval. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362605&req=5

f3-rado-49-01-41: Binding affinity of Herceptin® to HER-2. BT-474 cells were first incubated with unconjugated Herceptin® (5 × 10−6 to 5 × 10−2 mg/mL) and subsequently with FITC-Herceptin®. Fluorescence intensity on flow cytometry is plotted as a function of unlabeled Herceptin® concentration used for receptor saturation. Data expressed as mean ± SD calculated from three independent experiments and are fitted with Variable slope model (solid curve; confidence intervals, dotted curve) with a 95% confidence interval. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p < 0.05 (NS, non-significant).
Mentions: To gain insight into the effect of mHT on the immunological properties of Herceptin®, the binding affinity of this antibody to HER-2 receptors was assessed by a competition assay on BT-474 cells using FITC-coupled Herceptin®. Figure 3 indicates that low concentrations of unlabeled and native Herceptin® (i.e., below 5×10−5 mg/mL) did not change the binding of FITC-conjugated Herceptin® to HER-2 receptors, while a concentration range from 1×10−4 mg/mL to 5×10−2 mg/mL of this antibody induced a significant inhibition of the binding of FITC-coupled Herceptin®. Heated Herceptin® led to a similar inhibition of FITC-conjugated Herceptin® as native Herceptin®. Moreover, native and heated Herceptin® show comparable IC50 values, i.e., 3.1×10−4 mg/mL and 3.6×10−4 mg/mL (p > 0.05), respectively. These results indicate that mHT does not modify the recognition and the binding affinity of Herceptin® to HER-2 receptors.

Bottom Line: Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged.The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT.Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

View Article: PubMed Central - PubMed

Affiliation: Imaging Division, UMC Utrecht, Utrecht, the Netherlands.

ABSTRACT

Background: Mild hyperthermia (mHT) increases the tumor perfusion and vascular permeability, and reduces the interstitial fluid pressure, resulting in better intra-tumoral bioavailability of low molecular weight drugs. This approach is potentially also attractive for delivery of therapeutic macromolecules, such as antibodies. Here, we investigated the effects of mHT on the stability, immunological and pharmacological properties of Herceptin(®), a clinically approved antibody, targeting the human epidermal growth factor receptor 2 (HER-2) overexpressed in breast cancer.

Results: Herceptin(®) was heated to 37°C (control) and 42°C (mHT) for 1 hour. Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged. Immunological and pharmacological properties of Herceptin(®) were evaluated following mHT using HER-2 positive breast cancer cells (BT-474). Exposure of Herceptin(®) to mHT preserved recognition and binding affinity of Herceptin(®) to HER-2. Western-blot and cell proliferation assays on BT-474 cells showed that mHT left the inhibitory activities of Herceptin(®) unchanged.

Conclusions: The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT. Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

No MeSH data available.


Related in: MedlinePlus