Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs

Title:Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs

Volume: 25 Issue: 5

Author(s): Foroogh Nejatollahi*, Elham Nadimi, Ali Noorafshan, Setareh Moazen, Ali Mohammad Alizadeh, Solmaz Khalighfard and Amirhossein Sahebkar

Affiliation:

• Recombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract:

Purpose: We aimed to assess the effects of a cocktail comprising three specific anti- HER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis.

Methods: Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of each of the three antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 10¹¹ phage-scFv cocktail/ kg. The mice were then injected with 2×10⁶ SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides.

Results: The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20 mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05).

Conclusion: Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Cite this article as:

Nejatollahi Foroogh*, Nadimi Elham, Noorafshan Ali, Moazen Setareh, Alizadeh Mohammad Ali, Khalighfard Solmaz and Sahebkar Amirhossein, Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs, Current Protein & Peptide Science 2024; 25 (5) . https://dx.doi.org/10.2174/0113892037269645231031095145