Current Protein & Peptide ScienceCurrent Protein & Peptide ScienceCurrent Protein & Peptide Science1389-20371875-5550Bentham Science64562910.2174/0113892037269645231031095145Life SciencesResearch ArticleReduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvsNejatollahiForooghinfo@benthamscience.netNadimiElhaminfo@benthamscience.netNoorafshanAliinfo@benthamscience.netMoazenSetarehinfo@benthamscience.netAlizadehAliinfo@benthamscience.netKhalighfardSolmazinfo@benthamscience.netSahebkarAmirhosseininfo@benthamscience.netRecombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical SciencesRecombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical ScienceDepartment of Neuroscience,, City University in CanadaFaculty of Pharmaceutical Sciences, University of British ColumbiaCancer Research Center, Tehran University of Medical SciencesBiotechnology Research Center, Pharmaceutical Technology Institute,, Institute, Mashhad University of Medical Sciences0105202425540941811012025Copyright ©; 2024, Bentham Science Publishers2024Bentham Science Publishershttps://journals.eco-vector.com/1389-2037/article/view/645629

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-valup < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-valup < 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.

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