Evaluation of the anti-tumor effects of an anti-Human Epidermal growth factor receptor 2 (HER2) monoclonal antibody in combination with CD11b+/Gr-1+ myeloid cells depletion using a recombinant peptibody in 4 T1-HER2 tumor model.

INTRODUCTION: Clinical efficacy of Human Epidermal growth factor Receptor 2 (HER2) targeted strategies is limited due to impaired anti-tumor responses negatively regulated by immunosuppressive cells. We thus, investigated the inhibitory effects of an anti-HER2 monoclonal antibody (1 T0 mAb) in combination with CD11b+/Gr-1+ myeloid cells depletion in 4 T1-HER2 tumor model. METHODS: BALB/c mice were challenged with human HER2-expressing 4 T1 murine breast cancer cell line. A week post tumor challenge, each mouse received 50 µg of a myeloid cells specific peptibody every other day, or 10 mg/kg of 1 T0 mAb two times a week, and their combination for two weeks. The treatments effect on tumor growth was measured by calculating tumor size. Also, the frequencies of CD11b+/Gr-1+ cells and T lymphocytes were measured by flow cytometry. RESULTS: Peptibody treated mice indicated tumor regression and 40 % of the mice eradicated their primary tumors. The peptibody was capable to deplete notably splenic CD11b+/Gr-1+ cells as well as intratumoral CD11b+/Gr-1+ cells (P < 0.0001) and led to an increased number of tumor infiltrating CD8+ T cells (3.3 folds) and also that of resident tumor draining lymph nodes (TDLNs) (3 folds). Combination of peptibody and 1 T0 mAb resulted in enhanced expansion of tumor infiltrating CD4 + and CD8+ T cells which was associated with tumor eradication in 60 % of the mice. CONCLUSIONS: Peptibody is able to deplete CD11b+/Gr-1+ cells and increase anti-tumoral effects of the 1 T0 mAb in tumor eradication. Thus, this myeloid population have critical roles in development of tumors and their depletion is associated with induction of anti-tumoral responses.

Myeloid-derived suppressor cells (MDSCs) depletion by cabozantinib improves the efficacy of anti-HER2 antibody-based immunotherapy in a 4T1-HER2 murine breast cancer model.

BACKGROUND: Clinical trials using Cabozantinib have shown promising results in metastatic breast cancer. This efficacy mainly results from removing and/or polarization of tumor-promoting myeloid cells. Nevertheless, whether such myeloid-derived suppressor cells (MDSCs) depletion can be used to improve the efficacy of anti-HER2 antibodies in early breast cancer has not been defined yet. METHODS: BALB/c mice were inoculated with 4T1 and 4T1-HER2 murine tumor cell lines, and after 7 days, the mice were divided into different groups. Cabozantinib was orally administrated for 15 consecutive days, and anti-HER2 monoclonal antibody (mAb) 1 T0 was intraperitoneally injected twice a week. Tumor size was measured every other day. RESULTS: Our findings indicated that Cabozantinib combined with anti-HER2 mAb dramatically reduced tumor growth and increased tumor rejection (p = 0.0001). Flow cytometry analysis showed MDSC population decreased in TME, lymph nodes, and spleens by roughly 20%, 0.8%, and 35%, respectively. Myeloid suppressive phenotype was altered through inhibition of the expression of immunosuppressive factor Arg-1. Cytokine profiling of different groups indicated that the level of INF-γ was approximately two times higher than that in the control group, and IL-17 increased compared to the control group. However, IL-4 level was significantly reduced in the groups treated with Cabozantinib. These could bring about a 10% increase in CD8+ infiltration into the tumor bed and activation of tumor-draining lymph nodes and splenic T-lymphocytes. CONCLUSION: Collectively, our data provide pre-clinical evidence for using Cabozantinib to reshape the primary TME, which can enhance the effectiveness of anti-HER2 mAb immunotherapy in primary breast cancer.

Cloning, expression and characterization of a peptibody to deplete myeloid derived suppressor cells in a murine mammary carcinoma model.

BACKGROUND: Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we expressed and characterized a peptibody which had previously been defined by phage display technique capable of recognizing and depleting murine MDSCs. MATERIALS AND METHODS: Using splicing by overlap extension (SOE) PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. RESULTS: The purified peptibody appeared as a 70 KDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P < 0.05). CONCLUSION: In this study, a peptibody capable of depleting intratumoral MDSCs, was successfully expressed and purified. Our results imply that it could be considered as a potential tool for research on cancer immunotherapy.

Antioxidant nutrients can increase high-dose Methotrexate efficacy in 4T1 breast tumor Model: An experimental study on Vitamin E Succinate and Methyl-selenic acid.

BACKGROUND: We aimed to evaluate the anti-cancer and immune system enhancing properties of Vitamin E succinate (VES) and methylselenic acid (MSA) administration on 4T1 breast tumor model under high-dose methotrexate (HDMTX) therapy and folinic acid (FA) rescue. METHODS: Thirty six 4T1 mammary carcinoma bearing mice were randomly divided into six groups: control (untreated; n = 6), treatment-1 (T1 group; HDMTX; n = 6), T2 (T1 + FA; n = 6), T3 (T2 + MSA; n = 6), T4 (T2 + VES; n = 6) and T5 (T3 + VES; n = 6). On day 21 of the study, all surviving mice were sacrificed and primary tumors and peripheral tissues were examined for histological and gene expression assays. The expression of GATA Binding Protein-3 (GATA3), forkhead box-P3 (FOXP3), T-bet and Retinoic acid receptor-related orphan receptor γt (RORγt) were evaluated in tumors and spleens. Also, vascular endothelial growth factor-A (VEGF-A) and UL16-Binding Protein 1 (ULBP-1) expression were evaluated in tumors. RESULTS: The control, T4 and T5 groups were able to complete the entire 21-day study period. Also, significant tumor shrinkage was occurred in T4 group (P < 0.05). Suppression of splenic FOXP3 and GATA3 were observed in the mice receiving T4 and T5 regimens. Also, induction of tumoral FOXP3 and GATA3 were achieved in the T4 and T5 groups, respectively (P < 0.05). No metastasis occurred in T4 receiving group; while, lung and liver metastasis were observed in T5 group. CONCLUSION: In this study, high and fixed dose of MTX was used. Further studies are needed to optimize MTX dose along with FA, VES and MSA.