Abstract
The cyclic VHCDR3-derived peptide (Rb9) from RebMab200 antibody, directed to a NaPi2B phosphate-transport protein, displayed anti-metastatic melanoma activity at 50–300 μg intraperitoneally injected in syngeneic mice. Immune deficient mice failed to respond to the peptide protective effect. Rb9 induced increased CD8+ T and low Foxp3+ T cell infiltration in lung metastases and high IFN-γ and low TGF-β in lymphoid organs. The peptide co-localized with F-actin and a nuclear site in dendritic cells and specifically bound to MIF and CD74 in a dot-blot setting. Murine bone-marrow dendritic cells preincubated with Rb9 for 6 h were treated with MIF for short time periods. The modulated responses showed stimulation of CD74 and inhibition of pPI3K, pERK, and pNF-κB as compared to MIF alone. Rb9 in a melanoma-conditioned medium, stimulated the M1 type conversion in bone marrow-macrophages. Functional aspects of Rb9 in vivo were studied in therapeutic and prophylactic protocols using a melanoma metastatic model. In both protocols Rb9 exhibited a marked anti-melanoma protection. Human dendritic cells were also investigated showing increased expression of surface markers in response to Rb9 incubation. Rb9 either stimulated or slightly inhibited moDCs submitted to inhibitory (TGF-β and IL-10) or activating (LPS) conditions, respectively. Lymphocyte proliferation was obtained with moDCs stimulated by Rb9 and tumor cell lysate. In moDCs from cancer patients Rb9 exerted immunomodulatory activities depending on their functional status. The peptide may inhibit over-stimulated cells, stimulate poorly activated and suppressed cells, or cause instead, little phenotypic and functional alterations. Recently, the interaction MIF-CD74 has been associated to PD-L1 expression and IFN-γ, suggesting a target for melanoma treatment. The effects described for Rb9 and the protection against metastatic melanoma may suggest the possibility of a peptide reagent that could be relevant when associated to modern immunotherapeutic procedures.
Highlights
Cancer is a leading cause of human death with high incidence in low, middle and high-income countries [1, 2]
The anti-tumor protective effects of Rb9 against metastatic melanoma, which depends on a healthy immune response and immune-modulatory activation of murine or human dendritic cells, and the possible molecular mechanism of this response were further investigated in the present work as an important step to the development of new anticancer drugs
BmDCs from mice were stained for CD11c, CD11b, and CD74 after treatment with 50 μg/mL Rb9 for 48 h and 200 ng/mL LPS for 24 h, and Bone marrowderived macrophages (bmM s) were stained for F4/80, MHC-II, CD86, PD-L1, and CD206 in PBS, 0.5% BSA for 30 min using the different antibody-fluorophore conjugates from MACS, Miltenyi Biotec
Summary
Cancer is a leading cause of human death with high incidence in low, middle and high-income countries [1, 2]. High rates of resistance and relapse in anticancer treatment stimulate the search for additional agents, able to modulate dendritic cells and effector or regulatory T lymphocytes, memory T and B lymphocytes, which could improve the anti-infective or anti-tumor effectiveness of the immune response [18, 19]. The present work focus on the anti-tumor effect of an immunologically bioactive synthetic peptide, Rb9, derived from the complementarity determining region-3 (CDR3) of VH from a humanized monoclonal antibody (RebmAb 200) to NaPi2b transporter [22]. The anti-tumor protective effects of Rb9 against metastatic melanoma, which depends on a healthy immune response and immune-modulatory activation of murine or human dendritic cells, and the possible molecular mechanism of this response were further investigated in the present work as an important step to the development of new anticancer drugs
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