Activation of CD45 Reverses Tumor Suppression of Src Family Tyrosine Kinases in Leukocytes.

Introduction: Cancer cells apply multiple immunosuppressive mechanisms to evade immune-cell responses. The advent of immune-checkpoint inhibitors is a promising new strategy to treat solid tumors. This breakthrough is mainly based on monoclonal antibodies which block inhibitory molecules like the PD-1/PD-L1 axis to neutralize tumor immune evasion mechanisms. However, attempts to treat breast cancer with immunotherapy drugs have not shown much benefit for the vast majority of patients. The triple negative breast cancer (TNBC) is considered to be amongst the most aggressive subtype, generally with a worse outcome. Much effort is being invested to find a new generation of immune-modulatory strategies to address TNBC in particular.

Material and Methods and Results: We found a novel mechanism via which tumors induce immune suppression. C24D, a 24 amino acid homo-dimer peptide, reverses such tumor immunosuppression by binding and activating the CD45 receptor on leukocytes. Co-cultures of human TNBC cells with human peripheral blood mononuclear cells (PBMCs) resulted in the inhibition of the Src family of tyrosine kinases pathway in leukocytes. Addition of C24D to the immune-suppressed cells, induced Lck and Zap70 phosphorylation, resulting in TCR activation, tumor cell killing and IFNg secretion. The resulting increase of highly cytotoxic T and NK cell sub-populations: CD8⁺/CD69⁺, CD56⁺/CD69⁺, CD56⁺/CD57⁺ and CD8⁺/CD56⁺ in the C24D treated cultures, confirmed leukocytes activation.

In in vivo studies, intravenous treatment of C24D to nude mice engrafted with MDA-MB-231 human TNBC cells and transfused intravenously with human PBMCs resulted in inhibition of tumor growth compared with scrambled peptide treatment. In histological sections of the tumors extracted from the C24D treated mice, we found tumor infiltrated CD8⁺ and human activated CD56⁺ cells. Consequently, we found a significant increase in tumor apoptotic (caspase 3⁺) cells.

We demonstrated that the immune-activation effect of the C24D peptide on PBMCs occurs only in the presence of tumors. In the absence of tumors and in co-cultures with normal breast cells (MCF-10A), C24D does not activate Lck and ZAP70 in leukocytes. Moreover, no IFNg, TNFa and IL-2 was found in the supernatants of those cultures.

Conclusions: In this study we described a new mechanism of tumor immune suppression based on tumor inhibition of Lck and ZAP70 in T and NK cells. By binding to CD45 on leukocytes previously exposed to tumor cells, C24D peptide reverses tumor-induced immune-suppression, resulting in tumor cell killing. This study sets the stage for a new treatment modality of TNBC.