Engineered exosomes emerging from muscle cells break the tolerance towards tumor self-antigens

Researchers at the National Center for Global Health, Italy recently described a novel biotechnological platform for the production of unrestricted cytotoxic T lymphocyte (CTL) vaccines. It relies on in vivo engineering of exosomes, i.e., nanovesicles constitutively released by all cells, with full-length antigens of choice upon fusion with an exosome-anchoring protein referred to as Nefmut. They are produced upon intramuscular injection of a DNA vector and, when uploaded with a viral tumor antigen, were found to elicit an immune response inhibiting the tumor growth in a model of transplantable tumors. However, for a possible application in cancer immunotherapy, a number of key issues remained unmet. Among these, the researchers have now investigated: (i) whether the immunogenic stimulus induced by the engineered exosomes can break immune tolerance, and (ii) their effectiveness when applied in human system.

As a model of immune tolerance, the researchers considered mice transgenic for the expression of activated rat HER2/neu which spontaneously develop adenocarcinomas in all mammary glands. When these mice were injected with a DNA vector expressing the product of fusion between Nefmut and the extracellular domain of HER2/neu, antigen-specific CD8+ T lymphocytes became readily detectable. This immune response associated with a HER2-directed CTL activity and a significant delay in tumor development. On the other hand, through cross-priming experiments, they demonstrated the effectiveness of the engineered exosomes emerging from transfected human primary muscle cells in inducing antigen-specific CTLs. They propose their CTL vaccine platform as part of new immunotherapy strategies against tumors expressing self-antigens, i.e., products highly expressed in oncologic lesions but tolerated by the immune system.

Nef-specific CTL activity elicited by human DCs co-cultivated with
muscle cells expressing Nef^mut-based DNA vectors

a Scheme of the cross-priming assays. SKMC were transfected and, 48 h later, put in co-culture with iDCs which, after additional 24 h, were isolated and matured. Autologous PBLs were then added to mDCs, and the co-cultivation was carried out for 7 days. Afterwards, the PBL stimulation was repeated, and after additional 7 days, CD8⁺ T lymphocytes were isolated and tested in CTL assays through co-cultivation with syngeneic target cells. b Western blot analysis of cell lysates from either parental or Nef^mut stably transfected MCF-7 cells. Filters were incubated with either anti-Nef or anti-β-actin Abs. Arrow signs the relevant protein product. Molecular markers are given in kDa. c CTL assay carried out by co-cultivating primed CD8⁺ T lymphocytes with MCF-7 cells expressing or not Nef^mut in a 10:1 cell ratio. Results were presented as mean values +SD calculated from triplicate conditions of three independent experiments. *p < 0.05. Mean value of background conditions (i.e., co-culture of naïve CD8⁺ T lymphocytes with MCF-7), 11.9 ± 5

These researchers have established a novel, exosome-based method to produce unrestricted CTL vaccines. This strategy is effective in breaking the tolerance towards tumor self-antigens. The method is also useful to elicit antigen-specific CTL immunity in humans. These findings open the way towards the use of this antitumor strategy in clinic.