Background Anti-inflammatory (M2) tumour-associated macrophages (TAMs) exert protumoural roles through angiogenesis, immunosuppression and resistance to therapies.¹ M2 TAMs express the mannose receptor, CD206,² excellent marker for targeted therapies. We have previously identified a peptide called mUNO² that specifically binds to CD206 on M2 TAMs. Aiming to dissect the role of CD206^high M2 TAMs in the tumour progression and immunosuppression, we depleted them using an mUNO and doxorubicin (Adriamycin®)-containing polymer-drug nanoconjugate (St-PGA-DOX-mUNO, ‘OximUNO’) where the polymer backbone is branched polyglutamic acid (St-PGA).³

Materials and Methods We compared OximUNO with free DOX and the untargeted nanoconjugate St-PGA-DOX. To study the in vitro cytotoxicity of the nanoconjugates, we used M2 and M1 skewed macrophages derived from human blood buffy coat. To study the in vivo homing of nanoconjugates we used an orthotopic triple negative breast cancer (TNBC, 4T1 cells) model and a TNBC experimental metastases model in immunocompetent mice. For in vivo therapeutic efficacy studies, we used orthotopic and experimental metastases models of TNBC, and administered the compounds intraperitoneally (i.p.).

Results In vitro, OximUNO showed 39% higher toxicity to the primary human M2 macrophages than St-PGA-DOX, and 31% lower toxicity to the M1 macrophages than St-PGA-DOX. In vivo, OximUNO showed no change in creatinine or alanine aminotransferase values, indicating no toxic effects to the kidneys or liver. Compared to control St-PGA, i.p.-administered St-PGA-mUNO, showed improved homing to M2 TAMs in both orthotopic and experimental metastases models with low accumulation in the liver. In the orthotopic treatment study, only OximUNO significantly reduced the tumour volume and showed 56% and 38% less lung metastases than DOX and St-PGA-DOX, respectively. Additionally, DOX and St-PGA-DOX produced a significant bodyweight loss whereas OximUNO did not. Importantly, OximUNO treatment resulted in 2-5-fold increase in the ratio of CD8⁺/FOXP3⁺ expression, suggesting a shift in the immune landscape towards an immunostimulatory profile. In the experimental metastases model, OximUNO monotherapy resulted in the highest reduction of lung metastases, and this effect correlated with a significant reduction in CD206^high M2 TAMs; whereas no significant effect on M2 TAMs population was observed with DOX or untargeted nanoconjugate.

Conclusions Our data suggests that the elimination of CD206^high M2 TAMs with OximUNO suppresses spontaneous and experimental metastases in safe manner, shifts immune landscape towards immunostimulatory and could therefore be a potential treatment option for TNBC patients.

References

1. Hughes R, Qian B-Z, Rowan C, Muthana M, Keklikoglou I, Olson OC, et al. Perivascular M2 macrophages stimulate tumor relapse after chemotherapy. Cancer Res 2015;75(17):3479–91.

2. Scodeller P, Simón-Gracia L, Kopanchuk S, Tobi A, Kilk K, Säälik P, et al. Precision targeting of tumor macrophages with a CD206 binding peptide. Sci Rep 2017;7(1):14655.

3. Duro-Castano A, Nebot VJ, Niño-Pariente A, Armiñán A, Arroyo-Crespo JJ, Paul A, et al. Capturing ‘extraordinary’ soft-assembled charge-like polypeptides as a strategy for nanocarrier design. Adv Mater 2017;29(39):1702888.

Disclosure Information A. Lepland: None. A. Malfanti: None. U. Haljasorg: None. S. Dordevic: None. L. Salumäe: None. P. Peterson: None. T. Teesalu: None. M.J. Vicent: None. P. Scodeller: None.