Immune checkpoint blockade in melanoma by new targeted Doxorubicin immunoliposomes

Introduction: Programmed Cell Death Ligand 1(PD-L1), expressed in most solid tumors, binds to T-cells through its receptor PD-1, leading to the immunoresistance development and tumor progression [1]. Anti- PD-L1 monoclonal... [ view full abstract ]

Introduction: Programmed Cell Death Ligand 1(PD-L1), expressed in most solid tumors, binds to T-cells through its receptor PD-1, leading to the immunoresistance development and tumor progression [1]. Anti-
PD-L1 monoclonal antibody (mAb) blocks this ligand promoting antitumor activity of T-cells [2]. Moreover, Doxorubicin (Dox) liposomes improve the therapeutic effect over free drug. Both therapies can be combined in a single formulation of Dox liposomes decorated with anti-PD-L1 Fab’ fragments. Thus, the aim of this work is to develop and characterize Dox targeted liposomes and evaluate its antitumor effect in vitro and in vivo.

Methods: Dox liposomes (LPDOX) were prepared by film hydration method [3] and drug loaded by sulphate gradient [4]. DSPE-PEG2000-Mal micelles were coupled to anti-PD-L1-Fab' fragments and incorporated in preformed LPDOX with post insertion method, obtaining targeted liposomes (LPDOXFab’). After physicochemical characterization, drug release was measured in FBS at 37 ºC for 1h. B16 OVA melanoma mouse cell line was used for in vitro/in vivo studies. In vitro cytotoxicity (IC50) was obtained after 24h of drug exposure and Dox cell uptake was measured by FACS and confocal microscopy after 4h of treatment. Finally, in vivo antitumor efficacy was studied in tumour bearing mice.


Results: LPDPXFab’ and LPDOX were around 130 nm, PDI < 0.1 and 95±7.5% of encapsulation. Ligand conjugation was 40±7.4%. Drug release reached 20% after 1h in FBS. Targeted liposomes were able to bind specifically PD-L1, showing no internalization. IC50 values for both formulations were similar after 24h of exposure. There was a delay in melanoma tumor growth with no significant differences in both liposomes. However, survival for LPDOXFab’ was higher, suggesting a benefit that deserves to be explored.


Conclusion: LPDPXFab’ have been successfully developed using the post-insertion method. In vitro and in vivo studies revealed similar efficacy for both formulations. However, the difference in the survival suggests an improvement of the targeted formulation that will be deeper examined in further studies.


[1] Crit Rev Oncol Hematol. 2014 Jan; 89(1):140-65.
[2] Eur J Cancer. 2013 Sep; 49(14):2968-71.
[3] Eur J Pharm Biopharm. 2012 Jun; 81(2):273-80.
[4] J Control Release. 2012 Jun 10; 160(2): 117-34.