Liposomes targeted to MHC-restricted antigen improve drug delivery and antimelanoma response

Abstract

Purpose: Melanoma is the most aggressive form of skin cancer. Chemotherapy at a late stage fails due to low accumulation in tumors, indicating the need for targeted therapy.

Materials and methods: To increase drug uptake by tumor cells, we have targeted doxorubicin-containing liposomes using a T-cell receptor (TCR)-like antibody (scFv G8 and Hyb3) directed against melanoma antigen A1 (MAGE-A1) presented by human leukocyte antigen A1 (M1/A1). With the use of flow cytometry and confocal microscopy, we have tested our formulation in vitro. In vivo pharmacokinetics was done in tumor-free nu/nu mice, while biodistribution and efficacy study was done in nu/nu mice xenograft.

Results: We demonstrated two to five times higher binding and internalization of these immunoliposomes by M1⁺/A1⁺ melanoma cells in vitro in comparison with nontargeted liposomes. Cytotoxicity assay showed significant tumor cell kill at 10 µM doxorubicin (DXR) for targeted vs nontargeted liposomes. In vivo pharmacokinetics of nontargeted and targeted liposomes were similar, while accumulation of targeted liposomes was 2- to 2.5-fold and 6.6-fold enhanced when compared with nontargeted liposomes and free drug, respectively. Notably, we showed a superior antitumor activity of MAGE-A1-targeted DXR liposomes toward M1⁺/A1⁺ expressing tumors in mice compared with the treatment of M1^-/A1⁺ tumors. Our results indicate that targeted liposomes showed better cytotoxicity in vitro and pharmacokinetics in vivo.

Conclusion: Liposomes decorated with TCR-mimicking scFv antibodies effectively and selectively target antigen-positive melanoma. We showed that DXR-loaded liposomes coupled to anti-M1/-A1 scFv inflict a significant antitumor response. Targeting tumor cells specifically promotes internalization of drug-containing nanoparticles and may improve drug delivery and ultimately antitumor efficacy. Our data argue that targeting MAGE in A1 context, by nanosized carriers decorated with TCR-like antibodies mimicking scFv, can be used as a theragnostic platform for drug delivery, immunotherapy, and potentially imaging, and diagnosis of melanoma.

Keywords: MAGE-A1; TCR-mimicking scFv; antigen A1; chemotherapy; immunotherapy; melanoma; targeted liposomes.

Figure 1

Stability of liposomal formulation in HEPES and 100% FBS at 37°C over time. Notes: The leakage of encapsulated DXR from liposomes (DXR-L, DXR-L scFv G8, and DXR-L scFv Hyb3) in different media and incubation times is shown. In black, measurements in HEPES are shown and in color, measurements done in FBS are shown: blue, DXR-L; purple, DXR-L scFv G8; and green, DXR-L scFv Hyb3. (A) Stability of DXR liposomes during 1 hour of incubation in HEPES and 100% FBS at 37°C. Lines represent the continuous drug fluorescence measured (at every second). (B) Stability of DXR liposomal formulations up to 24 hours in HEPES and 100% FBS at 37°C. Dots correspond to sampling time points and bars correspond to the standard error mean. These experiments were done with three independent batches of each formulation. Abbreviations: DXR, doxorubicin; DXR-L, DXR-loaded liposome.

Figure 2

Interaction of liposomal formulations with tumor cells in vitro. Notes: Fluorescence signal of liposomes on cells is shown. Flow cytometry was performed on all four cell lines at 37°C for 2 hours and CF signal (lipids) (A) and DXR signal (B) was recorded. Cells were incubated with DXR-Ls and allowed to bind and/or internalize. Bars represent the average and standard error mean of three independent experiments of all cell lines tested. *P<0.05. (C) Live-cell confocal imaging of cells exposed to all the DXR-L formulations for 1 hour and incubated for additional 24 hours with medium. (i) MZ2Mel43 (M1⁺/A1⁺), (ii) G43 (M1⁺/A1⁺), (iii) Mel78 (M1⁻/A1⁺), and (iv) Mel2A (M1⁻/A1⁺) cells. Nuclei are stained with Hoechst and are shown in blue; liposomes had CF-PE in the bilayer and are shown in green, DXR in red, and bright field in black and white contrast. Abbreviations: DXR, doxorubicin; DXR-Ls, DXR-loaded liposomes.

Figure 3

Melanoma cell survival after exposure to liposomal formulations. Notes: (A) M1⁺/A1⁺ (MZ2Mel43 and G43) and M1⁻/A1⁺ (Mel2A and Mel78) melanoma cell lines were exposed to free DXR and DXR-L formulations (nontargeted and targeted) for 10, 30, 60, and 120 minutes. Medium was refreshed and cells were incubated until 72 hours. Data are presented as the mean percentage and standard error mean of three independent batches (i) MZ2Mel43 (M1⁺/A1⁺) cells, (ii) G43 (M1⁺/A1⁺), (iii) Mel78 (M1⁻/A1⁺), and (iv) Mel2A (M1⁻/A1⁺) cells. DXR concentration (µM) is on x-axis and cell survival in percentage is on y-axis. Mann–Whitney U test was used to compare treatments with each other. **P<0.01. (B) Cellular viability curves from (A) were used to calculate IC₅₀ values (µM) for various melanoma cell lines and treatments. The concentration >50 is indicated in case IC₅₀ was not reached even at 50 µM. Abbreviations: DXR, doxorubicin; DXR-L, DXR-loaded liposome.

Figure 4

Blood circulation profile of immunoliposomes in nontumor-bearing mice. Notes: Mice were systemically injected with DXR-L formulations at 4 mg/kg DXR dose. Blood was drawn from tail vein at different time intervals depicted in dots. Amount of DXR (µM) was as determined in plasma after liposome injection (as a measure of loss of liposome integrity). DXR release (y-axis) over 24 hours is presented against time in hours (x-axis). Data are presented as mean value and standard error mean of n=6 mice. Abbreviations: DXR, doxorubicin; DXR-L, DXR-loaded liposome.

Figure 5

Tissue distribution of immunoliposomes in tumor-bearing mice. Notes: Quantification of DXR in different organs at 6 hours following treatment with free DXR or DXR-L formulations at 4 mg/kg DXR dose in tumor-bearing mice. Tumors were derived from G43 (M1⁺/A1⁺) or Mel78 (M1^–/A1⁺) cell lines. Data are represented as mean values and standard mean error of n=3–6 and displayed per organ. (A) Tumor, (B) liver, (C) kidney, (D) spleen, (E) lung, and (F) heart. Abbreviations: DXR, doxorubicin; DXR-L, DXR-loaded liposome.

Figure 6

DXR-L scFvs show significant and sustained antimelanoma effect. Notes: Tumor growth in tumor-bearing mice derived from melanoma cell lines following treatment with PBS, free DXR, or DXR-Ls. G43 (M1⁺/A1⁺) tumors were treated with 4 (A) and 2 mg/kg (B) DXR dose. Mel78 (M1^–/A1⁺) tumors were treated with 4 (C) and 2 mg/kg (D) DXR dose. Mice were injected four times with 4 mg/kg DXR-Ls or eight times with 2 mg/kg DXR-Ls. Data are represented as mean tumor size index values (tumor volume at any given point in comparison with initial tumor volume) and SD of n=4–7. Starting size of all tumors was 100 mm³. *P<0.05; **P<0.01. (E) Tumor size indexes after treatment with immunoliposomes, summary of tumor responses from A to D per tumor type and treatments (PBS, free DXR, and DXR-L formulations). Data are represented as average of tumor size index values ± SD of n=4–7 individual mice after first and fourth treatments. The percentage of mice with a tumor size index above 5 is listed (%). Statistical significance was calculated by Mann–Whitney U test comparing G43 tumor vs Mel78 tumor data according to treatment, dose, and time point. Abbreviations: DXR, doxorubicin; DXR-Ls, DXR-loaded liposomes.