Tumor-specific anti-nucleosome antibody improves therapeutic efficacy of doxorubicin-loaded long-circulating liposomes against primary and metastatic tumor in mice

Abstract

The efficacy of drug delivery systems can be significantly enhanced by making them target-specific via the attachment of various ligands to their surface. We attempted to enhance tumor accumulation and therapeutic effect of doxorubicin-loaded long-circulating liposomes (Doxil, ALZA Corp.) by coupling to their surface the anticancer monoclonal antibody 2C5 (mAb 2C5) with nuclesome (NS)-restricted activity, that can recognize the surface of various tumor but not normal cells via the surface-bound nucleosomes released from the apoptotically dying neighboring tumor cells and specifically targets pharmaceutical carriers to tumor cells in vitro and in vivo. Antibody coupling to PEGylated doxorubicin-liposomes was performed by the "post-insertion" technique. The pharmacokinetics of plain and immuno-targeted Doxil-mimicking liposomes, as well as their accumulation in primary Lewis lung carcinoma (LLC) tumors in mice was followed by real-time gamma-scintigraphy upon liposomal membrane labeling with (111)In. Therapeutic action of various liposomal formulations was followed by registering primary tumor growth, determining tumor weigh upon mice sacrifice, and by counting the number of metastases in the liver and lungs. 2C5 antibody-targeted liposomes demonstrate significantly enhanced accumulation in LLC tumors. Targeted doxorubicin-loaded PEG-liposomes were significantly more effective in inhibiting tumor growth and metastatic process in the LLC tumor models in mice. Our results clearly show the remarkable capability of 2C5-targeted Doxil to specifically deliver its cargo into various tumor manifestations (solid and metastatic) significantly increasing the efficacy of therapy.

Figure 1

Whole body imaging of LLC-tumor-bearing mice 4 hr after injection with ¹¹¹In-labeled liposomes. Panel I - calculated radiologic signal intensity. Panel II – Gamma-scintigraphic images of mice injected with: (A) Control Doxil^®-mimicking liposomes; (B) Non-specific IgG-modified Doxil^®-mimicking liposomes; (C) mAb 2C5-modified Doxil^®-mimicking liposomes. Circles indicate tumor locations.

Figure 2

Single dose pharmacokinetics of various ¹¹¹In-labeled Doxil^®-mimicking liposomal preparations in healthy BALB/C mice. (n = 5, results indicated ± SD)

Figure 3

Therapeutic activity, expressed as tumor volumes (A) and post-mortem tumor weight (B) of 2C5-modified Doxil^® against control preparations in LLC-implanted mice. Arrows indicate treatment schedule, 2 mg/kg/q 5 days (n= 8–10), * (P = 0.05, Vs. corresponding IgG-Doxil^®), non-parametric Kruskal-Wallis with Tukey’s HSD Post-Hoc test. (Results presented as mean± SD).

Figure 4

Schematics of tumor targeting and uptake of nucleosome-specific monoclonal antibody 2C5-modified Doxil^® liposomes, for both primary site and metastatic cells.