Malignant mesothelioma is a deadly tumor that currently has no curative treatment option [2
]. A few earlier studies using non-targeted liposomes were found useful for treating mesothelioma in the clinic [31
]. Moreover, current developments using immunoliposomes (ILs) have shown promise for targeted anti-cancer therapies [35
]. In this report we have focused our attention on developing ILs anchored with internalizing (M1) scFvs and investigated both their in vitro
and in vivo
tumor targeted binding and internalization towards two different subtypes of human mesothelioma, derived from epithelioid (M28) and sarcomatoid (VAMT-1) origins.
The composition of the liposomes and the method of their fabrication, as well as their stability are some of the important parameters that have to be tailored for particular drug delivery application [41
]. In this regard, our liposomes showed good in vitro
stability in PBS or serum at 37°C up to 72 h and their size remained unaltered (~100 nm) on storage up to 1 month at 4°C (data not shown).
For the IL preparation, we used a method of first conjugating the scFv at the distal end of PEG2000
chains (before insertion onto the surface of liposomes) instead of direct labeling of the scFv onto the liposomes (), based on favorable results reported earlier [44
]. For the scFv conjugation reaction, ideally we want to achieve close to 100 % coupling efficiencies without altering its binding affinity. We thus engineered the scFv to contain a cysteine tag that could conveniently be reduced and conjugated to DSPE-PEG functionalized with a maleimide linker (DSPE-PEG2000
A schematic of multifunctional sterically stabilized long circulating immunoliposome
We used a simple yet effective post-insertion technique for conjugating the DSPE-PEG2000
-scFv onto the surface of the liposomes, to form targeted ILs [48
]. In this regard, our room temperature insertion technique helped preserve the affinity of the scFvs, contrary to the post-insertion method which usually involves exposing the antibody to elevated temperatures (usually ~60°C for 1 h), where the potential exists, for detrimental effects on the antibody, such as reduction in its binding affinity for its target antigen [27
It has been predicted [49
] and experimentally demonstrated that the apparent affinity of multivalent ILs can be several fold higher than that of their monovalent counterparts [50
]. We thus first optimized the scFv density on the surface of liposomes ( and ). We observed that increasing the antibody density on the liposome surface increased the binding affinity, and in turn the internalizing ability of the ILs to the target cells (), consistent with what was observed for similar systems [51
]. While IL binding to cells with different amounts of surface antigen may vary, antibody densities of ~50 scFv per
vesicle was found optimal in our case (). Our current findings and prior reports indicate that larger amounts of bound antibody may not be necessary for efficient target binding of scFvs to cells [53
]. More interestingly, a detailed in vitro
cell binding and internalization study using the optimized ILs (111
) revealed not only efficient and selective binding but also rapid internalization of the ILs by both epithelioid (M28) and sarcomatoid (VAMT-1) mesothelioma cells, but not by the non-targeted BPH-1 cells ( and ). In contrast, we observed that the in vitro
cell binding and internalization of 111
In-CL under varied lipid concentrations was comparably much lower (<40%) in all the three (M28, VAMT-1 and BPH-1) cell lines, under identical conditions, even after 48 h of incubation (data not shown).
Although the findings from the in vitro cell studies using 111In-IL-M1(50) are very encouraging, it is essential to test the ILs in vivo on animal tumor models for successful translation into clinics. As a next step in this direction, we tested the 111In-IL-M1(50) on xenograft mice models bearing both epithelioid (M28) and sarcomatoid (VAMT-1) mesothelioma. The live animal imaging using SPECT/CT and ex vivo biodistribution results corroborated the in vitro findings. For instance, we observed that the uptake of 111In-IL-M1(50) in both M28 and VAMT-1 tumors was strikingly high in the time frame of our study, higher than all other organs/tissues studied (except the liver 7.97±0.34) ( and ) and the tumors could be clearly visualized by SPECT/CT (with values of 4.01±0.39 and 4.69±0.72 % ID/g for M28 and VAMT-1 tumors respectively at 48 h after injection). From the and , it can be noted that for the time frame from 24 h to 48 h the clearance of the 111In-IL-M1(50) from most of the non-target organs and tissues occurred rapidly whereas the tumor uptake remained high, even at 48 h time point. This result may be attributed to the efficient binding and rapid internalization of the 111In-IL-M1(50) into the tumor cells.
It should be noted that we intentionally used a low % of PEG in our liposome formulations in order distinguish the internalizing property and the ‘active tumor targeting’ ability of the 111
in comparison to the 111
In-CL. From our results it is clear that the 111
showed much higher tumor targeted uptake in comparison to 111
In-CL (, and , ). It can also be observed that the lower % PEGylation of 111
may have attributed at least in part for their faster clearance from the blood as early as 24 h (), thus providing a good contrast for early tumor imaging by SPECT/CT (). However the lower % PEGylation also resulted in shorter plasma circulation half-life of 111
. Hence, further optimization of the ILs using higher % PEG may render longer plasma half-life to the ILs thereby facilitating their preferential accumulation in the target tumors by passive targeting strategies or the EPR-effect [15
]. Furthermore, the stealth ILs may also help evade the RES, thereby reducing its in non-target uptake in organs such as the liver and the spleen [55
Although we have addressed some of the critical issues of the ILs in vitro
in cell cultures and in vivo on
animal models, there still remain several questions regarding their effective use in the clinical settings. For instance while our cell studies and animal model demonstrates the ability of the ILs to target and internalize into the cells and implanted tumors in vivo
, their ability to reach the target tissue/cell in the clinical setting may vary or depend on whether the target is accessible from the vasculature [53
]. Also their in vivo
stability and half life, tendency to evoke immune response or ability to effectively evade RES and reach their target organ/tissue are some of the other important factors that needs to be evaluated.