Page 237 - Haematologica March 2020

CXCR4-targeted nanocarrier to DLBCL cells
Discussion
A huge limitation for the clinical translation of nanomedicines in oncology is the fact that only 0.7-5.0% of the administered dose reaches the tumor.23,24 In contrast, our biodistribution studies show a very high level of T22- GFP-H6 uptake in tumor tissue (86.1% of the total emitted fluorescence) compared to the combined fluorescence emitted by all normal tissues (13.9% of total tumor+non- tumor fluorescence), including the spleen, liver, kidney, heart, lung and BM. We have recently reported a similar finding for the same nanocarrier in a SC colorectal cancer (CRC) model.25 These data are consistent with the fast biodistribution half-life for the nanocarrier in blood (approx. 20 min) and the detection of the full length pro- tein in the 10min-5h period in SC CXCR4+ DLBCL tumors. Unexpectedly, we found that most of the prote- olytic metabolism of T22-GFP-H6 occurs in tumor tissues, whereas clearance in liver or kidney is negligible, being detectable in these organs at 10 min, probably by access- ing the fenestrated vessels during a short time period, but being unable to reach their parenchyma. Our data are in dramatic contrast to the reported biodistribution of most nanocarriers studied so far, regardless of whether this was targeted actively or passively.
Nowadays, most nanocarriers that transport medicinal drugs in clinical trials, or that are available on the market, use passive targeting (e.g. liposomal doxorubicin or albu- min-paclitaxel). They enhance the drug antitumor effect because its particulate size increases its permeability and retention in the tumor (EPR effect). Nevertheless, 50-80% of these nanocarriers accumulate in the liver.26 Although still at an initial stage, active nanocarrier targeting is being developed to selectively deliver antitumor drugs to tumor cells through specific surface receptors.27 Regarding B-cell lymphoma therapy, the use of doxorubicin-loaded meso- porous silica nanoparticles bound to rituximab, for target- ing CD20+ B cells, demonstrated a significant increase in doxorubicin tumor uptake and higher inhibition of tumor growth than free doxorubicin.28 Moreover, additional tar- geted and non-targeted therapeutic nanoparticles are cur- rently being evaluated for treatment of B-cell malignancies; however, no efficacy data are available yet because Phase I clinical assays to test their tolerability are still ongoing.29
The strategy we have used here with the actively target- ed T22-GFP-H6 nanocarrier achieves selective and enhanced biodistribution to tumor tissue with no toxicity in the non-tumor organs. One possible explanation for the enhanced T22-GFP-H6 tumor uptake relates to the nature of the nanocarrier material. While our nanocarrier is made of self-assembled proteins, most, if not all, nanocarriers showing limited biodistribution to tumor are either inor- ganic (gold, silica, iron oxide, quantum dots) or organic (dendrimers, liposomes polymers, hydrogels) rather than protein-based.23,24 Once administered in blood, non-pro- tein-based nanocarriers are covered by a protein corona that changes the conformation of the nanocarrier surface30 and undergo intensive phagocytosis by resident macrophages in clearance organs.31 A completely different protein drug delivery system is represented by the targeted antibody-drug conjugates (ADC), which have lower load- ing capacity and flexibility for encapsulating various cargos and display a less controllable drug release kinetics com- pared to nanocarriers.32 Consequently, in clinical studies, only 0.001-0.01% of the injected antibody dose reaches
the tumor;33 thus, although ADC are standard treatment in some neoplasias, protein nanocarriers could offer an enor- mous opportunity to improve drug delivery to tumors.
Our results on nanocarrier biodistribution in the SC tumor model demonstrate a specific co-localization of the nanocarrier together with the CXCR4 receptor in the cell membrane followed by their internalization, via endocy- tosis, to reach the cytosol of CXCR4+ DLBCL cells. Once inside the cytosol, the structure of the nanocarrier elicits endosomal escape and delivery of the materials into the cytoplasm, before its ultimate intracellular proteolysis.16
Furthermore, the efficacy of a T22-GFP-H6 nanocarrier that targets CXCR4+ DLBCL cells appears to be exclusive- ly dependent on the overexpression of CXCR4 receptor in the membrane of tumor cells. This notion is currently sup- ported by two main findings: on the one hand, T22-GFP- H6 displays a tumor uptake significantly higher than that achieved in the same SC tumor when CXCR4 is inhibited by AMD3100 in the competition assay. On the other hand, T22-GFP-H6 administration to mice bearing CXCR4+ SC SUDHL-2 tumors shows significantly higher uptake than CXCR4– SC SUDHL-2 tumors. Moreover, we confirmed the capacity of T22-GFP-H6 to internalize in CXCR4+ mouse cells, similar to our findings in CXCR4+ human cells. Thus, the high T22-GFP-H6 tumor uptake, and its low uptake in non-tumor organs, is necessarily related to the huge CXCR4 overexpression in DLBCL lymphoma cells and the negligible or low CXCR4 expres- sion in normal organs, including BM mouse hematopoietic cells.
Importantly, in the disseminated CXCR4+ DLBCL mouse model, involving BM and LN, this nanocarrier also shows a high tumor uptake in the organs affected by CXCR4+ lymphoma cells, while displaying low biodistrib- ution to normal tissues (with low or null CXCR4 expres- sion). Unlike low molecular weight drugs that passively diffuse to all cells in the body, the biodistribution of the nanocarrier, or drug-loaded nanocarriers, is limited by their size; thus, it becomes highly dependent on the phys- iology and anatomy of specific organs in the body. Nanocarriers are unable to access organs irrigated by ves- sels with continuous endothelia and unable to penetrate membranes, unless they are actively targeted for endocy- tosis.34 Our protein nanocarrier can accumulate in the sinu- soids of BM and LN infiltrated with tumor cells because they display vascular beds with discontinuous endotheli- um and 100-200nm fenestrations that allow the transport of macromolecules, including nanocarriers.35-37 Moreover, as we have showed in the SC mouse model, T22-GFP-H6 also has the capacity to internalize specifically in the CXCR4+ DLBCL cells, here localized in BM and LN in the DLBCL disseminated model. Even though there is no con- sistent EPR effect in hematologic neoplasias,20 the struc- ture of the vessels in the sinusoids of the DLBCL niches and the active targeting to CXCR4 allow T22-GFP-H6 accumulation and internalization in the tumor niches that are infiltrated by CXCR4+ DLBCL cells.
Given the high selectivity that T22-GFP-H6 achieves in targeting CXCR4+ DLBCL cells within the tumor, we used the SC CXCR4+ Toledo model to test the antitumor activ- ity of T22-DITOX-H6, a therapeutic nanoparticle derived from this nanocarrier that incorporates the diphtheria cytotoxic domain. This therapeutic nanoparticle induced a high level of apoptotic cell death in tumor tissue without toxicity, since it did not induce any macroscopic or histo-
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