Development and optimization of targeted radionuclide tumor therapy using folate based radiopharmaceuticals

The folate receptor (FR) has been used for a quarter of a century as a tumor-associated target for selective delivery of drugs and imaging agents to cancer cells. While several folic acid radioconjugates have been successfully employed for imaging purposes in (pre)clinical studies, a therapeutic application of folic acid radioconjugates has not yet reached the critical stage which would allow a clinical translation. Due to a substantial expression of the FR in the proximal tubule cells, radiofolates accumulate in the kidneys which are at risk of damage by particle-radiation. To improve this situation, we aimed to develop and evaluate strategies for the performance of FR-targeted radionuclide therapy by decreasing the renal uptake of radiofolates and thereby reducing potential nephrotoxic effects. Two different strategies were investigated. First, the combination of radiofolates with chemotherapeutic agents such as pemetrexed (PMX) and 5-fluorouracil (5-FU) and secondly, an approach based on radioiodinated folates and their partial in vivo deiodination. It was known from the previous work in our group that PMX reduces the kidney uptake of radiofolates up to ~ 4-fold. Thus, we evaluated the combined application of a therapeutic radiofolate with the antifolate PMX as an option to perform FR-targeted radionuclide therapy. A first therapy study was performed in nude mice bearing FR-positive cervical KB tumors. Injections of subtherapeutic amounts of PMX and the $^{177}$Lu-labeled DOTA-folate ( $^{177}$Lu-EC0800, 20 MBq) showed a 50% prolonged survival time when compared to control animals. To evaluate potential nephrotoxic effects of such a therapy, an additional experiment was performed in mice without tumors. Therapeutic amounts of $^{177}$Lu-EC0800 were injected into the animals either with or without pre-injection of subtherapeutic amounts of PMX. The mice which received only $^{177}$Lu-EC0800 showed significantly increased levels of blood plasma parameters such as blood urea nitrogen (5-fold increased) and creatinine (10-fold increased) when compared to control animals after 6 months. In the same time range SPECT/CT studies using $^{99m}$Tc-DMSA showed an 85% lower uptake in the kidneys compared to controls which confirmed an impaired kidney function. In contrast, analysis of plasma parameters and the results of the $^{99m}$Tc-DMSA studies showed consistently intact kidney function in animals which received PMX in addition to $^{177}$Lu-EC0800. PMX is also known as a radiosensitizing agent. Therefore, we investigated the combined application of $^{177}$Lu-EC0800 and PMX in vitro which showed a pronounced synergistic effect in FR-positive KB and ovarian cancer cell lines, IGROV-1 and SKOV-3. It could also be shown in vivo that the therapeutic efficacy of $^{177}$Lu-EC0800 was enhanced by therapeutic amounts of PMX using two different tumor mouse models. A novel DOTA-folate radioconjugate ( $^{177}$Lu-cm09) was also investigated in vitro in combination with the radiosensitizing agent 5-FU in two different cancer cell lines. The simultaneous application of $^{177}$Lu-cm09 and 5-FU resulted in a synergistic anticancer effect in KB and IGROV-1 tumor cells similar to the results obtained with $^{177}$Lu-EC0800 and PMX. To reduce the cell viability in the combined treatment to 75%, a 5-fold lower quantity of 5-FU was needed compared to the single treatment in both cell lines. The amount of $^{177}$Lu-cm09 was reduced 6-fold in KB and 3-fold in IGROV-1cells. These findings demonstrated the radio sensitizing potential of 5-FU, which is routinely used in combination with external radiotherapy in the clinic. In the second approach we studied three different radioiodinated folate conjugates. Biodistribution studies performed in tumor-bearing nude mice showed specific accumulation of all $^{125}$I-radiofolates in KB tumor xenografts (~ 3 % ID/g; 4 h p.i.). The tumor-to-kidney ratio of $^{125}$I-tyrosine-click-folate was increasing over time (from 0.12; 1 h p.i. to 0.27; 24 h p.i). With these findings the hypothesis was confirmed that partial deiodination of radioiodinated folate conjugates would improve tumor-to- background contrast over time. An 11-fold increase in the tumor-to-background contrast was achieved with $^{125}$I-tyrosine-click-folate if the mice were pre-injected with PMX. The introduction of an albumin-binding entity in the structure improved the tumor-to-background ratio further and resulted in an unprecedented high tumor-to-kidney ratio of approximately 2 (24 h p.i.). The SPECT/CT image showed uptake of radioactivity in FR-positive tumors and in the thyroid gland. Based on these findings the more promising approach that may allow a therapeutic application of folate radioconjugates in the future is the combination of therapeutic radiofolates ( $^{177}$Lu-EC0800, $^{177}$Lu-cm09) with chemotherapeutics (PMX, 5-FU) that can also act as radiosensitizing agents. If the kidney uptake reducing effect of PMX could be confirmed in patients, this therapy concept would be promising for the treatment of non-small cell lung cancer because it shows frequent FR- overexpression and because PMX is already approved for the therapy.