Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.

Promising biochemical responses to ²²⁵Ac-PSMA-617, even in patients that are refractory to beta-particle radiation, illustrate the potential of targeted alpha therapy (TAT) for the treatment of metastatic castrate-resistant prostate cancer. However, side effects such as xerostomia are severe and irreversible. In order to fully harness the potential of TAT, it is necessary to increase the therapeutic index of the targeted radioligands. One emerging strategy is to Increase clearance half-life through enhanced binding to serum albumin. We have evaluated the albumin-binding PSMA-targeting ligand RPS-074 in a LNCaP xenograft model to determine its potential value to the treatment of prostate cancer. Methods: ²²⁵Ac-RPS-074 was evaluated in male BALB/c mice bearing LNCaP xenograft tumors. A biodistribution study was performed over 21 d to determine the dosimetry in tumors and normal tissue. The dose response was measured in groups of 7 mice using 37 kBq, 74 kBq, and 148 kBq ²²⁵Ac-RPS-074 and compared to positive and negative control groups. Mice were sacrificed when tumor volume exceeded 1500 mm³. Results: ²²⁵Ac-RPS-074 was labeled in >98% radiochemical yield and showed high (>10 %ID/g) and sustained accumulation in LNCaP tumors from 24 h to beyond 14 d. Signal in blood and highly vascularized tissues was evident over the first 24 h p.i., and cleared by 7 d. The tumor-to-kidney ratio was 4.3 ± 0.7 at 24 h and 62.2 ± 9.5 at 14 d. A single injection of 148 kBq induced a complete response in 6/7 tumors with no apparent toxic effects. Treatment with 74 kBq induced a partial response in 7/7 tumors, but from 42 d, 6/7 experienced significant regrowth. The 37 kBq group experienced a survival benefit relative to the negative control, but not compared to the positive control group. Conclusion: A single dose of 148 kBq ²²⁵Ac-RPS-074 induced a complete response in 86% of tumors, with tumor-to-normal tissue ratios that predict an improved therapeutic index. The use of the macropa chelator enabled quantitative radiolabeling and may facilitate the clinical translation of this promising targeted alpha therapeutic.

PSMA-targeted radiotherapy of prostate cancer (PCa) has emerged recently as a promising approach to the treatment of disseminated disease. A small number of ligands have been evaluated in patients, and while early tumor response is encouraging, relapse rate is high, and these compounds localize to the parotid, salivary and lacrimal glands as well as the kidney, leading to dose-limiting toxicities and adverse events affecting quality of life. We envision that dual targeting ligands displaying high affinity for PSMA and appropriate affinity for human serum albumin (HSA) may demonstrate a higher therapeutic index and be suitable for treatment of PCa by targeted alpha therapy (TAT). Methods: Six novel urea-based ligands with varying affinities for PSMA and HSA were synthesized, labeled with ¹³¹I and evaluated by in vitro binding and uptake assays in LNCaP cells. Four compounds were advanced for further evaluation in a preclinical model of PCa. The compounds were compared to MIP-1095, a PSMA ligand currently in clinical evaluation. Results: The compounds demonstrated affinity for PSMA on the order of 4-40 nM, and affinity for HSA in the range 1-53 µM. Compounds with relatively high affinity for HSA (≤ 2 µM) showed high and sustained blood pool activity and reduced uptake in the kidneys. ¹³¹I-RPS-027, with IC50 (PSMA) = 15 nM and Kd (HSA) = 11.2 µM, clears from the blood over the course of 48h, shows good tumor uptake (10 %ID/g) and retention, and greater than a 5-fold decrease in kidney uptake relative to MIP-1095. The tumor-to-kidney ratio of ¹³¹I-RPS-027 is greater than 3:1 at 24h post injection increasing to 7:1 by 72h. Conclusion: RPS-027 shows dual targeting to PSMA and albumin, resulting in high tumor uptake, highly favorable tissue distribution and a promising therapeutic profile in a preclinical model of prostate cancer. In comparison to existing ligands proposed for targeted therapy of prostate cancer, RPS-027 has tumor-to-tissue ratios that predict a significant reduction in side effects during therapy. Using iodine/radioiodine as a surrogate for the radiohalogen ²¹¹At, we therefore propose dual targeting ligands such as RPS-027 as next generation radiopharmaceuticals for TAT using ²¹¹At.

Despite significant gains in the treatment of metastatic castration-resistant prostate cancer by radioligands targeting prostate-specific membrane antigen (PSMA), 30% of patients never respond to therapy. One possible explanation is insufficient dose delivery to the tumor because of suboptimal pharmacokinetics. We have recently described RPS-063, a trifunctional ligand targeting PSMA with high uptake in LNCaP xenograft tumors but also in kidneys. We aimed to use structural modifications to increase the tumor-to-kidney ratio through increased albumin binding and tumor uptake and reduction of kidney activity. Methods: Four structurally related trifunctional PSMA-targeting small molecules were prepared by either varying the albumin-binding group or inserting a polyethylene glycol 8 linker into a common structure. The compounds were ranked by PSMA affinity and albumin affinity and were radiolabeled with ⁶⁸Ga and ¹⁷⁷Lu. Tissue kinetics were determined in male BALB/C nu/nu mice bearing LNCaP xenograft tumors. Results: Each of the compounds binds PSMA with a half-maximal inhibitory concentration of no more than 10 nM. The albumin-binding group had a minimal effect on PSMA affinity but changed albumin affinity by an order of magnitude. However, the addition of a polyethylene glycol 8 spacer weakened affinity for albumin in each case. Increased affinity for albumin corresponded with delayed blood clearance and modified uptake kinetics in the tumor and kidney. Uptake of ¹⁷⁷Lu-RPS-072 (34.9 ± 2.4 %ID/g) and ¹⁷⁷Lu-RPS-077 (27.4 ± 0.6 %ID/g) increased up to 24 h after injection, and washout by 96 h was not significant. As a result, the area under the curve (AUC) in the tumor was in the following order: ¹⁷⁷Lu-RPS-072 > ¹⁷⁷Lu-RPS-077 > ¹⁷⁷Lu-RPS-063 > ¹⁷⁷Lu-RPS-071. Increased linker length corresponded to more rapid clearance from kidneys. Consequently, the ratio of tumor AUC and kidney AUC was 4.7 ± 0.3 for ¹⁷⁷Lu-RPS-072. Conclusion: The tumor AUC and tumor-to-kidney ratio of ¹⁷⁷Lu-RPS-072 are significantly enhanced compared with any small molecule investigated in a LNCaP xenograft model to date. In comparison to other PSMA-targeting radioligands that have been evaluated in a PC3-PIP model, activity in kidneys is reduced and activity in tumors compares favorably when the different PSMA expression levels in LNCaP and PC3-PIP cells are considered. RPS-072 therefore exhibits an increased therapeutic index, shows the potential to increase the dose delivered to tumors, and is a highly promising candidate for targeted radioligand therapy.

Objectives: A GMP Ge-68/Ga-68 generator that utilizes modified Dodecyl-3,4,5-trihydroxybenzoate hydrophobically bound to a Octadecyl silica resin (C-18) as an adsorbent has been developed that allows for very dilute HCl (0.05 N) to efficiently elute metal impurity free ⁶⁸Ga3+ ready for peptide labeling. We characterized the performance of this generator system over a year in conjunction with the production of ⁶⁸Ga labeled DOTATOC and PSMA-HBED-CC intended for clinical studies and established protocols for batch release. Methods: A 2040 MBq self-shielded ITG ⁶⁸Ge/⁶⁸Ga generator provided metal-free ⁶⁸GaCl3 ready for peptide labeling in the iQS Fluidic Labeling Module following elution with 4 mL of 0.05N HCl. The compact system was readily housed in a laminar flow cabinet allowing an ISO class-5 environment. ⁶⁸Ga labeling of peptides using GMP kits was performed in 15-20 min and the total production time was 45-50 min. Batch release quality control specifications were established to meet IND submission and IRB approval standards. Results: Over a period of 12 months, ⁶⁸Ga elution yields from the generator averaged 80% (range 72.0%-95.1%) and ⁶⁸Ge breakthrough was 98% with a minimum specific activity of 12.6 GBq/µmole and 42 GBq/µmole respectively. The radionuclidic (⁶⁸Ge) impurity was ≤0.00001% (under the detection limit). Final sterile, pyrogen-free formulation was provided in physiological saline with 5-7% ethanol. Conclusion: The GMP certified ITG ⁶⁸Ge/⁶⁸Ga generator system was studied for a year. The generator system is contained within the iQS Fluidic Labeling Module and it is compact, self-shielded and easy to operate using simple manual techniques. The system provides radiolabeled peptides with high (>98%) radiochemical purity and greater than 80% radiochemical yield. The ⁶⁸Ge levels in the final drug products were under the detection limits at all times. ⁶⁸Ga-DOTATOC and ⁶⁸Ga-PSMA-HBED-CC investigational radiopharmaceuticals are currently being studied clinically under INDs submitted to the U.S. FDA.