Evaluation of a Radio-IMmunoStimulant (RIMS) in a Syngeneic Model of Murine Prostate Cancer and ImmunoPET Analysis of T-cell Distribution

Abstract

An immunosuppressive tumor microenvironment and tumor heterogeneity have led to the resilience of metastatic castrate resistant prostate cancer (mCRPC) to current treatments. To address these challenges, we developed and evaluated a new drug paradigm, Radio-IMmunostimulant (RIMS), in a syngeneic model of murine prostate cancer. RIMS-1 was generated using a convergent synthesis employing solid phase peptide and solution chemistries. The prostate-specific membrane antigen (PSMA) inhibitory constant for natLu-RIMS-1 was determined, and radiolabeling with 177Lu generated 177Lu-RIMS-1. The TLR 7/8 agonist payload release from natLu-RIMS-1 was determined using a cathepsin B assay. The biodistribution of 177Lu-RIMS-1 was evaluated in a bilateral xenograft model in NCru nude mice bearing PSMA­(+) (PC3-PiP) and PSMA(−) (PC3-Flu) tumors at 2, 24, and 72 h. The therapeutic effect of 177Lu-RIMS-1 was evaluated in C57BL/6J mice bearing RM1-PGLS (PSMA-positive, green fluorescent protein-positive, and luciferase-positive) tumors and compared to that of 177Lu-PSMA-617 at the same total administered radioactivity of 57 MBq and molar activity of 5.18 MBq/nmol. natLu-RIMS-1 and vehicle were evaluated as the controls. Immuno-positron emission tomography (PET) using 89Zr-DFO-anti-CD3 was used to visualize T-cell distribution during treatment. 177Lu-RIMS-1 was quantitatively radiolabeled at >99% radiochemical purity and maintained a high affinity toward PSMA (K i = 3.77 ± 0.5 nM). Cathepsin B efficiently released the entire immunostimulant payload in 17.6 h. 177Lu-RIMS-1 displayed a sustained uptake in PSMA­(+) tumor tissue up to 72 h (2.65 ± 1.03% ID/g) and was not statistically different (P = 0.1936) compared to 177Lu-PSMA-617 (3.65 ± 0.59% ID/g). All animals treated with 177Lu-RIMS-1 displayed tumor growth suppression and provided a median survival of 30 days (P = 0.0007) while 177Lu-PSMA-617 provided a median survival of 15 days, which was not statistically significant (P = 0.3548) compared to the vehicle group (14 days). ImmunoPET analysis revealed 2-fold more tumor infiltrating T-cells in 177Lu-RIMS-1-treated animals compared to 177Lu-PSMA-617-treated animals; 177Lu-RIMS-1 improves therapeutic outcomes in a syngeneic model of mouse prostate cancer and elicits greater T-cell infiltration to the tumor compared to 177Lu-PSMA-617. These results support further investigation of the RIMS paradigm as the first example of a single molecular entity combining radiotherapy and immunostimulation.

Metal-Mediated, Autolytic Amide Bond Cleavage: A Strategy for the Selective, Metal Complexation-Catalyzed, Controlled Release of Metallodrugs

“…The construct includes a doubly functionalized NOTA chelator that incorporates a cleavable G−S (glycine−serine) cap by amidation; a PSMA-targeting peptide was introduced via backbone functionalization of the macrocycle in accordance with methods established by us previously (Figure 7A). 54 We then probed radiochemical labeling and autocleavage efficiency with the radioactive isotope 67 Ga. The direct, insolution radiolabeling to produce [ 67 Ga]Ga (10) resulted in high radiochemical yield (>90%) at pH 5.0 and at 37 °C.…”

“…In contrast with the human-derived PSMA-expressing cell lines PC-3 PiP (500,000 receptor copies per cell), RM-1 only expresses approximately 80,000 copies of the PSMA receptor per cell. 56,57 This induces a greater sensitivity to the molar activity of radiopharmaceuticals, with low molar activity reducing probe uptake in target tissues. We conducted radiosynthesis of [ 68 Ga]Ga-8 using MMAAC followed by administration to tumor-bearing mice, positron emission tomography (PET) imaging at 90 min postinjection, and biodistribution analysis at 2 h postinjection.…”

“…We prepared the NOTA-linked short peptide conjugate 10 , targeting the prostate-specific membrane antigen (PSMA). The construct includes a doubly functionalized NOTA chelator that incorporates a cleavable G–S (glycine–serine) cap by amidation; a PSMA-targeting peptide was introduced via backbone functionalization of the macrocycle in accordance with methods established by us previously (Figure A) …”

Construction of the Bioconjugate Py‐Macrodipa‐PSMA and Its In Vivo Investigations with Large 132/135La3+ and Small 47Sc3+ Radiometal Ions

“…Specifically, the well‐known “Lys‐urea‐Glu” moiety was used. This targeting vector, which was prepared by solid‐phase peptide synthesis, [27] has high affinity for the PSMA, a membrane protein that is overexpressed on the surface of various subtypes of prostate cancer cells [28] . The naphthyl and tranexamic acid moieties present in py‐macrodipa‐PSMA, are also employed by the recently FDA‐approved radiotherapeutic [ 177 Lu]Lu 3+ ‐PSMA‐617 (Pluvicto™) [29] and confer additional enhancement to the PSMA target [30] …”

“…The Fmoc-trans-nap-Lys-urea-Glu-Wang resin (200 mg, 0.16 mmol) was prepared via established solid-phase chemistry. [27] Fmoc-β-Lalanine (199 mg, 0.64 mmol) was coupled to the pre-loaded resin using PyBOP (167 mg, 0.32 mmole) in the presence of DIPEA (112 μL, 0.64 mmol) over a 12 h time period. The resin was then treated with a mixture of 20 % piperidine in DMF to remove Fmoc group.…”