SBPD-1 binds with high affinity to PSMA and contains a cathepsin B cleavable linker.
To achieve a specific, high-affinity interaction with PSMA we used the low-molecular-weight (LMW) scaffold Lys-Glu-Urea-DSS originally developed in our laboratory32. The synthetic tubulin inhibitor MMAE was conjugated to the Lys-Glu-Urea-DSS via a cathepsin B cleavable valine-citrulline linker (SBPD-1) or non-cleavable linker (SBPD-2), as a control to determine the utility of the linker (Fig. 1a).
Synthesis of SBPD-1 began with known amine 1,33 which on treatment with previously reported Lys-Glu-Urea-DSS32 in the presence of diisopropylethylamine afforded 2 in 85% yield. Compound 2 was further converted into activated carbonate 3 in 45% yield by treating with bis(4-nitrophenyl) carbonate and subsequent reaction with MMAE, followed by deprotection to realize target conjugate SBPD-1 in 20% combined yield (Supplementary Fig. S1).
Synthesis of SBPD-2 began with previously reported Lys-Glu-Urea-DSS32, which on treatment with L-glutamic acid α-tert-butyl ester in the presence of diisopropylethylamine in DMF, afforded 4 in 70% yield. Compound 4 was subsequently reacted with MMAE followed by deprotection to provide target conjugate SBPD-2 in 20% combined yield (Supplementary Fig. S2).
PSMA inhibitory capacity, a surrogate for affinity, was measured according to a previously described assay34. Both conjugates, SBPD-1 and SBPD-2, demonstrated high affinity to PSMA with Ki values of 8.84 nM (95% CI, 5.00–15.63) and 3.0 nM (95% CI, 1.94–4.67), respectively. We tested if SBPD-1 could release MMAE when incubated with recombinant cathepsin B in vitro and found that MMAE was efficiently released (80%) within 3 h of incubation (Fig. 1b).
SBPD-1 selectively kills PSMA-expressing PC cells in vitro.
We evaluated the cytotoxicity of SBPD-1 and SBPD-2 in PSMA-expressing PC3 PIP and PSMA-negative PC3 flu cells in vitro35,36. SBPD-1 demonstrated IC50 values of 3.9 nM (95% CI, 2.8–5.5 nM) and 151.1 nM (95% CI, 104.1–219.3 nM) for PSMA + PC3 PIP and PSMA- PC3 flu cells, respectively, indicating selectivity for PSMA-expressing cells. The IC50 value of 151.1 nM for PSMA- PC3 flu cells suggests release of some MMAE to enable non-selective cell kill in vitro. SBPD-2 demonstrated IC50 values of 4.8 µM (95% CI, 0.8–28.5 µM) and 5.8 µM (95% CI, 0.7–47.2 µM) for PSMA + PC3 PIP and PSMA- PC3 flu cells, respectively, indicating a lack of potency regardless of PSMA expression and the need for cleavage of MMAE from the targeting moiety. MMAE alone proved exquisitely potent in both cell lines, demonstrating an IC50 value of 39.2 pM (95% CI, 19.5–78.7 pM) and 40.0 pM (95% CI, 21.2–75.4 pM) for PSMA + PC3 PIP and PSMA- PC3 flu cells, respectively (Fig. 2).
SBPD-1 selectively kills PSMA-expressing PC xenografts in vivo.
Prior to in vivo potency we evaluated the stability of SBPD-1 and SBPD-2 in murine serum. While 90% of SBPD-2 remained intact for 48 h in murine serum, SBPD-1 was metabolized more quickly (Fig. 3b). While more than 80% of SBPD-1 was intact in serum at 8 h of incubation, less than half represented parent compound at 24 h, and the majority of the prodrug was fully degraded by 48 h of incubation (Fig. 3a). It has been reported that the valine-citrulline linker is stable in human and monkey serum but that it can be hydrolyzed in mouse plasma via extracellular carboxylesterase 1c37,38. Based on those stability results, we applied small, fractionated doses for the murine efficacy study to avoid systemic toxicity that could affect the overall survival of the test animals.
To evaluate efficacy in preclinical models of human PC, we initially employed xenograft tumor models derived from PSMA + PC3 PIP and PSMA- PC3 flu cells in NOD/SCID/IL2Rγnull (NSG) mice. Three weeks after injection of the cells, the average tumor volume reached 62.4 (±11.6) mm3, and mice were treated with 20, 40 and 80 µg/kg of SBPD-1 via daily intraperitoneal (IP) injection for 30 days, n = 5. We monitored tumor growth and overall animal welfare (Fig. 4a). Animals were scored ‘dead’ when the tumor reached 4-times its original volume (Fig. 4b). Tumors in non-treated, control mice for both tumor types, in PSMA + PC3 PIP mice treated with 20 µg/kg and in PSMA- PC3 flu mice with all three doses, grew rapidly and all animals so treated were euthanized on day 20 post-initiation of treatment (Fig. 4b). The median survival time for non-treated groups of animals harboring either PSMA + PC3 PIP or PSMA-PC3 flu tumors was 15 days. For animals harboring PSMA + PC3 PIP tumors, the median survival time of the group treated with 20 µg/kg was 17 days. The median survival times for group harboring PSMA-PC3 flu tumors treated with 20, 40, and 80 µg/kg were 15, 15, and 20 days, respectively. Doses of 40 and 80 µg/kg delivered to animals harboring PSMA + PC3 PIP tumors cleared the tumors such that they were undetectable by the completion of treatment (Fig. 4a). Approximately one week was required to be able to re-measure previously undetectable tumors in the group treated at 40 µg/kg. Two weeks were required for re-appearance of tumors in animals treated with the 80 µg/kg dose. In animals harboring PSMA + PC3 PIP tumors, both the 40 and 80 µg/kg doses provided significant survival benefits as the median survival times were 54 days [P = 0.003, Log-rank (Mantel-Cox) test] and 69 days (P = 0.003), respectively (Fig. 4b). Urine protein level and specific gravity measured for all test animals on Days 9 and 20 were normal, indicating that no acute renal toxicity occurred at any dose tested. (Supplementary Table. S1)
SBPD-1 is effective in an experimental metastatic model of PSMA-expressing PC.
To evaluate efficacy of SBPD-1 on established metastatic tumors, we used a PSMA-expressing experimental metastatic model of human PC18. PSMA + PC3/ML/PSMA cells were administered to NSG mice intravenously (IV) and tumors were allowed to establish for four weeks. PC3/ML/PSMA cells express firefly luciferase as an imaging reporter to allow us to monitor tumor development via weekly bioluminescence imaging (BLI). Mice were treated with 40, 80 and 160 µg/kg of SBPD-1 via daily IP injection for 30 days, n = 5. We increased the doses to compensate for the lower expression of PSMA on PC3/ML/PSMA cells compared with that of PSMA + PC3 PIP tumors (Supplementary Figure. S3). The 40 µg/kg dose did not show survival benefit to non-treated control mice, with median survival times of 47 days for each group. Mice treated at the 80 and 160 µg/kg dose levels, however, exhibited significant survival benefits, with median survival of 56 days [P = 0.003, Log-rank (Mantel-Cox) test] and 58 days (P = 0.003), respectively (Fig. 5, Supplementary Figure. S4).
SBPD-1 is non-toxic to C57BL/6 mice.
We evaluated potential toxicity of SBPD-1 in immunocompetent animals. We administered MMAE (80 µg/kg), SBPD-1 (160 µg/kg), and 5% DMSO to healthy C57BL/6 mice (n = 5). We monitored animals for 80 days after initiation of administration. As previously reported39, MMAE demonstrated severe toxicity as all treated mice required euthanasia during treatment due to weight loss (Fig. 6b). Mice injected with vehicle or SBPD-1 did not show any signs of toxicity and steadily gained weight (Fig. 6a). We removed lung, liver, kidneys, salivary and lacrimal glands from all tested animals at Day 80 after initiation of SBPD-1 treatment. Histopathological examination revealed no tissue damage (Fig. 6c). We also obtained peripheral blood from mice injected with vehicle, SBPD-1, and healthy untreated animals, and prepared serum for chemistry studies (n = 5). Blood urea nitrogen (BUN), creatinine, glucose, alkaline phosphatase (ALP), total protein (T-Pro), and alanine aminotransferase (ALT) analyses showed that animals injected with either vehicle or SBPD-1 did not show differences in these values compared with those from untreated mice (Supplementary Table S2). Complete blood counts from the mice also showed no abnormalities except for lower white blood cell count for mice injected with SBPD-1, which may have resulted from the relative instability of the cathepsin B linker in murine serum and subsequent bone marrow toxicity of MMAE37,40.