NASDAQ: PNT
PNT2001 EANM E-Poster
Summary
Accelerating Precision MedicineTM 1
Development and characterization of 225Ac-PNT2001, a next-generation PSMA radioligand
A. Vito1, M. Dornan1, T. Beale1, A. Felten1, M. Ernste1, Q. Nguyen2, S. Ahn3,4, A. P. Belanger3,4, S. Pan5, W. Wu5, Y. Liu5, J. H. Lai5, W. W. Bachovchin5, V.
Hergott1, J. McCann1, R. Hallett1
A.
Prostate-specific membrane antigen (PSMA) is a type II membrane protein B.
that is highly expressed in the majority of prostate tumors1. PSMA has
been successfully targeted with radioligands for both cancer imaging and
therapy, illustrated by the recent FDA approvals of gallium Ga 68
gozetotide, piflufolastat F 18, and lutetium Lu 177 vipivotide tetraxetan.
PNT2001 has been developed as a lead candidate compound for PSMA-
targeted radioligand therapy. PNT2001 displays high internalization in
prostate cancer cells and reduced kidney uptake in biodistribution
studies, properties which are hypothesized to translate into improved
therapeutic efficacy. Here, the in vivo performance of 177Lu-PNT2001 and
225Ac-PNT2001 was further studied using in vitro and in vivo assays.
1. Hawkey N. et al. Clinical Advances in Hematology and Oncology. 2022 Apr; 20(4):227-238. Fig 1. (A) Mechanism of action schematic of PSMA-
targeted radioligands. Created using BioRender.com.
Accelerating Precision MedicineTM (B) Structure of PNT2001.
2
Materials and Methods
The PNT2001 peptide precursor was synthesized using solid-phase peptide synthesis. 177Lu-PNT2001 was synthesized by heating
PNT2001 with 177LuCl3 (nca) in acetate buffer (pH 5.5) to 95°C for 10 minutes. The radiochemical purity was determined by radio-TLC
and radio-HPLC. 177Lu-PSMA-I&T was synthesized by heating PSMA-I&T with 177LuCl3 (nca) in ascorbate buffer (pH 5.5) to 90°C for 10
minutes and formulating to 30 mg/mL ascorbate, 25 mg/mL gentisic acid and 0.1 mg/mL DTPA. The radiochemical purity was
determined by radio-TLC and radio-HPLC. 225Ac-PNT2001 was synthesized by heating PNT2001 with 225Ac(NO3)3 in ascorbate buffer
(pH 5.5 – 6.0) to 90°C for 30 minutes and formulating in PBS with 0.1 mg/mL DTPA (pH 6 – 7). The radiochemical purity was
determined by radio-TLC. 177LuCl3 was purchased from commercial vendors. The 225Ac(NO3)3 used in this research was supplied by the
U.S. Department of Energy Isotope Program managed by the Office of Isotope R&D and Production.
The IC50 of natLu-PNT2001 for PSMA was measured using a competitive cell binding assay with LNCaP cells (n = 3). In vitro
internalization measurements were completed in LNCaP cells using 177Lu-PNT2001, 177Lu-PSMA-I&T and 177Lu-PSMA-617 and the data
were normalized to the reference compound [125I]I-BAKuE (n = 5). The biodistributions of 177Lu-PNT2001 and 177Lu-PSMA-I&T
were evaluated in CB17-SCID mice bearing LNCaP tumors (150-500 mm3). Mice were injected with the radioligand vial tail vein
(approx. 5-6 MBq, 160 pmol), euthanized 24 post-injection, and the activity of the tissues was quantified using a γ-counter to
determine the %ID/g in each tissue (n = 4). To conduct in vivo blocking studies CB17-SCID mice bearing LNCaP tumors were injected
via the tail vein with 177Lu-PSMA-62 (approx. 5-6 MBq, 160 pmol) both with and without a saturating amount of the PSMA inhibitor 2-
PMPA (6.4 nmol). Static µSPECT/CT images were acquired 24 h post-injection (n = 2). The efficacy of 225Ac-PNT2001 was measured
using LNCaP-tumor bearing NSG mice with tumors measuring approximately 200 mm3 or 800 mm3. The mice were treated with
vehicle or a single dose of 225Ac-PNT2001 (10, 20 or 40 kBq) (n = 5). The efficacy of 225Ac-PNT2001 and 225Ac-PSMA-I&T were
measured in a luciferase-enabled PSMA+ C4-2 model of metastatic prostate cancer. Mice were injected via tail vein with vehicle or a
single dose of the radioligand (40 kBq) and in vivo bioluminescent imaging was used to assess tumor burden (n = 10).
Accelerating Precision MedicineTM 3
Results
PNT2001 shows high affinity for PSMA (IC50 = 3.1 ± 0.2 nM), and increased tumor cell internalization compared to that of current first-
generation ligands (approximately 3.4-fold higher than PSMA-I&T) (Figure 2). 177Lu-PNT2001 has improved biodistribution properties
with low off-target tissue uptake in the kidneys (14.45 ± 2.15 %ID/g), high tumor retention (13.03 ± 1.04 %ID/g), and rapid renal
clearance, with an improved tumor:kidney ratio at 24 h post-injection (Figure 3).
% Internalization300 %ID/g 40
(relative control)
200 30
177Lu-PNT2001
100 177Lu-PSMA-I&T
0 20
177 Lu-PSM1A7-7I<u-PNT2001 Tumor:Kidney Ratio (X:1):
177Lu-PNT2001 0.902
Fig 2. PNT2001 shows improved
cellular internalization to PSMA- 10 177Lu-PSMA-I&T 0.117
I&T, measured with LNCaP cells
(n=3), relative to [125I]I-BAKuE 0
reference compound
Fig 3. Biodistribution of 177Lu-PNT2001 and 177Lu-PSMA-I&T at 24 h post-injection. LNCaP tumors were
implanted subcutaneously on the left flank and mice were treated with a single dose of compound (10
MBq).
Accelerating Precision MedicineTM
Blood
Heart
Lungs
Liver
PStaoSncplmreaeceahns
IKindtnesetiysne
Adrenal
Muscle
Bone
Tumor
4
Results
An in vivo PSMA competition binding study with saturating concentrations of 2-PMPA demonstrated target-specific binding of 177Lu-
PNT2001 using µSPECT/CT imaging (Figure 4).
AB
In Vivo: Ex Vivo: In Vivo: Ex Vivo:
T: 14.3 %ID/g T: 12.4 %ID/g T: 3.5 %ID/g T: 4.9 %ID/g
K: 13.7 %ID/g K: 11.3 %ID/g K: 2.6 %ID/g K: 2.9 %ID/g
Fig 4. Post-mortem µSPECT/CT imaging of LNCaP tumor-bearing CB17-SCID mice at 24 h post-injection of 160 pmol of 177Lu-PNT2001 (A). Co-injection with 6.4 nmol
2-PMPA as competitor (B) shows specific blocking of 177Lu-PNT2001.
Accelerating Precision MedicineTM 5
Results
In vivo efficacy in PSMA+ LNCaP-tumor bearing mice showed strong and dose-responsive efficacy with 225Ac-PNT2001 (Figure 5).
A B C D
30 3000 3000
2000
Mouse Weight (g) 20 Tumor volume (mm3) 2000 Tumor volume (mm3) 1000 Percent Survival 100 **
**
0
50
10 1000 ***
*** ***
*
0 0 Vehicle
0 20 40 60 80 100 0 20 40 60 80 100 (10kBq) 0
(20kBq) 0 20 40 60 80 100
Time post-injection (d) Time post-injection (d) (40kBq) Time post-injection (d)
Vehicle 225Ac-PNT2001 (10kBq) Vehicle 225Ac-PNT2001 (10kBq) 222222555AAAccc---PPPNNNTTT222000000111 Vehicle 225Ac-PNT2001 (10kBq)
225Ac-PNT2001 (20kBq) 225Ac-PNT2001 (40kBq) 225Ac-PNT2001 (20kBq) 225Ac-PNT2001 (40kBq) 225Ac-PNT2001 (20kBq) 225Ac-PNT2001 (40kBq)
Fig 5. A single dose of 225Ac-PNT2001 slows tumor growth and improves survival outcomes. NSG mice bearing subcutaneous LNCaP tumors on the flank were treated
with vehicle, 225Ac-PNT2001 (10 kBq), 225Ac-PNT2001 (20 kBq), or 225Ac-PNT2001 (40 kBq). (A) Average body weights for each group. (B) Average tumor volumes for
each group. Graphing stops when the first mouse from a group reaches endpoint. (C) Tumor volumes on day 35. Each symbol represents an individual mouse within
the group. (D) Kaplan-Meier survival curves of each group. *p<0.05, **p<0.01, ***p<0.001.
Accelerating Precision MedicineTM 6
Results
A single dose of 40 kBq as monotherapy controlled aggressive LNCaP tumor growth, and multiple mice achieved durable tumor
control with long-term survival >100 days (Figure 6). Mice tolerated treatment well at all dose levels.
A B C D
30 2000 2000 ns 100
20 1000 ns **
10 1000 ***
Mouse Weight (g) 50
Tumor volume (mm3) ns
0 **
Tumor volume (mm3) 0
222255AAcc--PPNNTT22000011Pr((eV24ce00ukkhriBBsclqoqer))0
Percent Survival
0 0 20 40 60 80 100
0 20 40 60 80 0 20 40 60 80 Time post-injection (d)
Time post-injection (d) Time post-injection (d)
Vehicle 225Ac-PNT2001 (20kBq) Vehicle 225Ac-PNT2001 (20kBq) Vehicle 225Ac-PNT2001 (20kBq)
Precursor 225Ac-PNT2001 (40kBq) Precursor 225Ac-PNT2001 (40kBq) Precursor 225Ac-PNT2001 (40kBq)
Fig 6. A single dose of 225Ac-PNT2001 slows tumor growth and improves survival outcomes in large tumors. NSG mice bearing subcutaneous LNCaP tumors on the 7
flank were treated with vehicle, precursor, 225Ac-PNT2001 (20 kBq), or 225Ac-PNT2001 (40 kBq). (A) Average body weights for each group. (B) Average tumor
volumes for each group. Graphing stops when the first mouse from a group reaches endpoint. (C) Tumor volumes on day 21. Each symbol represents an individual
mouse within the group. (D) Kaplan-Meier survival curves of each group. **p<0.01; ***p<0.001; ns, not significant.
Accelerating Precision MedicineTM
Results
Further in vivo efficacy was conducted in a challenging metastatic model of prostate cancer (luciferase-enabled PSMA+ C4-2 model),
in which 225Ac-PNT2001 significantly outperformed 225Ac-PSMA-I&T and controlled the formation of metastatic lesions (Figure 7).
A B C D E
30Mouse weight (g) 2×10 8 3×10 8BLI signal (p/sec/cm2/sr) 100
BLI signal (p/sec/cm2/sr) 1.5×10 8
20 2×10 8 Percent Survival 50
1×10 8
10 5×10 7 1×10 8 **T*o* Vehicle and
0 0 **** PSMA-I&T
0 20 40 60 80 ****
Time post-injection (d) ****
0 22252A5cA-c-PPSNMTA2-I0&0T1 ((44V00ekkhiBBclqq))e 0
0 20 40 60 80
Time post-injection (d) 0 20 40 60 80 100 120
Time post-injection (d)
Vehicle 225Ac-PNT2001 (40kBq) Vehicle 225Ac-PNT2001 (40kBq) Vehicle 225Ac-PNT2001 (40kBq)
225Ac-PSMA-I&T (40kBq) 225Ac-PSMA-I&T (40kBq) 225Ac-PSMA-I&T (40kBq)
Fig 7. A single dose of 225Ac-PNT2001 or 225Ac-PSMA-I&T slows tumor growth and improves survival outcomes. NSG mice bearing metastatic C4-2 tumors were
treated with vehicle, 225Ac-PNT2001 (40 kBq), or 225Ac-PSMA-I&T (40 kBq). Tumor cells expressed luciferase and could therefore be imaged to assess tumor burden
based on the correlative bioluminescence (BLI) signal. (A) Body weights for each group. (B) Average BLI signal for each group. Graphing stops when the first mouse
from a group reaches endpoint. (C) Average BLI signal for each mouse on day 29. Each symbol represents an individual mouse within the group. (D) Representative
BLI images on day 29 for each group. (E) Kaplan-Meier survival curves of each group. ****p<0.0001
Accelerating Precision MedicineTM 8
Conclusion
In this study PNT2001 has been shown to be a potent next-generation PSMA ligand with improved cell internalization using in vitro
studies.
In vivo biodistribution studies have shown that 177Lu-PNT2001 exhibits elevated tumor targeting and reduced kidney uptake,
properties well suited for actinium-225 alpha therapy.
Compelling therapeutic efficacy results using a single dose has been demonstrated in multiple preclinical tumor models.
Clinical studies assessing the safety, biodistribution and efficacy of 225Ac-PNT2001 are warranted.
The authors would like to acknowledge Scintomics for their work on the cellular internalization and Lu-177 biodistribution (Figures 2-
4) as well as Dr. Christine Mona and Dr. Pauline Jeanjean for their work on the metastatic efficacy study (Figure 7).
Accelerating Precision MedicineTM 9