Synthesis and Preclinical Evaluation of Two Novel 68Ga-Labeled Bispecific PSMA/FAP-Targeted Tracers with 2-Nal-Containing PSMA-Targeted Pharmacophore and Pyridine-Based FAP-Targeted Pharmacophore

Some bispecific radiotracers have been developed to overcome the limitations of monospecific tracers and improve detection sensitivity for heterogeneous tumor lesions. Here, we aim to synthesize two bispecific tracers targeting prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are key markers expressed in prostate cancer. A pyridine-based FAP-targeted ligand was synthesized through multi-step organic synthesis and then connected to the 2-Nal-containing PSMA-targeted motif. The Ki(PSMA) values of Ga-complexed bispecific ligands, Ga-AV01084 and Ga-AV01088, were 11.6 ± 3.25 and 28.7 ± 6.05 nM, respectively, and the IC50(FAP) values of Ga-AV01084 and Ga-AV01088 were 10.9 ± 0.67 and 16.7 ± 1.53 nM, respectively. Both [68Ga]Ga-AV01084 and [68Ga]Ga-AV01088 enabled the visualization of PSMA-expressing LNCaP tumor xenografts and FAP-expressing HEK293T:hFAP tumor xenografts in PET images acquired at 1 h post-injection. However, the tumor uptake values from the bispecific tracers were still lower than those obtained from the monospecific tracers, PSMA-targeted [68Ga]Ga-PSMA-617 and FAP-targeted [68Ga]Ga-AV02070. Further investigations are needed to optimize the selection of linkers and targeted pharmacophores to improve the tumor uptake of bispecific PSMA/FAP tracers for prostate cancer imaging.


Introduction
As the second most common cancer and the fifth leading cause of cancer death in men worldwide, prostate cancer had an estimated 1,410,000 new cases and 375,304 deaths in 2020 [1].Based on the recent cancer statistics by Siegel et al. [2], the 5-year relative survival rates of localized and regional prostate cancer are >99%; however, they have decreased significantly to 32% for metastatic prostate cancer patients.Therefore, early and accurate detection is important to further improve the survival rates of prostate cancer patients.Recently, the application of radiopharmaceuticals for the non-invasive detection and treatment of prostate cancer has shown positive outcomes, such as improving positive lesion detection rate and prolonging imaging-based progression-free survival [3,4].One of the promising radiotracers for metastatic prostate cancer diagnosis, which has been approved by the US FDA, is [ 68 Ga]Ga-PSMA-11, which targets prostate-specific membrane antigen (PSMA) [5,6].PSMA is a type II transmembrane glycoprotein, also known as folate hydrolase 1 and glutamate carboxypeptidase II [7,8].PSMA has been found to be highly expressed in prostate tumors and the neovasculature of other types of cancer, such as blood uptake.Furthermore, we found that Ga-AV02070 (Figure 1A), which has a carbonyl group at the para position to the pyridine nitrogen, has a better binding affinity to FAP and a higher tumor uptake compared to Ga-AV02053, which has a carbonyl group at the meta position to the pyridine nitrogen.Hence, the pharmacophore of AV02070 is a potential candidate for the design of FAP-targeted tracers.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 15 leading to much higher tumor-to-background contrast ratios.Our results suggest that pyridine-based FAPIs are more hydrophilic than quinoline-based FAPIs and have potential to help reduce blood uptake.Furthermore, we found that Ga-AV02070 (Figure 1A), which has a carbonyl group at the para position to the pyridine nitrogen, has a better binding affinity to FAP and a higher tumor uptake compared to Ga-AV02053, which has a carbonyl group at the meta position to the pyridine nitrogen.Hence, the pharmacophore of AV02070 is a potential candidate for the design of FAP-targeted tracers.In this paper, we report the design, synthesis, and evaluation of two bispecific PSMA/FAP-targeted radiotracers, [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 (Figure 1B).The PSMA-binding motif of AV01084 and AV01088 was based on the 2-Nal-containing PSMA-targeted tracer, [ 68 Ga]Ga-PSMA-617 (Figure 1A) [32], and their FAP-targeted motif was derived from our pyridine-based FAP-targeted radiotracer, [ 68 Ga]Ga-AV02070 (Figure 1A).The difference between the two tracers is the position of the DOTA chelator linked to the lysine, which is the ε-amino group in AV01084 and the α-amino group in AV01088.We performed an in vitro competition binding assay, PET imaging, and ex vivo biodistribution studies in preclinical PSMA-expressing LNCaP and FAP-expressing HEK293T:hFAP tumor models to evaluate the potential of [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 for prostate cancer imaging.The results were then compared with In this paper, we report the design, synthesis, and evaluation of two bispecific PSMA/FAP-targeted radiotracers, [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 (Figure 1B).The PSMA-binding motif of AV01084 and AV01088 was based on the 2-Nal-containing PSMA-targeted tracer, [ 68 Ga]Ga-PSMA-617 (Figure 1A) [32], and their FAP-targeted motif was derived from our pyridine-based FAP-targeted radiotracer, [ 68 Ga]Ga-AV02070 (Figure 1A).The difference between the two tracers is the position of the DOTA chelator linked to the lysine, which is the ε-amino group in AV01084 and the α-amino group in AV01088.We performed an in vitro competition binding assay, PET imaging, and ex vivo biodistribution studies in preclinical PSMA-expressing LNCaP and FAP-expressing HEK293T:hFAP tumor models to evaluate the potential of [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 for prostate cancer imaging.The results were then compared with those of the corresponding monospecific tracers, [ 68 Ga]Ga-PSMA-617 and [ 68 Ga]Ga-AV02070.

Binding Affinity and Lipophilicity
The binding affinities of Ga-AV01084, Ga-AV01088, and Ga-AV02070 to PSMA were measured by a cell-based competition binding assay using PSMA-expressing LNCaP prostate cancer cells and were compared to that of the previously published Ga-PSMA-617 (Ki = 1.23 ± 0.08 nM) [33].These ligands inhibited the binding of [ 18 F]DCFPyL to LNCaP cells in a dose-dependent manner (Figure 2A) and the calculated Ki(PSMA) values To synthesize AV01084, compound 5 was coupled to Lys(Lys(ivDde)-Gly-tranexamic acid-2-Nal)-urea-Glu(OtBu)-OtBu, followed by removal of the ivDde group at the Lys side chain and subsequent coupling with the DOTA chelator.To synthesize AV01088, DOTA chelator was first coupled to Lys(Lys(ivDde)-Gly-tranexamic acid-2-Nal)-urea-Glu(OtBu)-OtBu, followed by the deprotection of the amino group at the Lys side chain and coupling with compound 5.The DOTA-conjugated ligands were then cleaved off from resin and purified by HPLC (Table S1).AV01084 and AV01088 were obtained in 12% and 7.2% yields, respectively.

Binding Affinity and Lipophilicity
The binding affinities of Ga-AV01084, Ga-AV01088, and Ga-AV02070 to PSMA were measured by a cell-based competition binding assay using PSMA-expressing LNCaP prostate cancer cells and were compared to that of the previously published Ga-PSMA-617 (K i = 1.23 ± 0.08 nM) [33].These ligands inhibited the binding of [ 18 F]DCFPyL to LNCaP cells in a dose-dependent manner (Figure 2A) and the calculated K i (PSMA) values for Ga-AV01084, Ga-AV01088, and Ga-AV02070 were 11.6 ± 3.25, 28.7 ± 6.05, and >1000 nM, respectively (n = 3).

PET Imaging, Ex Vivo Biodistribution, and Blocking Studies
Representative PET images acquired at 1 h post-injection using [ 68 Ga]Ga-AV01084, [ 68 Ga]Ga-AV01088, [ 68 Ga]Ga-PSMA-617, and [ 68 Ga]Ga-AV02070 are provided in Figure 3. High uptake of all tracers in the kidneys and bladder indicated that they were excreted primarily through the renal pathway.LNCaP tumor xenografts were clearly visualized by [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 but not visualized by [ 68 Ga]Ga-AV02070 (Figure 3A).HEK293T:hFAP tumor xenografts were also visualized by the bispecific tracers ([ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088) but not visualized by [ 68 Ga]Ga-PSMA-617 (Figure 3B).The bispecific tracers had bone and joint uptake, which is commonly seen for FAP-targeted tracers.A high kidney uptake could be observed in mice injected with the Enzyme inhibition assays were performed using Suc-Gly-Pro-AMC as the FAP substrate to determine the binding affinities of Ga-AV01084, Ga-AV01088, and Ga-PSMA-617 to human FAP, and their data were compared to that of the previously published Ga-AV02070 (IC 50 = 17.1 ± 4.6 nM) [31].The human FAP enzymatic activity on the substrate was inhibited by these ligands in a dose-dependent manner (Figure 2B).The calculated IC 50 values for Ga-AV01084, Ga-AV01088, and Ga-PSMA-617 were 10.9 ± 0.67, 16.7 ± 1.53 and >1000 nM, respectively (n = 3).
Therefore, in this report, we selected the pharmacophores of [ 68 Ga]Ga-PSMA-617 (in brown, Figure 1) and [ 68 Ga]Ga-AV02070 (in blue, Figure 1) for constructing our bispecific PSMA/FAP tracers as they are more hydrophilic and have a high affinity for PSMA and FAP, respectively.We separated the two pharmacophores with a Lys-Gly linker (Figure 1B).The PSMA-targeted pharmacophore (Lys(tranexamic acid-2-Nal)-urea-Glu and the linker (Lys-Gly) were constructed directly on solid phase using commercially available amino acids.For AV01084, the pyridine-based FAP-targeted ligand and the DOTA chelator were linked to the α-amino group and side-chain of Lys, respectively.While for AV01088, the DOTA chelator and the pyridine-based FAP-targeted ligand were coupled to the α-amino group and side-chain of Lys, respectively.This allowed us to investigate the effect of the position of the DOTA chelator on the binding affinity and biodistribution of the bispecific tracers.
The enzymatic assay (Figure 2B) showed that the FAP-binding affinities of Ga-AV01084 (IC 50 = 10.9 ± 0.67 nM) and Ga-AV01088 (IC 50 = 16.7 ± 1.53 nM) were comparable or even slightly better than that of the previously reported Ga-AV02070 (IC 50 = 17.1 ± 4.60 nM) [31].We also measured the FAP-binding affinity of Ga-PSMA-617 to determine whether the PSMA-targeted pharmacophore has any effect on the overall FAP binding of our bispecific ligands or not.The very weak binding affinity of Ga-PSMA-617 (IC 50 > 1000 nM) indicates that the potent FAP-binding affinity of our bispecific ligands is mainly due to the binding of the AV02070 pharmacophore.
The PSMA-binding affinities of the bispecific ligands (K i = 11.6-28.7 nM) were inferior to that of Ga-PSMA-617 (K i = 1.23 ± 0.08) [33].However, when compared to the bispecific tracers from our previous report (K i = 20.1-54.4nM), changing Ala(9-Anth) to 2-Nal and changing quinoline to pyridine significantly increased the binding affinity of bispecific PSMA/FAP tracers to PSMA.This supports our hypothesis that there might be an interaction between the pharmacophores that interferes with the overall binding of the bispecific ligands to PSMA.Moreover, Ga-AV02070 has a very minimal binding affinity to PSMA (K i > 1000 nM), indicating that the PSMA-binding affinity of our bispecific ligands is mainly due to the binding of the PSMA-617 pharmacophore.
Unlike the improved uptake in LNCaP tumor xenografts, the uptake of [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 (1.20-1.90%ID/g) in HEK293T:hFAP tumor xenografts remains significantly lower than that of the monospecific counterpart, [ 68 Ga]Ga-AV02070 (7.93 ± 1.88 %ID/g, p < 0.05).One possible reason is the addition of the succinic acid linker, which might have interfered with the binding of our tracers to FAP, thus decreasing the tumor uptake.Previous studies have shown the importance of piperazine-based linkers for maintaining the good tumor uptake of FAP-targeted tracers [34].Therefore, incorporating a piperazine-based linker can also be considered for future modifications to improve the uptake of bispecific PSMA/FAP tracers to the FAP-expressing tumors.
Compared to the monospecific tracers, the bispecific tracers have higher blood, bone, and muscle uptake.However, the current modified tracers have significantly decreased blood retention (0.79-2.26 %ID/g) when compared to the bispecific PSMA/FAP tracers in our previous report (5.75-11.94%ID/g) [28].This leads to better LNCaP tumor-to-blood contrast ratios for [ 68 Ga]Ga-AV01084 (4.09 ± 0.77) and [ 68 Ga]Ga-AV01088 (4.10 ± 0.88) (Table S4) than our previously reported bispecific PSMA/FAP tracers (0.48-0.89) [28].The decreased blood retention might be attributed to the increased hydrophilicity of the new bispecific tracers shown by their low LogD 7.4 values (<−3.60).This is consistent with previous findings that more hydrophilic radiotracers will have faster pharmacokinetics and clearance and, hence, a better tumor-to-background contrast ratio [35].In addition, there are no significant differences in the tumor uptake or the tumor-to-background contrast ratios between [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088, which indicates that the position of the DOTA chelator at the lysine linker does not have a crucial effect on the pharmacokinetics of the tracers.Previously, Wang et al. [27] reported a 68 Ga-labeled bispecific PSMA/FAP tracer, [ 68 Ga]Ga-FAPI-PSMA (Figure 7), consisting of a PSMA-targeted pharmacophore from PSMA-617 (in brown, Figure 7) and an FAP-targeted pharmacophore from FAPI-04 (in blue).It would be difficult to directly compare the performance of our bispecific radiotracers with [ 68 Ga]Ga-FAPI-PSMA as different tumor models were used for evaluation: PSMAexpressing LNCaP tumors and FAP-expressing HEK293T:hFAP tumors were used in our study, and PSMA-expressing 22Rv1 tumors and FAP-expressing U87 MG tumors were used by Wang et al. [27].[ 68 Ga]Ga-FAPI-PSMA was shown to achieve superior tumor uptake (SUVmax = 1.67 for U87 MG tumor xenografts; SUVmax = 1.32 for 22Rv1 tumor xenografts) when compared to those of the monospecific tracers, [ 68 Ga]Ga-FAPI-04 (SUVmax = 0.45 for U87 MG tumor xenografts) and [ 68 Ga]Ga-PSMA-617 (SUVmax = 0.25 for 22Rv1 tumor xenografts).This demonstrates the potential of utilizing bispecific PSMA/FAP tracers to improve the tumor uptake.However, similar to our results, [ 68 Ga]Ga-FAPI-PSMA also had higher background uptake compared to the monospecific tracers.
vious findings that more hydrophilic radiotracers will have faster pharmacokinetics and clearance and, hence, a better tumor-to-background contrast ratio [35].In addition, there are no significant differences in the tumor uptake or the tumor-to-background contrast ratios between [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088, which indicates that the position of the DOTA chelator at the lysine linker does not have a crucial effect on the pharmacokinetics of the tracers.
Previously, Wang et al. [27] reported a 68 Ga-labeled bispecific PSMA/FAP tracer, [ 68 Ga]Ga-FAPI-PSMA (Figure 7), consisting of a PSMA-targeted pharmacophore from PSMA-617 (in brown, Figure 7) and an FAP-targeted pharmacophore from FAPI-04 (in blue).It would be difficult to directly compare the performance of our bispecific radiotracers with [ 68 Ga]Ga-FAPI-PSMA as different tumor models were used for evaluation: PSMA-expressing LNCaP tumors and FAP-expressing HEK293T:hFAP tumors were used in our study, and PSMA-expressing 22Rv1 tumors and FAP-expressing U87 MG tumors were used by Wang et al. [27].[ 68 Ga]Ga-FAPI-PSMA was shown to achieve superior tumor uptake (SUVmax = 1.67 for U87 MG tumor xenografts; SUVmax = 1.32 for 22Rv1 tumor xenografts) when compared to those of the monospecific tracers, [ 68 Ga]Ga-FAPI-04 (SU-Vmax = 0.45 for U87 MG tumor xenografts) and [ 68 Ga]Ga-PSMA-617 (SUVmax = 0.25 for 22Rv1 tumor xenografts).This demonstrates the potential of utilizing bispecific PSMA/FAP tracers to improve the tumor uptake.However, similar to our results, [ 68 Ga]Ga-FAPI-PSMA also had higher background uptake compared to the monospecific tracers.Replacing the quinoline-based FAP-targeted pharmacophore with a pyridine-based FAP-targeted pharmacophore helps decrease the background uptake (blood, bone, and muscle) of the bispecific tracers; however, it also results in a significantly lower uptake in HEK293T:hFAP tumors.Therefore, future attempts on the use of a pyridine-based FAPtargeted pharmacophore for the design of bispecific PSMA/FAP tracers need to include the optimization of linkers such as incorporating a piperazine-based linker to continue improving FAP-binding affinity and tumor uptake.

Synthesis of Bispecific PSMA/FAP-Targeted Ligands
The procedures for the synthesis, purification, and characterizations of the bispecific PSMA/FAP ligands, AV01084 and AV01088, and their Ga-labeled analogs (both nat Ga and 68 Ga) are provided in the Supplementary Materials (Tables S1-S3 and Figures S1-S11).

Lipophilicity Measurement
The lipophilicity of 68 Ga-labeled tracers was determined by measuring their LogD 7.4 values following literature procedures [31,36].Briefly, 68 Ga-labeled tracer (50 µL) was mixed with 3 mL n-octanol and 3 mL PBS (pH 7.4) in a 15 mL falcon tube.After vortexing (1 min) and centrifugation (3000 rpm, 15 min), 1 mL from each phase was collected and counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 gamma counter.The LogD 7.4 value was calculated using the following equation: LogD 7.4 = log 10 [(counts in the n-octanol phase)/(counts in the buffer phase)].

In Vitro PSMA Competition Binding Assay
Binding affinities of Ga-labeled ligands to PSMA were measured following literature procedures using PSMA-expressing prostate cancer LNCaP cells [28,33].K i values were calculated using the nonlinear regression algorithm of GraphPad Prism 7.02 (San Diego, CA, USA) software.

Biodistribution and PET/CT Imaging Studies
Imaging and ex vivo biodistribution studies were performed following literature procedures [28,31,33] using male NRG (NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ) mice.These animal experiments were performed following the guidelines of the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia.Mice were sedated by isoflurane (2.5% in oxygen) and injected with 100 µL LNCaP (2 × 10 5 cells/mouse) or HEK293T:hFAP (8.5 × 10 6 cells/mouse) cells subcutaneously behind the left shoulder.When tumors reached ~5-8 mm in diameter (~3-4 weeks for HEK293T:hFAP tumors and ~4-5 weeks for LNCaP tumors), mice were used for PET/CT imaging and ex vivo biodistribution studies.
For PET imaging studies, each mouse was injected with the 68 Ga-labeled tracer (~4-6 MBq/mouse) through a tail vein while under anesthesia (2.5% isoflurane in oxygen).PET/CT imaging was conducted using a Siemens (Knoxville, TN, USA) Inveon micro PET/CT scanner.At 50 min post-injection, a 10 min CT scan was carried out for localization and attenuation correction, followed by a 10 min static PET imaging acquisition.
For biodistribution studies, the tumor-bearing mice were injected with ~2-4 MBq/mouse of the 68 Ga-labeled tracer.For blocking studies, the mice bearing LNCAP tumor xenografts were co-injected with 2-PMPA (500 µg/mouse), and the mice bearing HEK293T:hFAP tumor xenografts were co-injected with FAPI-04 (250 µg/mouse).The tumor-bearing mice were euthanized by CO 2 inhalation at 1 h post-injection, and organs/tissues of interest were collected and counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 gamma counter.

Statistical Analysis
Data analyses were conducted using the GraphPad Prism version 7.02 and Microsoft (Redmond, WA, USA) Excel 2016 software.All organs in the biodistribution studies of [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 in LNCaP and HEK293T:hFAP tumor-bearing mice were analyzed with one-way ANOVA and multiple t-tests.When the adjusted p-value was <0.05 using the Holm-Sidak method, a statistically significant difference was considered present.

Conclusions
Both bispecific tracers, [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088, were confirmed to have the ability to bind PSMA and FAP in vitro and in vivo.Both [ 68 Ga]Ga-AV01084 and [ 68 Ga]Ga-AV01088 have decreased PSMA-binding affinities but retain comparable binding affinities towards FAP when compared with the monospecific tracers.Compared with three of our previously reported bispecific PSMA/FAP tracers ([ 68 Ga]Ga-AV01017, [ 68 Ga]Ga-AV01030, and [ 68 Ga]Ga-AV01038), both [ 68 Ga]Ga-AV01084 and Ga-AV01088 have better PSMA-binding affinity and improved tumor uptake in PSMA-expressing xenografts and tumor-to-background (blood, muscle, and bone) contrast ratios.Further optimization on the selection of linkers should be explored to improve the binding affinity, pharmacokinetics, and tumor uptake of the bispecific PSMA/FAP tracers.

Figure 6 .
Figure 6.Comparison of [ 68 Ga]Ga-AV01088 with and without co-injection of 2-PMPA on the uptake in LNCaP tumor xenografts and major organs/tissues in mice at 1 h post-injection.Error bars indicate standard deviation.* p < 0.05, *** p < 0.001.

Figure 6 .
Figure 6.Comparison of [ 68 Ga]Ga-AV01088 with and without co-injection of 2-PMPA on the uptake in LNCaP tumor xenografts and major organs/tissues in mice at 1 h post-injection.Error bars indicate standard deviation.* p < 0.05, *** p < 0.001.