Synthesis and Preclinical Evaluation of Three Novel 68Ga-Labeled Bispecific PSMA/FAP-Targeting Tracers for Prostate Cancer Imaging

Tumor heterogeneity limits the efficacy and reliability of monospecific radiopharmaceuticals in prostate cancer diagnosis and therapy. To overcome this limitation and improve lesion detection sensitivity, we developed and evaluated three bispecific radiotracers that can target both prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are the two key proteins overexpressed in prostate cancer. Three FAP-targeting ligands with various linker lengths were synthesized through multistep organic synthesis, and then connected to the PSMA-targeting motif. IC50(PSMA) and IC50(FAP) values of Ga-complexed bispecific ligands, Ga-AV01017, Ga-AV01030, and Ga-AV01038 were 25.2–71.6 and 1.25–2.74 nM, respectively. The uptake values in PSMA-expressing LNCaP tumor xenografts were 4.38 ± 0.55, 5.17 ± 0.51, and 4.25 ± 0.86 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were lower than the monospecific PSMA-targeting tracer [68Ga]Ga-HTK03041 (23.1 ± 6.11 %ID/g). The uptake values in FAP-expressing HEK293T:hFAP tumor xenografts were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were also lower than the monospecific FAP-targeting tracer, [68Ga]Ga-FAPI-04 (12.5 ± 2.00 %ID/g). We observed that the bispecific tracers had prolonged blood retention, in which tracers with a longer linker tend to have a higher blood uptake and lower tumor uptake. Further investigations are needed to optimize the linker selection to generate promising bispecific PSMA/FAP-targeting tracers for prostate cancer imaging.


Introduction
Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein also known as glutamate carboxypeptidase II, has enzymatic functions to cleave terminal glutamate [1,2]. PSMA has been shown to be overexpressed in prostate tumor and the neovasculature of other type of cancer, such as colon and renal cancers [3], making it an attractive imaging and therapeutic target. Various PSMA-targeting radioligands have been developed and three of them have been approved by the US FDA: [ 18 F]DCFPyL and [ 68 Ga]Ga-PSMA-11 for prostate cancer imaging [4,5] and [ 177 Lu]Lu-PSMA-617 for prostate cancer radioligand therapy [6].
Although [ 177 Lu]Lu-PSMA-617 has been proven to be effective in treating metastatic castration-resistant prostate cancer (mCRPC) patients [7], some patients have lesions with heterogeneous expression, or even no expression of PSMA [8,9], limiting their eligibility in benefiting from PSMA-directed therapy. Moreover, studies have shown that lesions that are PSMA-negative, such as neuroendocrine prostate cancer (NEPC), are prone to being To expand the potential usage of bispecific PSMA/FAP-targeting ligands for radio therapeutic applications, we chose DOTA as our chelator, which has theranostic capabil ties to label both diagnostic isotopes, such as 68 Ga, and therapeutic isotopes, such as 177 Lu This will enable us to use the same ligands for diagnostic applications and ensure th pharmacokinetics of diagnostic and radiotherapeutic agents are comparable as they ar derived from the same PSMA/FAP-targeting ligands.
Here we report the design, synthesis, and evaluation of three 68 Ga-labeled DOTA conjugated bispecific PSMA/FAP tracers ( Figure 2). The PSMA binding motif of AV01017 AV01030, and AV01038 was based on our previously reported [ 68 Ga]Ga-HTK03041 [34 and their FAP-targeting motif was derived from [ 68 Ga]Ga-FAPI-04. The difference be tween these three tracers is the length of their linker between the quinoline and the triazol with ring null, -O-CH2-, and -O-(CH2)3-for 68 Ga-labeled AV01030, AV01038, and AV01017 respectively. Their potential of the tracers for prostate cancer imaging was evaluated b Here we report the design, synthesis, and evaluation of three 68 Ga-labeled DOTAconjugated bispecific PSMA/FAP tracers ( Figure 2). The PSMA binding motif of AV01017, AV01030, and AV01038 was based on our previously reported [ 68 Ga]Ga-HTK03041 [34], and their FAP-targeting motif was derived from [ 68 Ga]Ga-FAPI-04. The difference between these three tracers is the length of their linker between the quinoline and the triazole with ring null, -O-CH 2 -, and -O-(CH 2 ) 3 -for 68 Ga-labeled AV01030, AV01038, and AV01017, respectively. Their potential of the tracers for prostate cancer imaging was evaluated by 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 in mice. The results were then compared with those of the corresponding monospecific tracers, [ 68 Ga]Ga-HTK03041 and [ 68 Ga]Ga-FAPI-04.
The synthesis of compounds 5, 9, and 13 are depicted in Schemes 1-3, respectively, and detailed synthetic procedures and characterizations for the final products and intermediates are provided in the Supplementary Materials. For the preparation of compound 5 (Scheme 1), quininic acid was demethylated in 48% HBr solution, followed by esterification in methanol in the presence of thionyl chloride to obtain 1 in 47% yield. Mitsunobu coupling between compound 1 and 4-pentyn-1-ol resulted in 2 in 100% yield. Compound 3 was obtained in 74% yield by the hydrolysis of 2 with NaOH in a mixture of water and methanol. Esterification of compound 3 with tetrafluorphenol (TFP) led to compound 4 in 72% yield. Compound 5 was obtained in 97% yield by coupling the activated ester 4 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile.
Molecules 2023, 28, x FOR PEER REVIEW  5 of 17 for AV01017, Compound 9 for AV01030, and compound 13 for AV01038. The amino group at the Lys side chain was then deprotected and coupled with the DOTA chelator. The DOTA-conjugated ligands were then cleaved off from resin and purified by HPLC. The synthesis of compounds 5, 9, and 13 are depicted in Schemes 1-3, respectively, and detailed synthetic procedures and characterizations for the final products and intermediates are provided in the Supplementary Materials. For the preparation of compound 5 (Scheme 1), quininic acid was demethylated in 48% HBr solution, followed by esterification in methanol in the presence of thionyl chloride to obtain 1 in 47% yield. Mitsunobu coupling between compound 1 and 4-pentyn-1-ol resulted in 2 in 100% yield. Compound 3 was obtained in 74% yield by the hydrolysis of 2 with NaOH in a mixture of water and methanol. Esterification of compound 3 with tetrafluorphenol (TFP) led to compound 4 in 72% yield. Compound 5 was obtained in 97% yield by coupling the activated ester 4 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile. The synthesis of compound 9 is depicted in Scheme 2. The Sonogashira reaction was performed for coupling methyl 6-bromoquinoline-4-carboxylate and ethynyltrimethylsilane to obtain compound 6 in 62% yield. Silane deprotection and ester hydrolysis were performed simultaneously with LiOH in a mixture of water and methanol to afford compound 7 in 83% yield. Compound 7 was subsequently activated with tetrafluorophenol to afford the activated ester 8 in 71% yield. The desired compound 9 was obtained in 100% yield by coupling the activated ester 8 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile. for AV01017, Compound 9 for AV01030, and compound 13 for AV01038. The amino group at the Lys side chain was then deprotected and coupled with the DOTA chelator. The DOTA-conjugated ligands were then cleaved off from resin and purified by HPLC.
The synthesis of compounds 5, 9, and 13 are depicted in Schemes 1-3, respectively, and detailed synthetic procedures and characterizations for the final products and intermediates are provided in the Supplementary Materials. For the preparation of compound 5 (Scheme 1), quininic acid was demethylated in 48% HBr solution, followed by esterification in methanol in the presence of thionyl chloride to obtain 1 in 47% yield. Mitsunobu coupling between compound 1 and 4-pentyn-1-ol resulted in 2 in 100% yield. Compound 3 was obtained in 74% yield by the hydrolysis of 2 with NaOH in a mixture of water and methanol. Esterification of compound 3 with tetrafluorphenol (TFP) led to compound 4 in 72% yield. Compound 5 was obtained in 97% yield by coupling the activated ester 4 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile. The synthesis of compound 9 is depicted in Scheme 2. The Sonogashira reaction was performed for coupling methyl 6-bromoquinoline-4-carboxylate and ethynyltrimethylsilane to obtain compound 6 in 62% yield. Silane deprotection and ester hydrolysis were performed simultaneously with LiOH in a mixture of water and methanol to afford compound 7 in 83% yield. Compound 7 was subsequently activated with tetrafluorophenol to afford the activated ester 8 in 71% yield. The desired compound 9 was obtained in 100% yield by coupling the activated ester 8 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile. The synthetic procedures for the preparation of compound 13 are provided in Scheme 3. Mitsunobu coupling of compound 1 and 2-propyn-1-ol resulted in 10 in 100% yield. Compound 10 was hydrolyzed with NaOH in a mixture of water and methanol to obtain compound 11 in 73% yield. Activation of compound 11 with tetrafluorophenol resulted in compound 12 in 68% yield. Compound 13 was obtained in 100% yield by coupling the activated ester 12 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile.  Tables S2 and S3). Nonradioactive Ga-complexed AV01017, AV01030, and AV01038 were obtained in 25-89% yields, and their 68 Ga-labeled analogs were obtained in 43-60% decay-corrected radiochemical yields with > 74 GBq/µmol molar activity and > 99% radiochemical purity.
The synthesis of compound 9 is depicted in Scheme 2. The Sonogashira reaction was performed for coupling methyl 6-bromoquinoline-4-carboxylate and ethynyltrimethylsilane to obtain compound 6 in 62% yield. Silane deprotection and ester hydrolysis were performed simultaneously with LiOH in a mixture of water and methanol to afford compound 7 in 83% yield. Compound 7 was subsequently activated with tetrafluorophenol to afford the activated ester 8 in 71% yield. The desired compound 9 was obtained in 100% yield by coupling the activated ester 8 with (S)-1- The synthetic procedures for the preparation of compound 13 are provided in Scheme 3. Mitsunobu coupling of compound 1 and 2-propyn-1-ol resulted in 10 in 100% yield. Compound 10 was hydrolyzed with NaOH in a mixture of water and methanol to obtain compound 11 in 73% yield. Activation of compound 11 with tetrafluorophenol resulted in compound 12 in 68% yield. Compound 13 was obtained in 100% yield by coupling the activated ester 12 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile.

Discussion
Both PSMA and FAP are promising biomarkers of prostate cancer and many radioligands have been developed to target these two membrane proteins for imaging and therapy. However, the detection sensitivities of these radiotracers are strongly dependent on the expression levels and heterogeneities of these two biomarkers in different disease stages and between individuals [8,9]. Despite many effective PSMA-targeting radiotherapeutic agents being developed, patients with low to no PSMA expression are not eligible

Discussion
Both PSMA and FAP are promising biomarkers of prostate cancer and many radioligands have been developed to target these two membrane proteins for imaging and therapy. However, the detection sensitivities of these radiotracers are strongly dependent on the expression levels and heterogeneities of these two biomarkers in different disease stages and between individuals [8,9]. Despite many effective PSMA-targeting radiotherapeutic agents being developed, patients with low to no PSMA expression are not eligible for these emerging PSMA-targeted radioligand therapies and still have very limited treatment options. Since FAP and PSMA are both expressed in prostate cancer and other cancers such as pancreatic cancer [25,[35][36][37][38], the use of PSMA/FAP bispecific radioligands is expected to increase lesion detection sensitivity and treatment efficacy. The ultimate goal of this reported research is to develop 68 Ga-labeled PSMA/FAP bispecific tracers with comparable or higher tumor uptake when compared to the monospecific tracers, [ 68 Ga]Ga-FAPI-04 and [ 68 Ga]Ga-HTK03041, a PSMA-targeting tracer previously reported by our group [34].
To date there have been only two reports on the development of PSMA/FAP bispecific tracers for imaging ( Figure 1) [32,33]. Both reports used the NOTA chelator for radioisotope complexation: one for 64 Cu [32] and the other for Al 18 F [33]. To the best of our knowledge, there has been no report on the development of 68 Ga-labeled PSMA/FAP bispecific tracers despite the growing popularity and increased accessibility of clinical 68 Ga generators. Although NOTA can be used for labeling 68 Ga as well, it cannot be used for labeling the common and effective radiotherapeutic nuclides such as 90 Y and 177 Lu. Therefore, for the current research, we chose DOTA as the chelator for 68 Ga labeling as DOTA is a widely used chelator and is effective for labeling with many diagnostic (such as 68 Ga, 152 Tb, and 111 In) and radiotherapeutic radionuclides (such as 149 Tb, 90 Y, and 177 Lu).
We selected the pharmacophores of [ 68 Ga]Ga-FAPI-04 (in blue, Figure 2) and [ 68 Ga]Ga-HTK03041 (in brown, Figure 2) for the design of our PSMA/FAP bispecific tracers as they have high affinity for FAP and PSMA, respectively [26,34]. These two pharmacophores are separated by an azidoacetic-Lys-Gly linker ( Figure 2) to minimize the interaction of both pharmacophores as such interaction might interfere with the bindings of both pharmacophores to their respective targets. The PSMA-targeting pharmacophore (Lys(tranexamic acid-Ala(9-Anth))-urea-Glu and the linker (azidoacetic acid-Lys-Gly) was constructed directly on solid phase using the commercially available amino acids. While the DOTA chelator was coupled to the amino group on the Lys side chain, the alkyne-containing FAP-targeting motif was coupled to the azido group via the formation of a triazole ring by click chemistry. For AV01030, the triazole ring is directly linked to the quinoline ring of the FAP-targeting pharmacophore. For AV01038 and AV01017, there are additional -O-CH 2and -O-(CH 2 ) 3 -, respectively, to separate the triazole ring and the quinoline ring of the FAP-targeting pharmacophore. This allowed us to investigate the effect of the additional linker and its length on the binding affinity and pharmacokinetics of the resulting bispecific tracers.
The enzymatic assay ( Figure 3B) confirmed that the FAP binding affinities of our bispecific ligands (IC 50 = 1.25-2.74 nM) were comparable to that of Ga-FAPI-04 (IC 50 = 1.03 nM). To investigate if the PSMA-targeting pharmacophore has any effect on the overall FAP binding of our bispecific ligands, we also measured the FAP binding affinity of Ga-HTK03041. The very weak binding affinity of Ga-HTK03041 (IC 50 = 2010 ± 585 nM) suggests that the potent FAP binding affinity of our bispecific ligands is contributed mainly by the FAPI-04 pharmacophore.
Unlike the comparable FAP binding affinity of bispecific ligands and the monospecific Ga-FAPI-4, the PSMA binding affinities of bispecific ligands (IC 50 = 25.2-71.6 nM) were inferior to that of Ga-HTK03041 (IC 50 = 0.76 nM). Although Ga-FAPI-04 has minimal binding affinity to PSMA (IC 50 > 1000 nM), the addition of its pharmacophore and a linker clearly interferes the overall binding of the bispecific ligands to PSMA. Such interference seems to decrease with the increased linker length between the triazole ring and the FAP-targeting pharmacophore as the IC 50 (PSMA) values for Ga-AV01030, Ga-AV01038, and Ga-AV01017 are 71.6, 29.4, and 25.2 nM, respectively.
PET imaging and biodistribution studies (Figures 4-6 and Supplemental Tables S4 and S5) revealed that the bispecific tracers retained the characteristics of the monospecific tracers as high uptake was observed in common off-targets of PSMA-targeting (kidneys and salivary glands) and FAP-targeting tracers (joints and salivary glands). However, the bispecific tracers showed much lower uptake values in LNCaP and HEK293T:hFAP tumor xenografts when compared to those of the monospecific tracers, [ 68 Ga]Ga-HTK03041 and [ 68 Ga]Ga-FAPI-04, respectively. Unlike the fast blood clearance of the monospecific tracers, the bispecific tracers had a much longer retention in blood. It seems that the blood retention was positively correlated to the length of the linker between the triazole ring and the FAP-targeting pharmacophore as the blood uptake values for Ga-AV01030, Ga-AV01038, and Ga-AV01017 are 5.68-5.75, 7.07-7.45, and 9.24-11.9 %ID/g, respectively. A longer blood retention would prevent fast binding of the bispecific tracers to their targets: PSMA in the LNCaP tumor xenografts and FAP in the HEK293T:hFAP tumor xenografts. This is evident by the observations that with no additional linker between the triazole ring and the FAP-targeting pharmacophore, [ 68 Ga]Ga-AV01030 showed relatively higher tumor uptake and tumor-to-background (bone, muscle and blood) contrast ratios in both tested tumor models than those of [ 68 Ga]Ga-AV01017 and [ 68 Ga]Ga-AV01038.
It should be noted that evaluation by imaging and/or biodistribution studies in tumorbearing mice as in this report is a common practice in the development of radiopharmaceuticals. However, the obtained high tumor-to-background contrast either from imaging or biodistribution data might not be observed in the clinic especially for 68 Ga-labeled tracers. This is because compared with other positron emitters, 68 Ga has a higher average positron energy ( 68 Ga, 0.829 MeV; 18 F, 0.250 MeV; 64 Cu, 0.288 MeV; 89 Zr, 0.396 MeV), leading to poorer spatial resolution and difficulty in the visualization of small lesions (a few millimeters in size). In addition, the tumor xenografts used in the imaging and biodistribution studies are often derived from (genetically-modified) cancer cell lines overexpressing the targeted cancer markers, which might not be representative of cancer lesions encountered in the clinic.
Directly comparing the performance of our bispecific tracers with those reported previously is difficult as different tumor models were used for evaluation: PSMA-expressing LNCaP tumors and FAP-expressing HEK293T:hFAP tumors used in this report; PSMAexpressing PC3-PIP tumors and FAP-expressing U87 tumors used by Boinapally et al. [32]; PSMA-expressing 22Rv1 tumors and FAP-expressing A549-FAP tumors used by Hu et al. [33]. However, comparing the pharmacokinetics of our tracers and those reported previously shows that our tracers had significantly higher blood retention at 1 h post injection. The longer blood retention of our tracers could be due to the increased lipophilicity [39], which could be a result of (1) the replacement of 2-Nal with a more lipophilic Ala(9-Anth) in the PSMA-targeting pharmacophore; (2) the deletion of the hydrophilic piperazine linker in the FAP-targeting pharmacophore of FAPI-04; and/or (3) the use of click reaction for coupling the FAP-targeting pharmacophore as this forms a relatively lipophilic triazole ring instead of a hydrophilic amide bond.
It should be noted that the same diagnostic information obtained from the use of a bispecific tracer could be obtained by two separate scans using its corresponding monospecific tracers. However, the use of a bispecific tracer would save the time and overall cost by reducing two procedures (hospital visit, tracer preparation, tracer injection, PET scan, scan reading, and report writing) to one, and reduce patients' absorbed radiation dose from PET scanning. It should also be noted that compared to its corresponding monospecific tracers, a bispecific tracer would have a larger molecular size, and combining two different targeting vectors into a single molecule could potentially negatively impact both its pharmacokinetic properties and receptor binding affinity. However, with thorough investigations on the selection of targeting vectors, linkers, and potentially pharmacokinetic modifiers, it is likely to obtain an optimized bispecific tracer with higher tumor uptake and even higher tumor-to-background contrast ratios than both of its corresponding monospecific tracers. For example, the Chen group designed and evaluated gastrin-releasing peptide receptor (GRPR) and integrin α v β 3 bispecific radiotracers for imaging prostate cancer [40]. GRPR is highly expressed by prostate cancer cells, whereas integrin α v β 3 is expressed by tumor neovasculature. Their results showed that the GRPR/integrin α v β 3 bispecific tracer ([ 18 F]FB-BBN-RGD) achieved higher tumor uptake and tumor-to-background contrast ratios than both of its corresponding monospecific tracers ([ 18 F]FB-BBN for GRPR and [ 18 F]FB-RGD for integrin α v β 3 ).
Further modifications are needed to improve the binding affinities, pharmacokinetics, and tumor uptake of 68 Ga-labeled PSMA/FAP bispecific tracers by considering the linker's length, lipophilicity, and the use of less lipophilic PSMA-and/or FAP-targeting pharmacophores. This can be accomplished by the use of less lipophilic linkers such as PEG linkers and well as piperazine-based linkers which have been shown to be important for maintaining good tumor uptake of FAP-targeting tracers [41]. Similarly, the use of potent and less lipophilic pharmacophores should also be investigated such as (1) replacing Ala(9-Anth) in the PSMA-targeting pharmacophore with 2-Nal, (2) replacing the quinoline motif in the FAP-targeting pharmacophore with a more hydrophilic pyridine motif, and/or (3) replacing the 2-pyrrolidinecarbonitrile motif in the FAP-targeting pharmacophore with a more hydrophilic pyrrolidin-2-ylboronic acid.

Synthesis of Bispecific PSMA/FAP-Targeting Ligands
Detailed information for the synthesis, purification, and characterizations of AV01017, AV01030, and AV01038, and their nonradioactive Ga-complexed standards and 68 Galabeled analogs are provided in the supplemental information (Supplemental Tables S1-S3).

Cell Culture
The LNCaP cells obtained from ATCC (via Cedarlane, Burlington, ON, Canada) were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 • C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 . The cells were confirmed to be pathogen-free by the IMPACT Rodent Pathogen Test (IDEXX BioAnalytics). Cells were grown until 80-90% confluence and washed with sterile phosphate-buffered saline (PBS, pH 7.4) and collected after 1 min trypsinization. The cell concentration was counted in triplicate using a hemocytometer and a manual laboratory counter.

Cell Transfection
The HEK293T cells were obtained from ATCC. The FAP-expressing vector was constructed using Genome-CRISPRTM Human AAVS1 Safe Harbor Gene Knock-in Kits (GeneCopoeaiaTM) by inserting FAP-expressing gene into the AAVS1 vector. The cells were then transfected by the FAP-expressing vector following the EndofectinTM Transfection Reagent protocol. The cells underwent 3 serial dilutions and were sorted using fluorescence-activated cell sorting (FACS) to obtain FAP-expressing monoclonal colonies. HEK293T:hFAP cells were cultured in DMEM GlutaMAX™ medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 • C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 . Cells were grown until 80-90% confluence and washed with sterile PBS (pH 7.4) and collected.

In Vitro PSMA Competition Binding Assay
The PSMA binding assays were conducted following previously published procedures using LNCaP cells and [ 18 F]DCFPyL as the radioligand [34,42,43]. Data analyses of IC 50 were performed using the nonlinear regression algorithm of GraphPad Prism 7 (San Diego, CA, USA) software.

In Vitro FAP Fluorescence Assay
The half maximal inhibitory concentration (IC 50 ) values of the tested compounds for FAP were measured by in vitro enzymatic assay. The recombinant human FAP (Biolegend; 0.2 µg/mL, 50 µL) was added into a costar 96-well plate. PBS and varied concentrations (0.2 pM to 2 µM) of tested nonradioactive Ga-complexed standards were added to each well (in duplicate) containing the recombinant human FAP. After being incubated for 30 min at 37 • C, 50 µL of Suc-Gly-Pro-AMC (2 µM, Bachem) was added to each well. The fluorescent signals were acquired at 15, 30, 45, and 60 min using a FlexStation 3 Multi-Mode Microplate Reader with excitation at 380 nm and emission at 460 nm. The IC 50 (FAP) was calculated using a "nonlinear fit model" built-in model in GraphPad Prism 7.02 software

Ex Vivo Biodistribution and PET/CT Imaging Studies
Imaging and biodistribution studies were performed using male NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) mice following previously published procedures [44][45][46]. The experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. The mice were briefly sedated by inhalation of 2.5% isoflurane in oxygen, and 100 µL LNCaP (2 × 10 5 cells) or HEK293T:hFAP (8.5 × 10 6 cells) cells were inoculated subcutaneously behind the left shoulder. When the tumor grew to 5-8 mm in diameter over 3-4 weeks and 4-5 weeks for HEK293T:hFAP and LNCaP tumors, respectively, the mice were used for PET/CT imaging and biodistribution studies.
PET/CT imaging experiments were carried out using a Siemens (Knoxville, TN) Inveon micro PET/CT scanner. Each tumor-bearing mouse was injected with~4-6 MBq of 68 Ga-labeled tracer through a lateral caudal tail vein under 2.5% isoflurane in oxygen anesthesia, followed by recovery and free roaming in its cage during the uptake period. At 50 min post injection, a 10-min CT scan was conducted first for localization and attenuation correction after segmentation for reconstructing the PET images, followed by a 10-min static PET imaging acquisition.
For biodistribution studies, the mice were injected with the radiotracer (~2-4 MBq) as described above. At 1 h post injection, the mice were euthanized by CO 2 inhalation. Blood was withdrawn by cardiac puncture, and organs/tissues of interest were collected, weighed, and counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.

Statistical Analysis
Data were analyzed with the GraphPad Prism, version 7.02, and Microsoft (Redmond, WA, USA) Excel software. One way ANOVA and multiple t-tests were performed for all organs in the biodistribution studies of [ 68 Ga]Ga-AV01017, [ 68 Ga]Ga-AV01030, [ 68 Ga]Ga-AV01038, [ 68 Ga]Ga-HTK03041, and [ 68 Ga]Ga-FAPI-04 in LNCaP and HEK293T:hFAP tumor models. A statistically significant difference was considered present when the adjusted p value was less than 0.05 using the Holm-Sidak method.

Conclusions
Three novel 68 Ga-labeled PSMA/FAP bispecific tracers were designed, synthesized, and confirmed to have the ability to bind both PSMA and FAP in vitro and in vivo. Compared with the monospecific tracers, the bispecific tracers have decreased binding affinities towards PSMA, but retain comparable binding affinities towards FAP. The bispecific tracers have lower tumor uptake values compared to the monospecific tracers, in which the tracer with a longer linker tends to have a lower tumor uptake. This might be caused by the longer blood retention of the bispecific tracers. Further modifications will be explored to improve the binding affinity, pharmacokinetics, and tumor uptake to generate promising PSMA/FAP bispecific radioligands for imaging and radioligand therapy of mCRPC.