Medicine, Faculty of
DSpace
Molecules 28 (3): 1088 (2023)
BC Cancer Research Centre. Molecular Oncology
BC Cancer Agency
Verena, Arsyangela
Zhang, Zhengxing
Kuo, Hsiou-Ting
Merkens, Helen
Zeisler, Jutta
Wilson, Ryan
Bendre, Shreya
Wong, Antonio A. W. L.
Bénard, François
Lin, Kuo-Shyan
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, ⁶⁸Ga-AV01017, ⁶⁸Ga-AV01030, and ⁶⁸Ga-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.
Citation: Verena, A.; Zhang, Z.; Kuo,H.-T.; Merkens, H.; Zeisler, J.; Wilson,R.; Bendre, S.; Wong, A.A.W.L.;Bénard, F.; Lin, K.-S. Synthesis andPreclinical Evaluation of Three Novel68Ga-Labeled BispecificPSMA/FAP-Targeting Tracers forProstate Cancer Imaging. Molecules2023, 28, 1088. https://doi.org/10.3390/molecules28031088Academic Editor: João D. G. CorreiaReceived: 24 December 2022Revised: 15 January 2023Accepted: 19 January 2023Published: 21 January 2023Copyright: © 2023 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).moleculesArticleSynthesis and Preclinical Evaluation of Three Novel68Ga-Labeled Bispecific PSMA/FAP-Targeting Tracers forProstate Cancer ImagingArsyangela Verena 1 , Zhengxing Zhang 1, Hsiou-Ting Kuo 1, Helen Merkens 1, Jutta Zeisler 1, Ryan Wilson 1,Shreya Bendre 1, Antonio A. W. L. Wong 1 , François Bénard 1,2,3,* and Kuo-Shyan Lin 1,2,3,*1 Department of Molecular Oncology, BC Cancer Research Institute, Vancouver, BC V5Z1L3, Canada2 Department of Radiology, University of British Columbia, Vancouver, BC V5Z1M9, Canada3 Department of Functional Imaging, BC Cancer, Vancouver, BC V5Z4E6, Canada* Correspondence: fbenard@bccrc.ca (F.B.); klin@bccrc.ca (K.-S.L.); Tel.: +1-604-675-8208 (K.-S.L.)Abstract: Tumor heterogeneity limits the efficacy and reliability of monospecific radiopharmaceuticalsin prostate cancer diagnosis and therapy. To overcome this limitation and improve lesion detectionsensitivity, 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 keyproteins overexpressed in prostate cancer. Three FAP-targeting ligands with various linker lengthswere synthesized through multistep organic synthesis, and then connected to the PSMA-targetingmotif. 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 inPSMA-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 thanthe monospecific PSMA-targeting tracer [68Ga]Ga-HTK03041 (23.1 ± 6.11 %ID/g). The uptake valuesin FAP-expressing HEK293T:hFAP tumor xenografts were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83 %ID/gfor [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were alsolower than the monospecific FAP-targeting tracer, [68Ga]Ga-FAPI-04 (12.5 ± 2.00 %ID/g). Weobserved that the bispecific tracers had prolonged blood retention, in which tracers with a longerlinker tend to have a higher blood uptake and lower tumor uptake. Further investigations are neededto optimize the linker selection to generate promising bispecific PSMA/FAP-targeting tracers forprostate cancer imaging.Keywords: prostate-specific membrane antigen; fibroblast activation protein; bispecific radiotracers;PET/CT; Gallium-681. IntroductionProstate-specific membrane antigen (PSMA), a type II transmembrane glycoproteinalso known as glutamate carboxypeptidase II, has enzymatic functions to cleave terminalglutamate [1,2]. PSMA has been shown to be overexpressed in prostate tumor and theneovasculature of other type of cancer, such as colon and renal cancers [3], making it anattractive imaging and therapeutic target. Various PSMA-targeting radioligands havebeen developed and three of them have been approved by the US FDA: [18F]DCFPyL and[68Ga]Ga-PSMA-11 for prostate cancer imaging [4,5] and [177Lu]Lu-PSMA-617 for prostatecancer radioligand therapy [6].Although [177Lu]Lu-PSMA-617 has been proven to be effective in treating metastaticcastration-resistant prostate cancer (mCRPC) patients [7], some patients have lesions withheterogeneous expression, or even no expression of PSMA [8,9], limiting their eligibility inbenefiting from PSMA-directed therapy. Moreover, studies have shown that lesions thatare PSMA-negative, such as neuroendocrine prostate cancer (NEPC), are prone to beingMolecules 2023, 28, 1088. https://doi.org/10.3390/molecules28031088 https://www.mdpi.com/journal/moleculesMolecules 2023, 28, 1088 2 of 17more aggressive and metabolically active, shown by high [18F]FDG uptake [10]. Due to theintrapatient heterogeneous PSMA expression in prostate cancer, several PSMA-negativelesions could be missed by PSMA-targeting radioligands which could lead to worse overallsurvival [11,12]. Therefore, new strategies should be considered to improve lesion detectionfor this patient cohort.As a member of type II transmembrane serine proteases, fibroblast activation protein(FAP) acts on various hormones and extracellular matrix components [13,14]. FAP is foundto be highly expressed in activated fibroblasts, such as during wound healing [15], andis essentially absent or lowly expressed in normal adult cells [16]. Many studies havedemonstrated that FAP is expressed by the cancer-associated fibroblasts (CAFs) that occupythe tumor microenvironment (TME) [17,18], and is overexpressed in 90% of epithelialtumors, including colon and breast cancers [19,20]. FAP expression is associated withworse prognosis as FAP supports tumor migration [21], angiogenesis [22], metastasisvia matrix remodeling in TME [23], and immunomodulatory function [24]. A study byKesch et al. [25] showed that there is a significant rise in FAP expression throughout theprogression of prostate cancer, in which men with advanced CRPC have the highest numberof FAP-positive lesions. Seeing the potential of FAP as a diagnostic and therapeutic target,there have been many studies evaluating new FAP targeting for imaging and radioligandtherapy. For instance, [68Ga]Ga-FAPI-04, a quinoline-based FAP-targeting tracer [26], hasbeen shown to have faster pharmacokinetics, higher tumor uptake, and superior tumor-to-background contrast in patients with various cancers when compared with [18F]FDG [27].Although several novel FAP-targeting radiotherapeutic agents have been developed, suchas [90Y]Y-FAPI-04 [26], [177Lu]Lu-DOTA.SA.FAPi [28], and [177Lu]Lu-FAPI-46 [29], theirtumor-retention time and efficacy in clinical investigation have been disappointing.Previous studies have shown the potential of using bispecific radioligands targetingPSMA and other overexpressed proteins, such as gastrin-releasing peptide receptor (GRPR),to improve tumor targeting and increase the detection sensitivity of prostate cancer imag-ing [30,31]. Here, our goal is to develop PSMA/FAP bispecific radioligands with compara-ble or even higher tumor uptake compared to the PSMA- and FAP-targeting monospecifictracers. Previously, Boinapally et al. [32] reported two 64Cu-labelled PSMA/FAP bispecifictracers, [64Cu]Cu-FP-L1 and [64Cu]Cu-FP-L2 (Figure 1A), which showed high and specificuptake in both FAP- and PSMA-expressing tumor models. However, no head-to-headcomparison of their bispecific tracers with the FAP- or PSMA-targeting monospecific tracerwas reported. Hu et al. [33] reported the development of two 18F-labeled PSMA/FAPbispecific tracers, [18F]AlF-PSMA-FAPI-01 and [18F]AlF-PSMA-FAPI-02 (Figure 1B), andboth showed higher uptake in PSMA- and FAP-expressing tumor models when comparedwith the PSMA- and FAP-targeting monospecific tracers, respectively. Although promis-ing results were obtained from these two reports, their use of NOTA as the radioisotopechelator excludes the labeling of these reported PSMA/FAP bispecific ligands with thecommonly used radiotherapeutic metals such as 177Lu and 90Y.To expand the potential usage of bispecific PSMA/FAP-targeting ligands for radiother-apeutic applications, we chose DOTA as our chelator, which has theranostic capabilitiesto label both diagnostic isotopes, such as 68Ga, and therapeutic isotopes, such as 177Lu.This will enable us to use the same ligands for diagnostic applications and ensure thepharmacokinetics of diagnostic and radiotherapeutic agents are comparable as they arederived from the same PSMA/FAP-targeting ligands.Molecules 2023, 28, 1088 3 of 17Molecules 2023, 28, x FOR PEER REVIEW 3 of 17 Figure 1. Chemical structures of (A) Cu-FP-L1 and Cu-FP-L2; and (B) AlF-PSMA-FAPI-01 and AlF-PSMA-FAPI-02. The PSMA- and FAP-targeting pharmacophores are shown in brown and blue, re-spectively. To expand the potential usage of bispecific PSMA/FAP-targeting ligands for radio-therapeutic applications, we chose DOTA as our chelator, which has theranostic capabili-ties to label both diagnostic isotopes, such as 68Ga, and therapeutic isotopes, such as 177Lu. This will enable us to use the same ligands for diagnostic applications and ensure the pharmacokinetics of diagnostic and radiotherapeutic agents are comparable as they are derived from the same PSMA/FAP-targeting ligands. Here we report the design, synthesis, and evaluation of three 68Ga-labeled DOTA-conjugated bispecific PSMA/FAP tracers (Figure 2). The PSMA binding motif of AV01017, AV01030, and AV01038 was based on our previously reported [68Ga]Ga-HTK03041 [34], and their FAP-targeting motif was derived from [68Ga]Ga-FAPI-04. The difference be-tween these three tracers is the length of their linker between the quinoline and the triazole with ring null, -O-CH2-, and -O-(CH2)3- for 68Ga-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 Figure 1. Chemical structures of (A) Cu-FP-L1 and Cu-FP-L2; and (B) AlF-PSMA-F PI-01 andAlF-PSMA-FAPI-02. The PSMA- P-targeting pharmacophores are shown in brown and blue,respecti l .Here we report the design, synthesis, and evaluation of three 68Ga-labeled DOTA-conjugated bispecific PSMA/FAP tracers (Figure 2). The PSMA binding motif of AV01017,AV01030, and AV01038 was based on our previously reported [68Ga]Ga-HTK03041 [34],and their FAP-targeting motif was derived from [68Ga]Ga-FAPI-04. The difference betweenthese three tracers is the length of their linker between the quinoline and the triazole withring null, -O-CH2-, and -O-(CH2)3- for 68Ga-labeled AV01030, AV01038, and AV01017,respectively. Their potential of the tracers for prostate cancer imaging was evaluated byan in vitro competiti n binding assay, PET imaging, and ex vivo biodistribution studies inpreclinic l PSMA- xpressing LNCa and FAP-expressing HEK293T:hFAP tumor modelsMolecules 2023, 28, 1088 4 of 17in mice. The results were then compared with those of the corresponding monospecifictracers, [68Ga]Ga-HTK03041 and [68Ga]Ga-FAPI-04.Molecules 2023, 28, x FOR PEER REVIEW 4 of 17 in preclinical PSMA-expressing LNCaP and FAP-expressing HEK293T:hFAP tumor mod-els in mice. The results were then compared with those of the corresponding monospecific tracers, [68Ga]Ga-HTK03 [68 a]Ga-FAPI-04. Figure 2. Chemical structures of (A) PSMA-targeting [68Ga]Ga-HTK03041 and FAP-targeting [68Ga]Ga-HTK03041; and (B) bispecific PSMA/FAP-targeting 68Ga-labeled AV01017, AV01030, and AV01038. The PSMA- and FAP-targeting pharmacophores are shown in brown and blue, respec-tively. 2. Results 2.1. Synthesis of PSMA/FAP Bispecific Ligands AV01017, AV01030, and AV01038 were synthesized on solid phase. Detailed syn-thetic procedures and characterizations are provided in the Supplementary Materials (Supplementary Materials, Table S1). Briefly, Lys(azidoacetic acid-Lys(ivDde)-Gly-tranexamic acid-Ala(9-Anth))-urea-Glu(OtBu)-OtBu was first constructed on solid phase, followed by the click addition of the alkyne-containing FAP-targeting motif: compound 5 Figure 2. Chemical structures of (A) PSMA-targeting [68Ga]Ga-HTK03041 and FAP-targeting[68Ga]Ga-HTK03041; and (B) bispecific PSMA/FAP-targeting 68Ga-labeled AV01017, AV01030, andAV01038. The PS A- and FAP-targeting pharmacophores are shown in brown and blue, respectively.2. Results2.1. Synthesis of PSMA/FAP Bispecific LigandsAV01017, AV01030, and AV01038 were synthesized on solid phase. Detailed syntheticprocedures and characterizations are provided in the Supplementary Materials(Supplementary Materials, Table S1). Briefly, Lys(azidoacetic acid-Lys(ivDde)-Gly-tranexamicMolecules 2023, 28, 1088 5 of 17acid-Ala(9-Anth))-urea-Glu(OtBu)-OtBu was first constructed on solid phase, followed bythe click addition of the alkyne-containing FAP-targeting motif: compound 5 for AV01017,Compound 9 for AV01030, and compound 13 for AV01038. The amino group at the Lys sidechain was then deprotected and coupled with the DOTA chelator. The DOTA-conjugatedligands 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 interme-diates are provided in the Supplementary Materials. For the preparation of compound 5(Scheme 1), quininic acid was demethylated in 48% HBr solution, followed by esterifica-tion in methanol in the presence of thionyl chloride to obtain 1 in 47% yield. Mitsunobucoupling between compound 1 and 4-pentyn-1-ol resulted in 2 in 100% yield. Compound3 was obtained in 74% yield by the hydrolysis of 2 with NaOH in a mixture of water andmethanol. Esterification of compound 3 with tetrafluorphenol (TFP) led to compound 4 in72% 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 co pounds 5, 9, and 13 are depicted in Sche es 1–3, respectively, and detailed synthetic procedures and characterizations for the final products and inter-mediates are provided in the Supplementary Materials. For the preparation of compound 5 (Scheme 1), qui inic acid was demethylated in 48% HBr solution, followed by esterifi-catio in methanol in the pr sence of thionyl chloride to obtain 1 in 47% yield. its li t - t - - l r s lte i i i l . t i i i l l i it i i f t t l. fi ti f i fl r l ( ) l t i i l . i ( )- -( - i cet l)-4,4- ifl oropyrrolidine-2-carbonitrile. Scheme 1. Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(pent-4-yn-1-yloxy)quinoline-4-carboxamide (5). The synthesis of compound 9 is depicted in Scheme 2. The Sonogashira reaction was performed for coupling methyl 6-bromoquinoline-4-carboxylate and ethynyltrime-thylsilane 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 tetrafluorophe-nol 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-difluoropyrrol-idine-2-carbonitrile. 1. Synt esis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1- l)- -yloxy)q inoline-4-carboxa i e (5).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 inter-mediates are provided in the Supplementary Materials. For the preparation of compound 5 (Scheme 1), quininic acid was demethylated in 48% HBr solution, followed by esterifi-cation in methanol in the presence of thionyl chloride to obtain 1 in 47% yield. Mitsuno u coupling between compound 1 a d 4-pentyn-1-ol r sulted 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) l d to compound 4 in 72% yield. Compound 5 was obtain d in 97% yield by coupling the activated ester 4 with (S)-1-(2-aminoacetyl)-4,4-difl oropyrrolidine-2-carbonitrile. Scheme 1. Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(pent-4-yn-1-yloxy)quinoline-4-carboxamide (5). The synthesis of compound 9 is depicted in Scheme 2. The Sonogashira reaction was performed for coupling methyl 6-bromoquinoline-4-carboxylate and ethynyltrime-thylsilane 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 tetrafluorophe-nol 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-difluoropyrrol-idine-2-carbonitrile. Scheme 2. Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-ethynylquinoline-4-carboxamide (9).Molecules 2023, 28, 1088 6 of 17Molecules 2023, 28, x FOR PEER REVIEW 6 of 17 Scheme 2. Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-ethynylquino-line-4-carboxamide (9). 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 re-sulted in compound 12 in 68% yield. Compound 13 was obtained in 100% yield by cou-pling the activated ester 12 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboni-trile. Scheme 3. Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(prop-2-yn-1-yloxy)quinoline-4-carboxamide (13). Detailed syntheses and characterizations of nonradioactive Ga- and 68Ga-complexed AV01017, AV01030, and AV01038 are provided in the Supplementary Materials (Supple-mentary Materials, Tables S2 and S3). Nonradioactive Ga-complexed AV01017, AV01030, and AV01038 were obtained in 25–89% yields, and their 68Ga-labeled analogs were ob-tained in 43–60% decay-corrected radiochemical yields with > 74 GBq/µmol molar activity and > 99% radiochemical purity. 2.2. Binding Affinity The binding affinities of Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-HTK03041, and Ga-FAPI-04 to PSMA were measured by a cell-based binding assay using PSMA-express-ing LNCaP prostate cancer cells. The nonradioactive Ga-complexed standards, except Ga-FAPI-04, inhibited the binding of [18F]DCFPyL to LNCaP cells in a dose-dependent man-ner (Figure 3A). The calculated IC50(PSMA) values for Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-HTK03041, and Ga-FAPI-04 were 25.2 ± 10.7, 71.6 ± 23.0, 29.4 ± 25.2, 0.76 ± 0.12 and >1000 nM, respectively (n = 3). The binding affinities of Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-HTK03041, and Ga-FAPI-04 to human FAP were measured by an enzyme-inhibition assay using Suc-Gly-Pro-AMC as the FAP substrate. The human FAP enzymatic activity on the substrate was inhibited by Ga-complexed standards in a dose-dependent manner (Figure 3B). The cal-culated IC50(FAP) values for Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-HTK03041, and Ga-FAPI-04 were 1.25 ± 0.39, 2.74 ± 0.33, 2.31 ± 0.13, 2010 ± 585, and 1.03 ± 0.4 nM, respec-tively (n = 3). Sche e 3. Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(prop-2-yn-1-yloxy)quinoline-4-carboxamide (13).The synthesis of compound 9 is depicted in Scheme 2. The Sonogashira reaction wasperformed for coupling methyl 6-bromoquinoline-4-carboxylate and ethynyltrimethylsi-lane o obtain compound 6 in 62% yield. Silane deprotecti n and ester hydrolysis wereperformed simultaneously with LiOH in a mixture of water and m thanol to afford compoun 7 in 83% yield. Compoun 7 was subsequent y act vated with tetrafluorophenol toafford 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-carbonitr le.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. Com-pound 10 was hydrolyzed with NaOH in a mixture of water and methanol to obtaincompound 11 in 73% yield. Activation of compound 11 with tetrafluorophenol resultedin compound 12 in 68% yield. Compound 13 was obtained in 100% yield by coupling theactivated ester 12 with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile.Detailed syntheses and characterizations of nonradioactive Ga- and 68Ga-complexedAV01017, AV01030, and AV01038 are provided in the Supplementary Materials (Supplemen-tary Materials, Tables S2 and S3). Nonradioactive Ga-complexed AV01017, AV01030, andAV01038 were obtained in 25–89% yields, and their 68Ga-labeled analogs were obtained in43–60% decay-corrected radiochemical yields with >74 GBq/µmol molar activity and >99%radiochemical purity.2.2. Binding AffinityThe binding affinities of Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-HTK03041, andGa-FAPI-04 to PSMA were measured by a cell-based binding assay using PSMA-expressingLNCaP prostate cancer cells. The nonradioactive Ga-complexed standards, except Ga-FAPI-04, inhibited the binding of [18F]DCFPyL to LNCaP cells in a dose-dependent manner(Figure 3A). The calculated IC50(PSMA) values for Ga-AV01017, Ga-AV01030, Ga-AV01038,Ga-HTK03041, and Ga-FAPI-04 were 25.2 ± 10.7, 71.6 ± 23.0, 29.4 ± 25.2, 0.76 ± 0.12 and>1000 nM, respectively (n = 3).Molecules 2023, 28, 1088 7 of 17Molecules 2023, 28, x FOR PEER REVIEW 7 of 17 Figure 3. (A) Displacement curves of [18F]DCFPyL by Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-FAPI-04, and Ga-HTK03041 generated using PSMA-expressing LNCaP cells; (B) Fluorescence curve of FAP enzymatic activity on Suc-Gly-Pro-AMC substrate with inhibition by Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-FAPI-04, and Ga-HTK03041. 2.3. PET Imaging and Biodistribution Studies Representative PET images acquired at 1 h post injection using [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 are provided in Figure 4. All the radiotracers were excreted primarily through the renal path-way. All the bispecific tracers ([68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038) had significantly higher background and heart uptake compared to the mono-specific tracers ([68Ga]Ga-HTK03041 and [68Ga]Ga-FAPI-04). LNCaP tumor xenografts were clearly visualized by [68Ga]Ga-HTK03041 with an excellent contrast, barely visual-ized by the bispecific tracers ([68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038), and not visualized by [68Ga]Ga-FAPI-04 (Figure 4A). For HEK293T:hFAP tumor xeno-grafts, they were clearly visualized by [68Ga]Ga-FAPI-4 with an excellent contrast and by [68Ga]Ga-AV01030 with a good contrast, barely visualized by [68Ga]Ga-AV01017 and [68Ga]Ga-AV01038, and not visualized by [68Ga]Ga-HTK03041 (Figure 4B). All tracers, ex-cept [68Ga]Ga-HTK03041 have bone and joint uptake, which is commonly observed for FAP-targeting tracers. High thyroid uptake was also observed in [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038 PET images. There was a high kidney uptake in mice injected with the bispecific tracers and [68Ga]Ga-HTK03041, but not in mice in-jected with [68Ga]Ga-FAPI-04. Figure 3. (A) Displacement curves of [18F]DCFPyL by Ga-AV01017, Ga-AV01030, Ga-AV01038,Ga-FAPI-04, and Ga-HTK03041 generated using PSMA-expressing LNCaP cells; (B) Fluorescencecurve of FAP enzymatic activity on Suc-Gly-Pro-AMC substrate with inhibition by Ga-AV01017,Ga-AV01030, Ga-AV01038, Ga-FAPI-04, and Ga-HTK03041.The binding affinities of Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-HTK03041, andGa-FAPI-04 to human FAP were measured by an enzyme-inhibition assay using Suc-Gly-Pro-AMC as the FAP substrate. The human FAP enzymatic activity on the substratewas inhibited by Ga-complexed standards in a dose-dependent manner (Figure 3B). Thecalculated IC50(FAP) values for Ga-AV01017, Ga-AV01030, Ga-AV01038, Ga-HTK03041,and Ga-FAPI-04 were 1.25 ± 0.39, 2.74 ± 0.33, 2.31 ± 0.13, 2010 ± 585, and 1.03 ± 0.4 nM,respectively (n = 3).2.3. PET Imaging nd Biodistribution StudiesRepresentative PET images acquired at 1 h post injection using [68Ga]Ga-AV01017,[68Ga]Ga-AV01030, [68Ga] a-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 are pro-vided in Figure 4. All the radiotracers were excreted primarily through the renal pathwa .All the bispecific tracers ([68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038)had significantly higher background and heart upt ke compared to the monospecifi t ac rs([68Ga] -HTK03041 and [68Ga]Ga-FAPI-04). LNCaP tumor xenografts were clearly visual-ized by [68Ga]Ga-HTK03041 with an excellent contr st, barely isualized by the bispecifictracers ([68Ga]Ga-AV01017, [68 a]Ga-AV01030, [68Ga]Ga-AV01038), nd not visualized by[68Ga]Ga-FAPI-04 (Figure 4A). For HEK293T:hFAP tumor xenografts, they were cl arlyvisualized by [68Ga]Ga-FAPI-4 with an excellent contrast and by [68Ga]Ga-AV01030 witha good contrast, barely visualized by [68Ga]Ga-AV01017 and [68Ga]Ga-AV01038, and notvisualized by [68Ga]Ga-HTK03041 (Figure 4B). All tracers, except [68Ga]Ga-HTK03041 havebone and joint uptake, which is commonly observed for FAP-targeting tracers. High thyroiduptake was also observed in [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038Molecules 2023, 28, 1088 8 of 17PET images. There was a high kidney uptake in mice injected with the bispecific tracersand [68Ga]Ga-HTK03041, but not in mice injected with [68Ga]Ga-FAPI-04.Molecules 2023, 28, x FOR PEER REVIEW 8 of 17 Figure 4. Representative maximum intensity projection PET images of [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 acquired at 1 h post-injec-tion in mice bearing (A) LNCaP tumor xenografts and (B) HEK293T:hFAP tumor xenografts. t: tu-mor. Biodistribution studies were conducted at 1 h post injection with 68Ga-labeled AV01017, AV01030, AV01038, HTK03041, and FAPI-04 in LNCaP tumor-bearing mice (Figure 5 and Table S4, Supplementary Materials). The results were consistent with the observation from their PET images. Tumor uptake values for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 were 4.38 ± 0.55, 5.17 ± 0.51, 4.25 ± 0.86, 23.1 ± 6.11, and 3.15 ± 1.43 %ID/g, respectively. All the bispecific tracers have statistically significantly higher heart and blood uptake than the monospe-cific [68Ga]Ga-HTK03041 and [68Ga]Ga-FAPI-04 (blood uptake: 5.75–9.24 vs. 1.43–2.16 %ID/g, p < 0.05; heart uptake: 2.41–4.04 vs. 0.70–1.82 %ID/g, p < 0.05). Although not statis-tically significant, the bispecific tracers with a longer linker tend to have a higher blood uptake value (9.24 ± 1.55 %ID/g for [68Ga]Ga-AV01017; 7.07 ± 0.31%ID/g for [68Ga]Ga-AV01038; 5.75 ± 0.59 %ID/g for [68Ga]Ga-AV01030) and a lower tumor-to-blood ratio (0.48 ± 0.11 for [68Ga]Ga-AV01017; 0.60 ± 0.13 for [68Ga]Ga-AV01038; 0.89 ± 0.03 for [68Ga]Ga-AV01030). The bispecific tracers also had significantly higher uptake values in muscle and thyroid when compared to those of monospecific radiotracers (p < 0.05). Figure 4. Representative maximum intensity projection PET images of [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 acquired at 1 h post-injection in mice bearing (A) LNCaP tumor xenografts and (B) HEK293T:hFAP tumor xenografts.t: tumor.Biodistribution studies were conducted at 1 h post injection with 68Ga-labeled AV01017,AV01030, AV01038, HTK03041, and FAPI-04 in LNCaP tumor-bearing mice (Figure 5and Table S4, Supplementary Materials). The results were consistent with the obser-vation from their PET images. Tumor uptake values for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga] a- 01038, [68 a]Ga-HTK03041, and [68Ga]Ga-FAPI-04 were 4.38 ± 0.55,5.17 ± 0.51, 4.25 ± 0.86, 23.1 6.11, . . ID/ , respectively. All the bispecifitracers have i lly significantly higher heart and blood uptake than e monospecific[68Ga] -HTK03041 and [68Ga]Ga-FAPI 04 (blood uptake: 5.75–9.24 vs. 1.43–2.16 %ID/g,p < 0.05; heart upt ke: 2.41–4.04 vs. 0.70–1.82 %ID/g, p < 0.05). Although not statisticallysignificant, the bispecific tracers with a longer linker tend to have a higher blood uptakevalue (9.24 ± 1.55 %ID/g for [68Ga]Ga-AV01017; 7.07 ± 0.31%ID/g for [68Ga]Ga-AV01038;5.75 ± 0.59 %ID/g for [68Ga]Ga-AV01030) and a lower tumor-to-blood ratio (0.48 ± 0.11 for[68Ga]Ga-AV01017; 0.60 ± 0.13 for [68Ga]Ga-AV01038; 0.89 ± 0.03 for [68Ga]Ga-AV01030).The bispecific tracers also had significantly higher uptake values in muscle and thyroidwhen compared to those of monospecific radiotracers (p < 0.05).Biodistribution studies were also conducted at 1 h post injection with [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 in HEK293T:hFAP tumor-bearing mice (Figure 6 and Table S5, Supplementary Materi-als). Tumor uptake values for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038,Molecules 2023, 28, 1088 9 of 17[68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83,0.62 ± 0.19, and 12.5 ± 2.00 %ID/g, respectively. There was a very low tumor uptakein mice injected with [68Ga]Ga-HTK03041, showing very minimal FAP expression inthis tumor model. The uptake levels of these tracers on the major organs and tissuesare consistent with the trends observed in the LNCaP tumor-baring mice (Figure 5 andSupplemental Table S4).Molecules 2023, 28, x FOR PEER REVIEW 9 of 17 Figure 5. Biodistribution of [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 in LNCaP tumor-bearing mice. Biodistribution studies were also conducted at 1 h post injection with [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 in HEK293T:hFAP tumor-bearing mice (Figure 6 and Table S5, Supplementary Materials). Tumor uptake values for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83, 0.62 ± 0.19, and 12.5 ± 2.00 %ID/g, respectively. There was a very low tumor uptake in mice injected with [68Ga]Ga-HTK03041, showing very minimal FAP expression in this tumor model. The uptake levels of these tracers on the major organs and tissues are con-sistent with the trends observed in the LNCaP tumor-baring mice (Figure 5 and Supple-mental Table S4). Figure 5. Biodistribution of [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-TK03041, and [68 ]Ga-FAPI-04 in LNCaP tumor-bearing mice.Molecules 2023, 28, x FOR PEER REVIEW 10 of 17 Figure 6. Biodistribution of [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 in HEK293T:hFAP tumor-bearing mice. 3. Discussion Both PSMA and FAP are promising biomarkers of prostate cancer and many radi-oligands 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 radiothera-peutic 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 treat-ment options. Since FAP and PSMA are both expressed in prostate cancer and other can-cers such as pancreatic cancer [25,35–38], 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 68Ga-labeled PSMA/FAP bispecific tracers with comparable or higher tumor uptake when compared to the monospecific tracers, [68Ga]Ga-FAPI-04 and [68Ga]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 64Cu [32] and the other for Al18F [33]. To the best of our knowledge, there has been no report on the development of 68Ga-labeled PSMA/FAP bispecific tracers despite the growing popularity and increased accessibility of clinical 68Ga generators. Although NOTA can be used for labeling 68Ga as well, it cannot be used for labeling the common and effective radiotherapeutic nuclides such as 90Y and 177Lu. Therefore, for the current research, we chose DOTA as the chelator for 68Ga labeling as DOTA is a widely used chelator and is effective for labeling with many diagnostic (such as 68Ga, 152Tb, and 111In) and radiotherapeutic radionuclides (such as 149Tb, 90Y, and 177Lu). We selected the pharmacophores of [68Ga]Ga-FAPI-04 (in blue, Figure 2) and [68Ga]Ga-HTK03041 (in brown, Figure 2) for the design of our PSMA/FAP bispecific igure 6. Biodistribution of [68 ]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 in HEK293T:hFAP tumor-bearing mice.Molecules 2023, 28, 1088 10 of 173. DiscussionBoth PSMA and FAP are promising biomarkers of prostate cancer and many radioli-gands have been developed to target these two membrane proteins for imaging and therapy.However, the detection sensitivities of these radiotracers are strongly dependent on theexpression levels and heterogeneities of these two biomarkers in different disease stagesand between individuals [8,9]. Despite many effective PSMA-targeting radiotherapeuticagents being developed, patients with low to no PSMA expression are not eligible forthese emerging PSMA-targeted radioligand therapies and still have very limited treatmentoptions. Since FAP and PSMA are both expressed in prostate cancer and other cancers suchas pancreatic cancer [25,35–38], the use of PSMA/FAP bispecific radioligands is expectedto increase lesion detection sensitivity and treatment efficacy. The ultimate goal of thisreported research is to develop 68Ga-labeled PSMA/FAP bispecific tracers with comparableor higher tumor uptake when compared to the monospecific tracers, [68Ga]Ga-FAPI-04 and[68Ga]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 bispecifictracers for imaging (Figure 1) [32,33]. Both reports used the NOTA chelator for radioisotopecomplexation: one for 64Cu [32] and the other for Al18F [33]. To the best of our knowledge,there has been no report on the development of 68Ga-labeled PSMA/FAP bispecific tracersdespite the growing popularity and increased accessibility of clinical 68Ga generators.Although NOTA can be used for labeling 68Ga as well, it cannot be used for labeling thecommon and effective radiotherapeutic nuclides such as 90Y and 177Lu. Therefore, for thecurrent research, we chose DOTA as the chelator for 68Ga labeling as DOTA is a widelyused chelator and is effective for labeling with many diagnostic (such as 68Ga, 152Tb, and111In) and radiotherapeutic radionuclides (such as 149Tb, 90Y, and 177Lu).We selected the pharmacophores of [68Ga]Ga-FAPI-04 (in blue, Figure 2) and [68Ga]Ga-HTK03041 (in brown, Figure 2) for the design of our PSMA/FAP bispecific tracers as theyhave high affinity for FAP and PSMA, respectively [26,34]. These two pharmacophores areseparated by an azidoacetic-Lys-Gly linker (Figure 2) to minimize the interaction of bothpharmacophores as such interaction might interfere with the bindings of both pharma-cophores to their respective targets. The PSMA-targeting pharmacophore (Lys(tranexamicacid-Ala(9-Anth))-urea-Glu and the linker (azidoacetic acid-Lys-Gly) was constructed di-rectly on solid phase using the commercially available amino acids. While the DOTAchelator was coupled to the amino group on the Lys side chain, the alkyne-containingFAP-targeting motif was coupled to the azido group via the formation of a triazole ring byclick chemistry. For AV01030, the triazole ring is directly linked to the quinoline ring of theFAP-targeting pharmacophore. For AV01038 and AV01017, there are additional -O-CH2-and -O-(CH2)3-, respectively, to separate the triazole ring and the quinoline ring of theFAP-targeting pharmacophore. This allowed us to investigate the effect of the additionallinker and its length on the binding affinity and pharmacokinetics of the resulting bispecifictracers.The enzymatic assay (Figure 3B) confirmed that the FAP binding affinities of our bispe-cific ligands (IC50 = 1.25–2.74 nM) were comparable to that of Ga-FAPI-04 (IC50 = 1.03 nM).To investigate if the PSMA-targeting pharmacophore has any effect on the overall FAP bind-ing of our bispecific ligands, we also measured the FAP binding affinity of Ga-HTK03041.The very weak binding affinity of Ga-HTK03041 (IC50 = 2010 ± 585 nM) suggests that thepotent FAP binding affinity of our bispecific ligands is contributed mainly by the FAPI-04pharmacophore.Unlike the comparable FAP binding affinity of bispecific ligands and the monospecificGa-FAPI-4, the PSMA binding affinities of bispecific ligands (IC50 = 25.2–71.6 nM) wereinferior to that of Ga-HTK03041 (IC50 = 0.76 nM). Although Ga-FAPI-04 has minimalbinding affinity to PSMA (IC50 > 1000 nM), the addition of its pharmacophore and a linkerclearly interferes the overall binding of the bispecific ligands to PSMA. Such interferenceseems to decrease with the increased linker length between the triazole ring and the FAP-Molecules 2023, 28, 1088 11 of 17targeting pharmacophore as the IC50(PSMA) values for Ga-AV01030, Ga-AV01038, andGa-AV01017 are 71.6, 29.4, and 25.2 nM, respectively.PET imaging and biodistribution studies (Figures 4–6 and Supplemental Tables S4and S5) revealed that the bispecific tracers retained the characteristics of the monospecifictracers as high uptake was observed in common off-targets of PSMA-targeting (kidneysand salivary glands) and FAP-targeting tracers (joints and salivary glands). However, thebispecific tracers showed much lower uptake values in LNCaP and HEK293T:hFAP tumorxenografts when compared to those of the monospecific tracers, [68Ga]Ga-HTK03041 and[68Ga]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 retentionwas positively correlated to the length of the linker between the triazole ring and theFAP-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 longerblood retention would prevent fast binding of the bispecific tracers to their targets: PSMAin the LNCaP tumor xenografts and FAP in the HEK293T:hFAP tumor xenografts. This isevident by the observations that with no additional linker between the triazole ring and theFAP-targeting pharmacophore, [68Ga]Ga-AV01030 showed relatively higher tumor uptakeand tumor-to-background (bone, muscle and blood) contrast ratios in both tested tumormodels than those of [68Ga]Ga-AV01017 and [68Ga]Ga-AV01038.It should be noted that evaluation by imaging and/or biodistribution studies in tumor-bearing mice as in this report is a common practice in the development of radiopharmaceu-ticals. However, the obtained high tumor-to-background contrast either from imaging orbiodistribution data might not be observed in the clinic especially for 68Ga-labeled tracers.This is because compared with other positron emitters, 68Ga has a higher average positronenergy (68Ga, 0.829 MeV; 18F, 0.250 MeV; 64Cu, 0.288 MeV; 89Zr, 0.396 MeV), leading topoorer spatial resolution and difficulty in the visualization of small lesions (a few millime-ters in size). In addition, the tumor xenografts used in the imaging and biodistributionstudies are often derived from (genetically-modified) cancer cell lines overexpressing thetargeted cancer markers, which might not be representative of cancer lesions encounteredin the clinic.Directly comparing the performance of our bispecific tracers with those reported pre-viously is difficult as different tumor models were used for evaluation: PSMA-expressingLNCaP tumors and FAP-expressing HEK293T:hFAP tumors used in this report; PSMA-expressing 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 previouslyshows that our tracers had significantly higher blood retention at 1 h post injection. Thelonger blood retention of our tracers could be due to the increased lipophilicity [39], whichcould be a result of (1) the replacement of 2-Nal with a more lipophilic Ala(9-Anth) in thePSMA-targeting pharmacophore; (2) the deletion of the hydrophilic piperazine linker in theFAP-targeting pharmacophore of FAPI-04; and/or (3) the use of click reaction for couplingthe FAP-targeting pharmacophore as this forms a relatively lipophilic triazole ring insteadof a hydrophilic amide bond.It should be noted that the same diagnostic information obtained from the use of abispecific tracer could be obtained by two separate scans using its corresponding monospe-cific tracers. However, the use of a bispecific tracer would save the time and overall cost byreducing two procedures (hospital visit, tracer preparation, tracer injection, PET scan, scanreading, and report writing) to one, and reduce patients’ absorbed radiation dose fromPET scanning. It should also be noted that compared to its corresponding monospecifictracers, a bispecific tracer would have a larger molecular size, and combining two differenttargeting vectors into a single molecule could potentially negatively impact both its pharma-cokinetic properties and receptor binding affinity. However, with thorough investigationson the selection of targeting vectors, linkers, and potentially pharmacokinetic modifiers,it is likely to obtain an optimized bispecific tracer with higher tumor uptake and evenMolecules 2023, 28, 1088 12 of 17higher tumor-to-background contrast ratios than both of its corresponding monospecifictracers. For example, the Chen group designed and evaluated gastrin-releasing peptidereceptor (GRPR) and integrin αvβ3 bispecific radiotracers for imaging prostate cancer [40].GRPR is highly expressed by prostate cancer cells, whereas integrin αvβ3 is expressed bytumor neovasculature. Their results showed that the GRPR/integrin αvβ3 bispecific tracer([18F]FB-BBN-RGD) achieved higher tumor uptake and tumor-to-background contrastratios than both of its corresponding monospecific tracers ([18F]FB-BBN for GRPR and[18F]FB-RGD for integrin αvβ3).Further modifications are needed to improve the binding affinities, pharmacokinet-ics, and tumor uptake of 68Ga-labeled PSMA/FAP bispecific tracers by considering thelinker’s length, lipophilicity, and the use of less lipophilic PSMA- and/or FAP-targetingpharmacophores. This can be accomplished by the use of less lipophilic linkers such asPEG linkers and well as piperazine-based linkers which have been shown to be importantfor maintaining good tumor uptake of FAP-targeting tracers [41]. Similarly, the use ofpotent and less lipophilic pharmacophores should also be investigated such as (1) replacingAla(9-Anth) in the PSMA-targeting pharmacophore with 2-Nal, (2) replacing the quinolinemotif in the FAP-targeting pharmacophore with a more hydrophilic pyridine motif, and/or(3) replacing the 2-pyrrolidinecarbonitrile motif in the FAP-targeting pharmacophore witha more hydrophilic pyrrolidin-2-ylboronic acid.4. Materials and Methods4.1. Synthesis of Bispecific PSMA/FAP-Targeting LigandsDetailed information for the synthesis, purification, and characterizations of AV01017,AV01030, and AV01038, and their nonradioactive Ga-complexed standards and 68Ga-labeled analogs are provided in the supplemental information (Supplemental Tables S1–S3).4.2. Cell CultureThe LNCaP cells obtained from ATCC (via Cedarlane, Burlington, ON, Canada) werecultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), andstreptomycin (100 µg/mL) at 37 ◦C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIChumidified incubator containing 5% CO2. The cells were confirmed to be pathogen-freeby the IMPACT Rodent Pathogen Test (IDEXX BioAnalytics). Cells were grown until80–90% confluence and washed with sterile phosphate-buffered saline (PBS, pH 7.4) andcollected after 1 min trypsinization. The cell concentration was counted in triplicate usinga hemocytometer and a manual laboratory counter.4.3. Cell TransfectionThe HEK293T cells were obtained from ATCC. The FAP-expressing vector was con-structed using Genome-CRISPRTM Human AAVS1 Safe Harbor Gene Knock-in Kits(GeneCopoeaiaTM) by inserting FAP-expressing gene into the AAVS1 vector. The cellswere then transfected by the FAP-expressing vector following the EndofectinTM Trans-fection Reagent protocol. The cells underwent 3 serial dilutions and were sorted usingfluorescence-activated cell sorting (FACS) to obtain FAP-expressing monoclonal colonies.HEK293T:hFAP cells were cultured in DMEM GlutaMAX™ medium supplemented with10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 ◦C in a PanasonicHealthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO2. Cellswere grown until 80–90% confluence and washed with sterile PBS (pH 7.4) and collected.4.4. In Vitro PSMA Competition Binding AssayThe PSMA binding assays were conducted following previously published proceduresusing LNCaP cells and [18F]DCFPyL as the radioligand [34,42,43]. Data analyses of IC50were performed using the nonlinear regression algorithm of GraphPad Prism 7 (San Diego,CA, USA) software.Molecules 2023, 28, 1088 13 of 174.5. In Vitro FAP Fluorescence AssayThe half maximal inhibitory concentration (IC50) values of the tested compounds forFAP 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 at37 ◦C, 50 µL of Suc-Gly-Pro-AMC (2 µM, Bachem) was added to each well. The fluorescentsignals were acquired at 15, 30, 45, and 60 min using a FlexStation 3 Multi-Mode MicroplateReader with excitation at 380 nm and emission at 460 nm. The IC50(FAP) was calculatedusing a “nonlinear fit model” built-in model in GraphPad Prism 7.02 software4.6. Ex Vivo Biodistribution and PET/CT Imaging StudiesImaging and biodistribution studies were performed using male NOD.Cg-Rag1tm1MomIl2rgtm1Wjl/SzJ (NRG) mice following previously published procedures [44–46]. Theexperiments were conducted according to the guidelines established by the CanadianCouncil on Animal Care and approved by Animal Ethics Committee of the University ofBritish Columbia. The mice were briefly sedated by inhalation of 2.5% isoflurane in oxygen,and 100 µL LNCaP (2 × 105 cells) or HEK293T:hFAP (8.5 × 106 cells) cells were inoculatedsubcutaneously behind the left shoulder. When the tumor grew to 5–8 mm in diameterover 3–4 weeks and 4–5 weeks for HEK293T:hFAP and LNCaP tumors, respectively, themice 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 MBqof 68Ga-labeled tracer through a lateral caudal tail vein under 2.5% isoflurane in oxygenanesthesia, followed by recovery and free roaming in its cage during the uptake period. At50 min post injection, a 10-min CT scan was conducted first for localization and attenuationcorrection after segmentation for reconstructing the PET images, followed by a 10-minstatic 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 CO2 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 automaticgamma counter.4.7. Statistical AnalysisData 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 allorgans in the biodistribution studies of [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, [68Ga]Ga-AV01038, [68Ga]Ga-HTK03041, and [68Ga]Ga-FAPI-04 in LNCaP and HEK293T:hFAP tumormodels. A statistically significant difference was considered present when the adjusted pvalue was less than 0.05 using the Holm–Sidak method.5. ConclusionsThree novel 68Ga-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. Com-pared with the monospecific tracers, the bispecific tracers have decreased binding affinitiestowards PSMA, but retain comparable binding affinities towards FAP. The bispecific tracershave lower tumor uptake values compared to the monospecific tracers, in which the tracerwith a longer linker tends to have a lower tumor uptake. This might be caused by thelonger blood retention of the bispecific tracers. Further modifications will be explored toimprove the binding affinity, pharmacokinetics, and tumor uptake to generate promisingPSMA/FAP bispecific radioligands for imaging and radioligand therapy of mCRPC.Molecules 2023, 28, 1088 14 of 17Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28031088/s1. Detailed synthetic procedures and re-sults for the preparation of bispecific ligands and their natGa/68Ga-complexed analogs; Table S1:HPLC purification conditions and MS characterizations of DOTA-conjugated precursors; Table S2:HPLC purification conditions and MS characterizations of nonradioactive Ga-complexed standards;Table S3: HPLC conditions for the purification and quality control of 68Ga-labeled tracers; TableS4: Biodistribution and uptake ratios of 68Ga-labeled PSMA/FAP bispecific tracers, HTK03041and FAPI-04 in LNCaP tumor-bearing mice; Table S5: Biodistribution and uptake ratios of 68Ga-labeled PSMA/FAP bispecific tracers, HTK03041 and FAPI-04 in HEK293T:hFAP tumor-bearingmice [26,47–50].Author Contributions: Conceptualization, K.-S.L.; methodology, A.V., H.M., Z.Z., H.-T.K., S.B.,R.W., A.A.W.L.W. and J.Z.; validation, A.V., K.-S.L.; formal analysis, A.V., H.-T.K. and A.A.W.L.W.;investigation, A.V.; resources, H.M., F.B. and K.-S.L.; data curation, A.V. and K.-S.L.; writing—originaldraft preparation, A.V.; writing—review and editing, K.-S.L.; visualization, A.V.; supervision, K.-S.L.and F.B.; project administration, K.-S.L.; funding acquisition, K.-S.L. All authors have read and agreedto the published version of the manuscript.Funding: This research was supported by the Canadian Institutes of Health Research (PJT-162243,PJT-180299 and PJT-180300).Institutional Review Board Statement: The animal study protocol (A20-0113) was approved by theAnimal Ethics Committee of the University of British Columbia on 30 September 2020.Informed Consent Statement: Not applicable.Data Availability Statement: The data presented in this study are available in the SupplementaryMaterials.Acknowledgments: We thank Nadine Colpo for her help with the PET/CT imaging studies andJinhe Pan for his help in the production of [18F]DCFPyL for PSMA binding assays.Conflicts of Interest: François Bénard and Kuo-Shyan Lin are co-founders, shareholders, and consul-tants of Alpha-9 Theranostics Inc. The funder had no role in the design of the study; in the collection,analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish theresults.Sample Availability: Samples of the investigated compounds are available from A.V.References1. Pinto, J.T.; Suffoletto, B.P.; Berzin, T.M.; Qiao, C.H.; Lin, S.; Tong, W.P.; May, F.; Mukherjee, B.; Heston, W.D. Prostate-SpecificMembrane Antigen: A Novel Folate Hydrolase in Human Prostatic Carcinoma Cells. Clin. Cancer Res. 1996, 2, 1445–1451.[PubMed]2. Kaittanis, C.; Andreou, C.; Hieronymus, H.; Mao, N.; Foss, C.A.; Eiber, M.; Weirich, G.; Panchal, P.; Gopalan, A.; Zurita, J.; et al.Prostate-Specific Membrane Antigen Cleavage of Vitamin B9 Stimulates Oncogenic Signaling through Metabotropic GlutamateReceptors. J. Exp. Med. 2018, 215, 159–175. [CrossRef] [PubMed]3. Silver, D.A.; Pellicer, I.; Fair, W.R.; Heston, W.D.; Cordon-Cardo, C. Prostate-Specific Membrane Antigen Expression in Normaland Malignant Human Tissues. Clin. Cancer Res. 1997, 3, 81–85. [PubMed]4. Chen, Y.; Pullambhatla, M.; Foss, C.A.; Byun, Y.; Nimmagadda, S.; Senthamizhchelvan, S.; Sgouros, G.; Mease, R.C.; Pomper, M.G.2-(3-{1-Carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic Acid, [18F]DCFPyL, a PSMA-BasedPET Imaging Agent for Prostate Cancer. Clin. Cancer Res. 2011, 17, 7645–7653. [CrossRef]5. Eder, M.; Schäfer, M.; Bauder-Wüst, U.; Hull, W.-E.; Wängler, C.; Mier, W.; Haberkorn, U.; Eisenhut, M. 68Ga-ComplexLipophilicity and the Targeting Property of a Urea-Based PSMA Inhibitor for PET Imaging. Bioconjug. Chem. 2012, 23, 688–697.[CrossRef]6. Sartor, O.; de Bono, J.; Chi, K.N.; Fizazi, K.; Herrmann, K.; Rahbar, K.; Tagawa, S.T.; Nordquist, L.T.; Vaishampayan, N.; El-Haddad,G.; et al. Lutetium-177–PSMA-617 for Metastatic Castration-Resistant Prostate Cancer. N. Engl. J. Med. 2021, 385, 1091–1103.[CrossRef]7. Hofman, M.S.; Emmett, L.; Sandhu, S.; Iravani, A.; Joshua, A.M.; Goh, J.C.; Pattison, D.A.; Tan, T.H.; Kirkwood, I.D.; Ng, S.; et al.[177Lu]Lu-PSMA-617 versus Cabazitaxel in Patients with Metastatic Castration-Resistant Prostate Cancer (TheraP): A Randomised,Open-Label, Phase 2 Trial. Lancet 2021, 397, 797–804. [CrossRef]8. Isik, E.G.; Has-Simsek, D.; Sanli, O.; Sanli, Y.; Kuyumcu, S. Fibroblast Activation Protein–Targeted PET Imaging of MetastaticCastration-Resistant Prostate Cancer Compared With 68Ga-PSMA and 18F-FDG PET/CT. Clin. Nucl. Med. 2022, 47, e54. [CrossRef]Molecules 2023, 28, 1088 15 of 179. Paschalis, A.; Sheehan, B.; Riisnaes, R.; Rodrigues, D.N.; Gurel, B.; Bertan, C.; Ferreira, A.; Lambros, M.B.K.; Seed, G.;Yuan, W.; et al. Prostate-Specific Membrane Antigen Heterogeneity and DNA Repair Defects in Prostate Cancer. Eur. Urol.2019, 76, 469–478. [CrossRef]10. Michalski, K.; Ruf, J.; Goetz, C.; Seitz, A.K.; Buck, A.K.; Lapa, C.; Hartrampf, P.E. Prognostic Implications of Dual Tracer PET/CT:PSMA Ligand and [18F]FDG PET/CT in Patients Undergoing [177Lu]PSMA Radioligand Therapy. Eur. J. Nucl. Med. Mol. Imaging2021, 48, 2024–2030. [CrossRef]11. Seifert, R.; Seitzer, K.; Herrmann, K.; Kessel, K.; Schäfers, M.; Kleesiek, J.; Weckesser, M.; Boegemann, M.; Rahbar, K. Analysis ofPSMA Expression and Outcome in Patients with Advanced Prostate Cancer Receiving 177Lu-PSMA-617 Radioligand Therapy.Theranostics 2020, 10, 7812–7820. [CrossRef] [PubMed]12. Mannweiler, S.; Amersdorfer, P.; Trajanoski, S.; Terrett, J.A.; King, D.; Mehes, G. Heterogeneity of Prostate-Specific MembraneAntigen (PSMA) Expression in Prostate Carcinoma with Distant Metastasis. Pathol. Oncol. Res. 2009, 15, 167–172. [CrossRef]13. Park, J.E.; Lenter, M.C.; Zimmermann, R.N.; Garin-Chesa, P.; Old, L.J.; Rettig, W.J. Fibroblast Activation Protein, a Dual SpecificitySerine Protease Expressed in Reactive Human Tumor Stromal Fibroblasts. J. Biol. Chem. 1999, 274, 36505–36512. [CrossRef][PubMed]14. Fitzgerald, A.A.; Weiner, L.M. The Role of Fibroblast Activation Protein in Health and Malignancy. Cancer Metastasis Rev. 2020, 39,783–803. [CrossRef]15. Tillmanns, J.; Hoffmann, D.; Habbaba, Y.; Schmitto, J.D.; Sedding, D.; Fraccarollo, D.; Galuppo, P.; Bauersachs, J. FibroblastActivation Protein Alpha Expression Identifies Activated Fibroblasts after Myocardial Infarction. J. Mol. Cell. Cardiol. 2015, 87,194–203. [CrossRef] [PubMed]16. Levy, M.T.; McCaughan, G.W.; Abbott, C.A.; Park, J.E.; Cunningham, A.M.; Müller, E.; Rettig, W.J.; Gorrell, M.D. FibroblastActivation Protein: A Cell Surface Dipeptidyl Peptidase and Gelatinase Expressed by Stellate Cells at the Tissue RemodellingInterface in Human Cirrhosis. Hepatology 1999, 29, 1768–1778. [CrossRef] [PubMed]17. Dolznig, H.; Schweifer, N.; Puri, C.; Kraut, N.; Rettig, W.J.; Kerjaschki, D.; Garin-Chesa, P. Characterization of Cancer StromaMarkers: In Silico Analysis of an mRNA Expression Database for Fibroblast Activation Protein and Endosialin. Cancer Immun.2005, 5, 10.18. Teichgräber, V.; Monasterio, C.; Chaitanya, K.; Boger, R.; Gordon, K.; Dieterle, T.; Jäger, D.; Bauer, S. Specific Inhibition ofFibroblast Activation Protein (FAP)-Alpha Prevents Tumor Progression in Vitro. Adv. Med. Sci. 2015, 60, 264–272. [CrossRef]19. Coto-Llerena, M.; Ercan, C.; Kancherla, V.; Taha-Mehlitz, S.; Eppenberger-Castori, S.; Soysal, S.D.; Ng, C.K.Y.; Bolli, M.; von Flüe,M.; Nicolas, G.P.; et al. High Expression of FAP in Colorectal Cancer Is Associated With Angiogenesis and ImmunoregulationProcesses. Front. Oncol. 2020, 10, 979. [CrossRef]20. Tchou, J.; Zhang, P.J.; Bi, Y.; Satija, C.; Marjumdar, R.; Stephen, T.L.; Lo, A.; Chen, H.; Mies, C.; June, C.H.; et al. FibroblastActivation Protein Expression by Stromal Cells and Tumor-Associated Macrophages in Human Breast Cancer. Hum. Pathol. 2013,44, 2549–2557. [CrossRef]21. Yuan, Z.; Hu, H.; Zhu, Y.; Zhang, W.; Fang, Q.; Qiao, T.; Ma, T.; Wang, M.; Huang, R.; Tang, Q.; et al. Colorectal Cancer CellIntrinsic Fibroblast Activation Protein Alpha Binds to Enolase1 and Activates NF-KB Pathway to Promote Metastasis. Cell DeathDis. 2021, 12, 543. [CrossRef] [PubMed]22. Santos, A.M.; Jung, J.; Aziz, N.; Kissil, J.L.; Puré, E. Targeting Fibroblast Activation Protein Inhibits Tumor Stromagenesis andGrowth in Mice. J. Clin. Investig. 2009, 119, 3613–3625. [CrossRef] [PubMed]23. Balaziova, E.; Vymola, P.; Hrabal, P.; Mateu, R.; Zubal, M.; Tomas, R.; Netuka, D.; Kramar, F.; Zemanova, Z.; Svobodova, K.; et al.Fibroblast Activation Protein Expressing Mesenchymal Cells Promote Glioblastoma Angiogenesis. Cancers 2021, 13, 3304.[CrossRef] [PubMed]24. Kraman, M.; Bambrough, P.J.; Arnold, J.N.; Roberts, E.W.; Magiera, L.; Jones, J.O.; Gopinathan, A.; Tuveson, D.A.; Fearon, D.T.Suppression of Antitumor Immunity by Stromal Cells Expressing Fibroblast Activation Protein-Alpha. Science 2010, 330, 827–830.[CrossRef]25. Kesch, C.; Yirga, L.; Dendl, K.; Handke, A.; Darr, C.; Krafft, U.; Radtke, J.P.; Tschirdewahn, S.; Szarvas, T.; Fazli, L.; et al.High Fibroblast-Activation-Protein Expression in Castration-Resistant Prostate Cancer Supports the Use of FAPI-MolecularTheranostics. Eur. J. Nucl. Med. Mol. Imaging 2021, 49, 385–389. [CrossRef]26. Lindner, T.; Loktev, A.; Altmann, A.; Giesel, F.; Kratochwil, C.; Debus, J.; Jäger, D.; Mier, W.; Haberkorn, U. Development ofQuinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein. J. Nucl. Med. 2018, 59, 1415–1422.[CrossRef]27. Giesel, F.L.; Kratochwil, C.; Lindner, T.; Marschalek, M.M.; Loktev, A.; Lehnert, W.; Debus, J.; Jäger, D.; Flechsig, P.;Altmann, A.; et al. 68Ga-FAPI PET/CT: Biodistribution and Preliminary Dosimetry Estimate of 2 DOTA-Containing FAP-Targeting Agents in Patients with Various Cancers. J. Nucl. Med. 2019, 60, 386–392. [CrossRef]28. Ballal, S.; Yadav, M.P.; Kramer, V.; Moon, E.S.; Roesch, F.; Tripathi, M.; Mallick, S.; ArunRaj, S.T.; Bal, C. A Theranostic Approachof [68Ga]Ga-DOTA.SA.FAPi PET/CT-Guided [177Lu]Lu-DOTA.SA.FAPi Radionuclide Therapy in an End-Stage Breast CancerPatient: New Frontier in Targeted Radionuclide Therapy. Eur. J. Nucl. Med. Mol. Imaging 2021, 48, 942–944. [CrossRef]Molecules 2023, 28, 1088 16 of 1729. Liu, Y.; Watabe, T.; Kaneda-Nakashima, K.; Shirakami, Y.; Naka, S.; Ooe, K.; Toyoshima, A.; Nagata, K.; Haberkorn, U.;Kratochwil, C.; et al. Fibroblast Activation Protein Targeted Therapy Using [177Lu]FAPI-46 Compared with [225Ac]FAPI-46 in aPancreatic Cancer Model. Eur. J. Nucl. Med. Mol. Imaging 2022, 49, 871–880. [CrossRef]30. Lundmark, F.; Abouzayed, A.; Mitran, B.; Rinne, S.S.; Varasteh, Z.; Larhed, M.; Tolmachev, V.; Rosenström, U.; Orlova, A.Bispecific Radiotracer Targeting PSMA and GRPR for Imaging of Prostate Cancer—Optimization of the Affinity towards PSMAby Linker Modification in Murine Model. Pharmaceutics 2020, 12, 614. [CrossRef]31. Ma, X.; Wang, M.; Wang, H.; Zhang, T.; Wu, Z.; Sutton, M.V.; Popik, V.V.; Jiang, G.; Li, Z. Development of Bispecific NT-PSMAHeterodimer for Prostate Cancer Imaging: A Potential Approach to Address Tumor Heterogeneity. Bioconjug. Chem. 2019, 30,1314–1322. [CrossRef] [PubMed]32. Boinapally, S.; Lisok, A.; Lofland, G.; Minn, I.; Yan, Y.; Jiang, Z.; Shin, M.J.; Merino, V.F.; Zheng, L.; Brayton, C.; et al. Hetero-Bivalent Agents Targeting FAP and PSMA. Eur. J. Nucl. Med. Mol. Imaging 2022, 49, 4369–4381. [CrossRef] [PubMed]33. Hu, K.; Li, L.; Huang, Y.; Ye, S.; Zhong, J.; Yan, Q.; Zhong, Y.; Fu, L.; Feng, P.; Li, H. Radiosynthesis and Preclinical Evaluation ofBispecific PSMA/FAP Heterodimers for Tumor Imaging. Pharmaceuticals 2022, 15, 383. [CrossRef]34. Kuo, H.-T.; Lin, K.-S.; Zhang, Z.; Uribe, C.F.; Merkens, H.; Zhang, C.; Bénard, F. 177Lu-Labeled Albumin-Binder-ConjugatedPSMA-Targeting Agents with Extremely High Tumor Uptake and Enhanced Tumor-to-Kidney Absorbed Dose Ratio. J. Nucl. Med.2021, 62, 521–527. [CrossRef]35. Pereira, B.A.; Vennin, C.; Papanicolaou, M.; Chambers, C.R.; Herrmann, D.; Morton, J.P.; Cox, T.R.; Timpson, P. CAF Subpopula-tions: A New Reservoir of Stromal Targets in Pancreatic Cancer. Trends Cancer 2019, 5, 724–741. [CrossRef]36. Stock, K.; Steinestel, K.; Wiesch, R.; Mikesch, J.-H.; Hansmeier, A.; Trautmann, M.; Beller, N.; Rehkämper, J.; Wardelmann, E.;Heitkötter, B.; et al. Neovascular Prostate-Specific Membrane Antigen Expression Is Associated with Improved Overall Survivalunder Palliative Chemotherapy in Patients with Pancreatic Ductal Adenocarcinoma. BioMed. Res. Int. 2017, 2017, 2847303.[CrossRef] [PubMed]37. Poels, T.T.; Vuijk, F.A.; de Geus-Oei, L.-F.; Vahrmeijer, A.L.; Oprea-Lager, D.E.; Swijnenburg, R.-J. Molecular Targeted PositronEmission Tomography Imaging and Radionuclide Therapy of Pancreatic Ductal Adenocarcinoma. Cancers 2021, 13, 6164.[CrossRef] [PubMed]38. Kessel, K.; Seifert, R.; Weckesser, M.; Boegemann, M.; Huss, S.; Kratochwil, C.; Haberkorn, U.; Giesel, F.; Rahbar, K. Prostate-Specific Membrane Antigen and Fibroblast Activation Protein Distribution in Prostate Cancer: Preliminary Data on Immunohisto-chemistry and PET Imaging. Ann. Nucl. Med. 2022, 36, 293–301. [CrossRef] [PubMed]39. Yang, Y.; Ji, S.; Liu, S. Impact of Multiple Negative Charges on Blood Clearance and Biodistribution Characteristics of 99mTc-Labeled Dimeric Cyclic RGD Peptides. Bioconjug. Chem. 2014, 25, 1720–1729. [CrossRef]40. Li, Z.-B.; Wu, Z.; Chen, K.; Ryu, E.K.; Chen, X. 18F-Labeled BBN-RGD Heterodimer for Prostate Cancer Imaging. J. Nucl. Med.2008, 49, 453–461. [CrossRef]41. Imlimthan, S.; Moon, E.S.; Rathke, H.; Afshar-Oromieh, A.; Rösch, F.; Rominger, A.; Gourni, E. New Frontiers in CancerImaging and Therapy Based on Radiolabeled Fibroblast Activation Protein Inhibitors: A Rational Review and Current Progress.Pharmaceuticals 2021, 14, 1023. [CrossRef] [PubMed]42. Kuo, H.-T.; Pan, J.; Zhang, Z.; Lau, J.; Merkens, H.; Zhang, C.; Colpo, N.; Lin, K.-S.; Bénard, F. Effects of Linker Modificationon Tumor-to-Kidney Contrast of 68Ga-Labeled PSMA-Targeted Imaging Probes. Mol. Pharm. 2018, 15, 3502–3511. [CrossRef][PubMed]43. Kuo, H.-T.; Lepage, M.L.; Lin, K.-S.; Pan, J.; Zhang, Z.; Liu, Z.; Pryyma, A.; Zhang, C.; Merkens, H.; Roxin, A.; et al. One-Step18F-Labeling and Preclinical Evaluation of Prostate-Specific Membrane Antigen Trifluoroborate Probes for Cancer Imaging.J. Nucl. Med. 2019, 60, 1160–1166. [CrossRef] [PubMed]44. Lau, J.; Rousseau, E.; Zhang, Z.; Uribe, C.F.; Kuo, H.-T.; Zeisler, J.; Zhang, C.; Kwon, D.; Lin, K.-S.; Bénard, F. Positron EmissionTomography Imaging of the Gastrin-Releasing Peptide Receptor with a Novel Bombesin Analogue. ACS Omega 2019, 4, 1470–1478.[CrossRef]45. Bratanovic, I.J.; Zhang, C.; Zhang, Z.; Kuo, H.-T.; Colpo, N.; Zeisler, J.; Merkens, H.; Uribe, C.; Lin, K.-S.; Bénard, F. A Radiotracerfor Molecular Imaging and Therapy of Gastrin-Releasing Peptide Receptor-Positive Prostate Cancer. J. Nucl. Med. 2022, 63,424–430. [CrossRef]46. Lin, K.-S.; Pan, J.; Amouroux, G.; Turashvili, G.; Mesak, F.; Hundal-Jabal, N.; Pourghiasian, M.; Lau, J.; Jenni, S.; Aparicio, S.; et al.In Vivo Radioimaging of Bradykinin Receptor B1, a Widely Overexpressed Molecule in Human Cancer. Cancer Res. 2015, 75,387–393. [CrossRef]47. Jansen, K.; Heirbaut, L.; Verkerk, R.; Cheng, J.D.; Joossens, J.; Cos, P.; Maes, L.; Lambeir, A.-M.; De Meester, I.; Augustyns, K.; et al.Extended Structure–Activity Relationship and Pharmacokinetic Investigation of (4-Quinolinoyl)Glycyl-2-CyanopyrrolidineInhibitors of Fibroblast Activation Protein (FAP). J. Med. Chem. 2014, 57, 3053–3074. [CrossRef]48. North, A.J.; Karas, J.A.; Ma, M.T.; Blower, P.J.; Ackermann, U.; White, J.M.; Donnelly, P.S. Rhenium and Technetium-OxoComplexes with Thioamide Derivatives of Pyridylhydrazine Bifunctional Chelators Conjugated to the Tumour Targeting PeptidesOctreotate and Cyclic-RGDfK. Inorg. Chem. 2017, 56, 9725–9741. [CrossRef]Molecules 2023, 28, 1088 17 of 1749. Zhang, C.; Zhang, Z.; Lin, K.-S.; Pan, J.; Dude, I.; Hundal-Jabal, N.; Colpo, N.; Bénard, F. Preclinical Melanoma Imaging with68Ga-Labeled α-Melanocyte-Stimulating Hormone Derivatives Using PET. Theranostics 2017, 7, 805–813. [CrossRef]50. Kuo, H.-T.; Lin, K.-S.; Zhang, Z.; Zhang, C.; Merkens, H.; Tan, R.; Roxin, A.; Uribe, C.F.; Bénard, F. What a Difference a MethyleneMakes: Replacing Glu with Asp or Aad in the Lys-Urea-Glu Pharmacophore of PSMA-Targeting Radioligands to Reduce Kidneyand Salivary Gland Uptake. Theranostics 2022, 12, 6179–6188. [CrossRef]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.
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prostate-specific membrane antigen
fibroblast activation protein
bispecific radiotracers
PET/CT
Gallium-68
Synthesis and Preclinical Evaluation of Three Novel ⁶⁸Ga-Labeled Bispecific PSMA/FAP-Targeting Tracers for Prostate Cancer Imaging
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