Novel Generation of FAP Inhibitor-Based Homodimers for Improved Application in Radiotheranostics

Simple Summary

Radiopharmaceuticals targeting the fibroblast activation protein alpha (FAP) can be used in many different cancer types since FAP is highly expressed in the tumor microenvironment of almost all epithelial cancers. Monomeric radiotracers have shown great potential in molecular imaging (diagnosis), but the tumor retention time is relatively short (few hours). For effective radioligand therapy (RLT), the biological half-life of the radiotracer should ideally match the physical half-life of the important therapeutic radionuclides ¹⁷⁷Lu and ²²⁵Ac (6.7 and 9.9 days). The tumor retention was improved with the FAPi homodimer DOTAGA.(SA.FAPi)₂. In terms of optimizing, the new FAPi homodimers DO3A.Glu.(FAPi)₂ and DOTAGA.Glu.(FAPi)₂.were synthesized. DOTAGA.Glu.(FAPi)₂ showed superior radiolabeling properties (including successful ²²⁵Ac-labeling, higher hydrophilicity), in vitro affinity and selectivity compared to DOTAGA.(SA.FAPi)₂. In addition, significantly reduced uptake in the critical organs (liver, colon) compared to [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ was observed with [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ in a first patient study (medullary thyroid cancer) while maintaining high and prolonged tumor uptake.

Abstract

Radiopharmaceuticals based on the highly potent FAP inhibitor (FAPi) UAMC-1110 have shown great potential in molecular imaging, but the short tumor retention time of the monomers do not match the physical half-lives of the important therapeutic radionuclides ¹⁷⁷Lu and ²²⁵Ac. This was improved with the dimer DOTAGA.(SA.FAPi)₂, but pharmacological and radiolabeling properties still need optimization. Therefore, the novel FAPi homodimers DO3A.Glu.(FAPi)₂ and DOTAGA.Glu.(FAPi)₂. were synthesized and quantitatively radiolabeled with ⁶⁸Ga, ⁹⁰Y, ¹⁷⁷Lu and ²²⁵Ac. The radiolabeled complexes showed high hydrophilicity and were generally stable in human serum (HS) and phosphate-buffered saline (PBS) at 37 °C over two half-lives, except for [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ in PBS. In vitro affinity studies resulted in subnanomolar IC₅₀ values for FAP and high selectivity for FAP over the related proteases PREP and DPP4 for both compounds as well as for [^natLu]Lu-DOTAGA.Glu.(FAPi)₂. In a first proof-of-principle patient study (medullary thyroid cancer), [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ was compared to [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂. High uptake and long tumor retention was observed in both cases, but [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ significantly reduces uptake in non-target and critical organs (liver, colon). Overall, the novel FAPi homodimer DOTAGA.Glu.(FAPi)₂ showed improved radiolabeling in vitro and pharmacological properties in vivo compared to DOTAGA.(SA.FAPi)₂. [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ and [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ appear promising for translational application in patients.

1. Introduction

Fibroblast activation protein alpha (FAP), also called Seprase, is a S9B family serine protease and an integral type-II transmembrane glycoprotein [1]. It is mainly expressed on activated fibroblasts such as cancer-associated fibroblasts (CAFs), fibroblasts present during wound healing [2], at sites of inflammation (e.g., arthritis) [3] and fibrosis (e.g., liver cirrhosis) [4]. However, FAP is absent in resting fibroblasts of normal healthy tissue. CAFs behave as activated stromal fibroblasts and occupy a major part of the tumor microenvironment (TME) that further consists of vascular, inflammatory and immune cells as well as extracellular matrix components (ECM) [5]. The TME plays an essential role in tumorigenesis, tumor growth, metastasis and angiogenesis and can account for up to 90% of the total tumor mass [5,6,7,8]. Each epithelial tumor more than 1 mm in diameter consists of an extensive TME. More than 90% of these epithelial tumors strongly express FAP, including prostate, breast, lung, ovarian and colorectal cancer, among others [6].

FAP is only enzymatically active as a dimer and has unique dual enzymatic activity, both dipeptidyl peptidase and prolyl endopeptidase, whereas the similar serine proteases prolyl endopeptidase (PREP) and other post-proline dipeptidyl peptidases (DPP4, DPP8 and DPP9) only have one of these activities [8,9,10,11,12]. Natural substrates have been identified including several neuropeptides (neuropeptide Y, peptide YY etc.) [13], α₂-antiplasmin [4,14] and denatured type I collagen [15,16]. The actual in vivo role of FAP is not fully understood yet but seems to be non-essential in healthy tissue [8]. In contrast, FAP is considered to play an important role in fibroblast growth, tissue remodeling of the ECM, epithelial carcinogenesis, tumor growth (proliferation) and angiogenesis in malignant tissue [8,17,18,19,20,21,22].

Compared to other tumor targets that are vastly used in nuclear medicine such as prostate-specific membrane antigen (PSMA) or somatostatin receptor (SSTR), radiopharmaceuticals targeting FAP do not address the cancer cells directly but indirectly via its TME. PSMA inhibitor-based tracers can basically only be applied in prostate cancer and octreotide-based SSTR-targeting tracers for neuroendocrine tumors (NETs), while targeting FAP allows for a broader application in many different types of cancers, which makes it a more universal pan-tumor target. Jeremie Calais discussed FAP as potentially being “the next billion dollar nuclear theranostics target” following [¹⁷⁷Lu]Lu-PSMA-617 and [¹⁷⁷Lu]Lu-DOTA-TATE that has had a great impact in nuclear medicine in the last years [23].

The FAP inhibitor (FAPi) UAMC-1110 (Figure 1a) was the first small molecule that showed high affinity for FAP and high selectivity over PREP and the DPPs [24]. Since then, several FAPi-based radiopharmaceuticals have been synthesized [25,26,27,28,29,30,31,32,33,34]. Some of them have already been systematically investigated in human positron emission tomography (PET) studies such as FAPI-04, FAPI-46, and DOTA.SA.FAPi (Figure 1b) [35,36,37,38,39,40].

Most of them are conjugated with bifunctional chelators such as DOTA to enable PET imaging with ⁶⁸Ga. The rapid renal clearance of monomers such as the squaramide (SA)-based [⁶⁸Ga]Ga-DOTA.SA.FAPi or [⁶⁸Ga]Ga-FAPI-04 resulted in high-contrast PET images in various cancers even at early time points (10 min p.i.) [25,40]. However, the short tumor retention time and as such the biological half-life does not match the physical half-life of therapeutic radionuclides such as ¹⁷⁷Lu (t_1/2 = 6.7 d) or ²²⁵Ac (t_1/2 = 9.9 d), which limits the use of these compounds for radioligand therapy (RLT) with the beta minus particle emitter ¹⁷⁷Lu or targeted alpha-particle therapy (TAT) with the alpha emitter ²²⁵Ac.

Recently, peptides were introduced as FAP-targeting vectors in compounds such as FAP-2286, which showed prolonged retention in CAFs-expressing tissue and which represent a new approach regarding FAP radiotherapeutics with a similar approach to TOC or TATE used for peptide receptor radionuclide therapy by targeting SSTRs of NETs [41,42].

Another approach is to apply the dimeric concept, which has already shown, to improve accumulation and to prolong tumor retention time in the context of different targets (e.g., PSMA) beforehand [43,44,45,46,47]. This has led to the development of the first homodimeric structure DOTAGA.(SA.FAPi)₂ (see Figure 1c). Other groups have adapted this approach, and several other dimeric FAPi-based radiopharmaceuticals were published recently [48,49,50,51]. Zhao et al. developed the dimer DOTA-2P(FAPI)₂ based on the FAPI-46 structure, and the ⁶⁸Ga-labeled derivative was investigated in vivo [48,49]. Galbiati et al. developed the dimeric BiOncoFAP-DOTAGA [50]. The publication mainly focused on the ¹⁷⁷Lu-labeled therapeutic agent that showed promising results in preclinical studies. Both studies confirmed that the homodimeric concept is a successful strategy to target FAP and to prolong tumor retention as first demonstrated with DOTAGA.(SA.FAPi)₂.

The ¹⁷⁷Lu-labeled derivative [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ has already been investigated extensively in RNT of radioiodine-refractory differentiated thyroid cancer (RR-DTC) patients, among others (e.g., breast cancer) [39,52]. Much longer tumor residence times of up to one week could be measured via planar scintigraphy and single-photon emission computed tomography (SPECT) imaging [39]. The colon was identified as the critical organ regarding the radiation dose due to slow biliary excretion [39,52]. Furthermore, the linear molecular design with the central chelator may have resulted in steric hindrance during complexation, which led to slower reaction kinetics and/or lower radiochemical yields, in particular with ²²⁵Ac.

On the one hand, the new compounds DO3A.Glu.(FAPi)₂ and DOTAGA.Glu.(FAPi)₂ (Figure 1d,e) were designed, synthesized and investigated to improve radiolabeling, especially regarding ²²⁵Ac and therefore allowing for potential application in TAT. On the other hand, the pharmacokinetic and pharmacodynamic properties should be optimized while maintaining or even further improving the desired long tumor retention time. In contrast to the original linear dimer DOTAGA.(SA.FAPi)₂, these new FAPi dimers are designed in a branched structure where the two FAPi targeting vectors and the terminal chelator are coupled via a central glutamic acid (Glu) linker. The further use of squaramide as a linker or spacer was renounced with the intention to synthesize more hydrophilic compounds instead. This could accelerate clearance from non-target tissues as well as excretion to reduce the radiation dose for healthy organs such as colon and liver.

Following the organic synthesis of the precursors, the radiolabeling properties with different radiometals (⁶⁸Ga, ¹⁷⁷Lu, ²²⁵Ac and ⁹⁰Y) as well as the complex stabilities were investigated. In addition, the lipophilicity of the ⁶⁸Ga- and ¹⁷⁷Lu-labeled derivatives was evaluated. The new FAPi dimers were tested for their in vitro affinity for FAP as well as PREP, DPP4, DPP8, and DPP9. Beyond that, first proof-of-concept patient investigations were carried out with [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂, which demonstrated faster excretion kinetics and therefore reduced the radiation dose for healthy organs such as the colon and liver. All these studies were performed in comparison to DOTAGA.(SA.FAPi)₂.

2. Materials and Methods

2.1. Chemicals and Equipment

All basic chemicals were purchased from Sigma-Aldrich and Merck (Darmstadt, Germany), TCI (Eschborn, Germany), ABCR (Karlsruhe, Germany), Alfa Aesar by Thermo Fisher Scientific (Kandel, Germany), Acros Organics (Schwerte, Germany) and VWR (Bruchsal, Germany). Dry solvents were bought from Acros Organics, Alfa Aesar by Thermo Fischer Scientific and Merck and deuterated solvents from Deutero (Kastellaun, Germany). The chelators DOTA-tri(^tBu)ester 8 (2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid) and DOTA-GA(^tBu)₄ 5 (5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetra-azacyclododecan-1-yl)pentanoic acid) were obtained from CheMatech (Dijon, France). (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-car-boxamide (FAPi-NH₂ 1) was purchased from KE Biochem Co. (Shangai, China) or synthesized according to Figure S1.

Thin-layer chromatography (TLC) plates coated with silica gel 60 F₂₅₄ from Merck were used. For analysis, they were investigated via UV lamp (λ = 254 and 366 nm) as well as staining with potassium permanganate. Silica 60 (0.040–0.063 mm particle size) was used for column chromatography.

Nuclear magnetic resonance (NMR) spectra were recorded in deuterated solvents on an Avance II 400 spectrometer (400 MHz, 5 mm BBFO sample head with z-gradient and ATM, SampleXPress 60 sample changer) or an Avance III 300 spectrometer (300 MHz, 5 mm BBFO sample head with z-gradient and ATM, BACS 60 sample changer) from Bruker (Rheinstätten, Germany). MestReNova 14.2.0 software from Mestrelab Research (Santiago de Compostela, Spain) was used to analyze the spectra.

Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-LC/MS) was measured using Agilent Technologies 1220 Infinity LC coupled to an Agilent Technologies 6130B Single Quadruple LC/MS system with an Agilent Zorbax SB-C18 column (21 × 50 mm, 1.8 µm) with linear gradients of acetonitrile (MeCN)/Milli-Q^® water (H₂O) + 0.05% formic acid (HFo) and a flow rate of 0.5 mL/min.

High-pressure liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-HPLC-MS) measurements were performed using an Agilent Technologies G6545A Q-ToF with electron spray ionization coupled to a 1260 Infinity II HPLC system (Agilent Technologies) with G7111B 1260 Quaternary Pump, G7129A 1260 Vial Sampler, and G7116A Multicolumn Thermostat. Separation was performed using an Agilent Poroshell 120 EC-C8 column (2.1 × 100 mm, 2.7 µm) with H₂O + 2% MeCN/MeCN + 2% H₂O + 0.05% Hfo and a flow rate of 0.1 mL/min.

Semi-preparative reversed-phase high-pressure liquid chromatography (RP-HPLC) was performed using LaChrom HPLC (7000 series) from Merck Hitachi with L-7100 pump, L-7400 UV detector (λ = 254 nm), a D-7000 interface and autosampler. Separation was performed using a Phenomenex Synergi Max-RP C18 column (250 × 10 mm, 4 µm) and linear gradient of MeCN/H₂O + 0.1% trifluoacetic acid (TFA) and a flow rate of 5 mL/min.

For radiolabeling, only chemicals of the highest purity (TraceSelect if available) were used. Radio-TLCs were developed using 0.1 M citrate buffer (pH = 4.0). Additional TLCs were developed using 1 M ammonium acetate (pH = 4.0)/methanol (AmOAc/MeOH, 1:1). All radio-TLCs were evaluated using a CR-35 Bio Test-Imager from Duerr-ndt (Bietigheim-Bissingen, Germany) and AIDA software from Elysia-Raytest (Straubenhardt, Germany). Some radio-TLCs of ²²⁵Ac-labelings were additionally analyzed via high-resolution gamma spectroscopy with a HPGe detector. Analytical radio-HPLC was performed also using a Merck Hitachi LaChrom HPLC (7000 series). Separation was performed using Phenomenex Luna C18 column (250 × 4.6 mm, 5 µm) and linear gradient of MeCN/H₂O + 0.1% TFA and flow rate of 1 mL/min. The radio-HPLC is equipped with a Ramona analog radio detector from Elysia Raytest with its energy window set to 100–1200 keV for ⁶⁸Ga and 100–250 keV for ¹⁷⁷Lu.

2.2. Organic Synthesis

Figure 2 shows the synthesis scheme of the precursors DOTAGA.Glu.(FAPi)₂ 7 and DO3A.Glu.(FAPi)₂ 11.

2.2.1. Synthesis of Glu.(FAPi)₂

tert-Butyl ((S)-1,5-bis((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)-quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)carbamate (3, Boc-Glu.(FAPi)₂)

N-tert-Butoxycarbonyl-L-glutamic acid 2 (Boc-Glu-OH, 49.5 mg, 200 µmol, 1.00 eq), 1-hydroxybenzotriazole (HOBt, 70 mg, 518 µmol, 2.59 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC*HCl, 100 mg, 522 µmol, 2.61 eq) were dissolved in dry N,N-dimethylformamide (DMF, 4 mL), and N,N-diisopropylethylamine (DIPEA, 17.0 µL, 100 µmol, 0.50 eq) was added under argon atmosphere. The solution was stirred at room temperature (RT) for one hour. Then, a solution of FAPi-NH₂ 1 (173 mg, 400 µmol, 2.00 eq) and DIPEA (102 µL, 600 µmol, 3.00 eq) in DMF (2 mL) was added. After another 4 h at RT, further HOBt (13.5 mg, 100 µmol, 0.50 eq) and EDC*HCl (19.2 mg, 100 µmol, 0.50 eq) were added, and after another 30 min, a solution of FAPi-NH₂ 1 (43.4 mg, 100 µmol, 0.50 eq) and DIPEA (17.0 µL, 100 µmol, 0.50 eq) in DMF (1 mL) was added again. This addition of HOBt, EDC*HCl (0.50 eq each) followed by FAPi-NH₂ (28.5 mg, 66 µmol, 0.33 eq) and DIPEA (0.50 eq) was repeated one more time, and after 4 more hours, the solvent was removed in vacuo. After column chromatography (CHCl₃/MeOH, 100:10–15)) 3 was obtained as a yellowish solid (158 mg, 147 µmol, 74%). ¹H-NMR (400 MHz, DMF-d₇): δ [ppm] = 8.98 (t, J = 6.0 Hz, 1H), 8.83 (d, J = 4.3 Hz, 1H), 8.29–8.17 (m, 1H), 8.03–7.94 (m, 3H), 7.93–7.87 (m, 1H), 7.80–7.71 (m, 1H), 7.58 (d, J = 4.3 Hz, 1H), 7.52–7.43 (m, 2H), 7.34 (dd, J = 8.5, 2.0 Hz, 1H), 7.25 (dd, J = 8.4, 2.5 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 5.31 (dd, J = 9.6, 2.5 Hz, 1H), 4.53–3.70 (m, 11H), 3.37–3.01 (m, 8H), 2.33–2.25 (m, 2H), 2.16–1.83 (m, 4H), 1.78–1.66 (m, 2H), 1.62–1.44 (m, 2H), 1.42 (s, 9H), 1.40–1.37 (m, 6H). ¹⁹F-NMR (300 MHz, DMF-d₇): δ [ppm] = −74.48 (s). MS (ESI⁺): m/z (%) = 487.8 (100, [M−Boc+H]²⁺), 537.8 (73, [M+H]²⁺), 1074.4 (9, [M+H]⁺), 1075.4 (6, [M+H]⁺), calculated for C₅₂H₅₉F₄N₁₁O₁₀: 1073.44 [M].

(S)-2-Amino-N¹,N⁵-bis(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)-quinolin-6-yl)oxy)butyl)pentanediamide (4, Glu.(FAPi)₂)

Boc-Glu.(FAPi)₂ 3 (158 mg, 147 µmol, 1.00 eq) was dissolved in dry MeCN (3 mL) and under argon atmosphere and at 0 °C, 4 M hydrochloric acid (HCl) in 1,4-dioxane (300 µL, 1.20 mmol, 8.57 eq) was added. The reaction was allowed to warm to RT, and after 6 h, it was diluted with MeOH (20 mL), and the solvent was completely removed in vacuo. Glu.(FAPi)₂ 4 (136 mg, 140 µmol, 95%) was obtained as a yellowish solid. ¹H-NMR (300 MHz, MeOD): δ [ppm] = 8.84 z(dd, J = 8.4, 4.9 Hz, 2H), 8.01 (dd, J = 8.8, 2.6 Hz, 4H), 7.72 (dd, J = 6.6, 4.8 Hz, 2H), 7.55 (ddd, J = 9.3, 4.6, 2.7 Hz, 2H), 5.16 (dt, J = 9.3, 3.4 Hz, 2H), 4.35 (d, J = 2.1 Hz, 4H), 4.31–4.07 (m, 8H), 3.91 (t, J = 6.2 Hz, 1H), 3.58–3.36 (m, 2H), 3.11–2.71 (m, 6H), 2.47–2.37 (m, 2H), 2.17–2.00 (m, 2H), 2.00–1.87 (m, 4H), 1.86–1.70 (m, 4H). ¹⁹F-NMR (300 MHz, MeOD): δ [ppm] = −77.29 (s). MS (ESI⁺): m/z (%) = 325.6 (100, [M−Boc+H]³⁺), 487.8 (28, [M+H]²⁺), 974.3 (5, [M+H]⁺), calculated for C₄₇H₅₁F₄N₁₁O₈: 973.39 [M].

2.2.2. Synthesis of DOTAGA.Glu.(FAPi)₂

tri-tert-Butyl 2,2′,2″-(10-(5-(((S)-1,5-bis((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxo-ethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-1-(tert-butoxy)-1,5-dioxopentan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (6, DOTAGA(^tBu)₄.Glu.(FAPi)₂)

DOTAGA(^tBu)₄ 5 (56.1 mg, 80.0 µmol, 1.15 eq), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 30.3 mg, 80.0 µmol, 1.15 eq) and DIPEA (15.0 µL, 88 µmol, 1.25 eq) were dissolved in dry DMF (1 mL) under argon atmosphere and stirred at 30 °C for one hour. A solution of Glu.(FAPi)₂ (68.0 mg, 70.0 µmol, 1.00 eq) and DIPEA (30.0 µL, 175 µmol, 2.50 eq) in dry DMF (1 mL) was added. It was stirred for two days at 30 °C, and then, more HATU (15.2 mg, 40.0 µmol, 0.50 eq) was added. After another day of stirring at 30 °C, the solvent was removed in vacuo. A yellow oil was obtained, which was directly used in the next step without further purification. MS (ESI⁺): m/z (%) = 414.97 (13, [M+H]⁴⁺), 415.22 (12, [M+H]⁴⁺), 552.95 (100, [M+H]³⁺), 553.29 (97, [M+H]³⁺), 553.62 (51, [M+H]³⁺), 553.96 (18, [M+H]³⁺), 828.93 (82, [M+H]²⁺), 829.43 (78, [M+H]²⁺), 829.93 (40, [M+H]²⁺), 830.43 (15, [M+H]²⁺), 1656.85 (87, [M+H]⁺), 1657.85 (85, [M+H]⁺), 1658.85 (43, [M+H]⁺), 1659.86 (15, [M+H]⁺), calculated for C₈₂H₁₁₃F₄N₁₅O₁₇: 1655.84 [M].

2,2′,2″-(10-(4-(((S)-1,5-bis((4-((4-((2-((S)-2-Cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (7, DOTAGA.Glu.(FAPi)₂)

DOTAGA(^tBu)₄.Glu.(FAPi)₂ 6 was dissolved in TFA (850 µL), MeCN (100 µL), triisopropylsilane (TIPS, 50 µL) and H₂O (25 µL) and stirred at RT for 5 h. After co-distillation with MeOH (5 × 10 mL) and evaporation to dryness, the crude product was purified by semipreparative RP-HPLC (22–24% MeCN in 20 min, t_R = 17–18 min), and 7 was obtained as a yellow solid (28.0 mg, 19.5 µmol, 28%). MS (ESI⁺): m/z (%) = 358.85 (65, [M+H]⁴⁺), 369.05 (24, [M+MeCN+H]⁴⁺), 478.30 (100, [M+H]³⁺), 717.30 (6, [M+H]²⁺), 1432.40 (1, [M+H]⁺), 1454.70 (1, [M+Na]⁺), calculated for C₆₆H₈₁F₄N₁₅O₁₇: 1431.59 [M].

2.2.3. Synthesis of DO3A.Glu.(FAPi)₂

tri-tert-Butyl 2,2′,2″-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclodo-decane-1,4,7-triyl)triacetate (9, DOTA(^tBu)₃-NHS)

DOTA-tris(tert-butyl ester) 8 (573 mg, 1.00 mmol, 1.00 eq) and HBTU (417 mg, 1.10 mmol, 1.10 eq) were dissolved in dry MeCN (12 mL) and dry DMF (5 mL), and then, N-hydroxysuccinimide (NHS, 127 mg, 1.10 mmol, 1.10 eq) was added. After stirring at 30 °C for 6 h, 2-(1H-benzotriazol-1-yl-)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 190 mg, 501 µmol, 0.50 eq) and NHS (57.5 mg, 500 µmol, 0.50 eq) were added, and stirring was continued overnight. After all solvents were removed in vacuo, purification by column chromatography (dichloromethane/MeOH (100:10)) gave 9 as a colorless solid (643 mg, 961 μmol, 96%). ¹H-NMR (400 MHz, CDCl3): δ [ppm] = 2.97–2.77 (m, 12H), 2.66–2.02 (m, 16H), 1.44 (s, 27H). MS (ESI⁺): m/z (%) = 335.7 (100, [M+H]²⁺), 670.4 (50, [M+H]⁺), 671.4 (18, [M+H]⁺), calculated for C₃₂H₅₅N₅O₁₀: 669.39 [M].

tri-tert-Butyl 2,2′,2″-(10-(2-(((S)-1,5-bis((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxo-ethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (10, DOTA(tBu)₃.Glu.(FAPi)₂)

DOTA(^tBu)₃-NHS 9 (40.2 mg, 60.0 µmol, 1.20 eq) and Glu.(FAPi)₂ 4 (48.7 mg, 50.0 µmol, 1.00 eq) were dissolved in dry DMF (2 mL), and DIPEA (200 µL) was added. After stirring at 40 °C under argon atmosphere for one day, the solvents were completely removed in vacuo. A yellow oil was obtained and directly used in the next step without further purification. MS (ESI⁺): m/z (%) = 382.95 (22, [M+H]⁴⁺), 383.20 (19, [M+H]⁴⁺), 491.57 (34, [M−^tBu+H]³⁺), 491.90 (28, [M−^tBu+H]³⁺), 492.24 (13, [M−^tBu+H]³⁺), 510.26 (100, [M+H]³⁺), 510.59 (90, [M+H]³⁺), 510.93 (44, [M+H]³⁺), 511.26 (14, [M+H]³⁺), 764.88 (42, [M+H]²⁺), 765.38 (37, [M+H]²⁺), 765.89 (17, [M+H]²⁺), 1528.76 (25, [M+H]⁺), 1529.76 (22, [M+H]⁺), 1530.77 (10, [M+H]⁺), calculated for C₇₅H₁₀₁F₄N₁₅O₁₅: 1527.75 [M].

2,2′,2″-(10-(2-(((S)-1,5-bis((4-((4-((2-((S)-2-Cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (11, DO3A.Glu.(FAPi)₂)

DOTA(^tBu)₃.Glu.(FAPi)₂ 10 was dissolved in TFA (1.9 mL), TIPS (50 µL) and H₂O (50 µL) and stirred at RT for 8 h. After co-distillation with MeOH (4 × 10 mL), the solvents were completely removed under reduced pressure again. After semipreparative RP-HPLC (22–23% MeCN in 16 min, t_R = 14–15 min), 11 was obtained as a yellow solid (19.9 mg, 14.6 µmol, 29%). MS (ESI⁺): m/z (%) = 340.85 (42, [M+H]⁴⁺), 351.00 (57, [M+MeCN+H]⁴⁺), 361.35 (13, [M+2MeCN+H]⁴⁺), 454.15 (100, [M+H]³⁺), 468.00 (20, [M+MeCN+H]³⁺), 680.85 (9, [M+H]²⁺), calculated for C₆₃H₇₇F₄N₁₅O₁₅: 1359.57 [M].

2.2.4. Synthesis of ^natLu-Complexes

[^natLu]Lu-DOTAGA.Glu.(FAPi)₂ (^natLu-7)

DOTAGA.Glu.(FAPi)₂ 7 (8.6 mg, 6.0 µmol, 1.00 eq) was dissolved in 500 µL 1 M HEPES (pH = 5.5), 100 µL 1 M ammonium acetate (pH = 5.5) and 200 µL EtOH. Then, 0.1 M LuCl₃ (150 µL, 15.0 µmol, 2.50 eq) solution in 1 M ammonium acetate (pH = 5.5) was added and shaken at 90 °C for 6 h. Subsequent semi-preparative RP-HPLC (22–24% MeCN in 12 min, t_R = 11.5 min) yielded [^natLu]Lu.DOTAGA.Glu.(FAPi)₂ as a yellow solid (5.3 mg, 3.3 µmol, 55%). MS (ESI⁺): m/z (%) = m/z (%) = 535.50 (100, [M+H]³⁺), 802.95 (36, [M+H]²⁺), calculated for C₆₆H₇₈F₄LuN₁₅O₁₇: 1603.50 [M].

[^natLu]Lu-DO3A.Glu.(FAPi)₂ (^natLu-11)

DO3A.Glu.(FAPi)₂ 11 (2.8 mg, 2.0 µmol, 1.00 eq) was dissolved in 500 µL of 1 M HEPES buffer (pH = 5.5). Then, 0.1 M LuCl₃ solution (40 µL, 4.0 µmol, 2.00 eq) was added and shaken at 90 °C for 4 h. Subsequent semi-preparative RP-HPLC (20–25% MeCN in 20 min, t_R = 14–15 min) yielded [^natLu]Lu-DO3A.Glu.(FAPi)₂ as a yellow solid (0.7 mg, 0.46 µmol, 23%). MS (ESI⁺): m/z (%) = 511.55 (100, [M+H]³⁺), 766.75 (14, [M+H]²⁺), calculated for C₆₃H₇₄F₄LuN₁₅O₁₅: 1531.48 [M].

2.3. Radiosynthesis

2.3.1. ⁶⁸Ga-Radiolabeling

Gallium-68 was eluted from a ITM (Isotope Technologies Munich AG, Germany) ⁶⁸Ge/⁶⁸Ga-generator with 5 mL of 0.05 M HCl (TraceSelect). For the labeling of DOTAGA.Glu.(FAPi)₂ 7, the eluate (100–400 MBq in 0.5–2.0 mL) was directly used. The equivalent volume of 1 M 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES, pH = 4.5) was added, followed by precursor 7 (10–40 nmol, 10–40 µL 1 mM stock solution).

For DO3A.Glu.(FAPi)₂ 11, cationic post processing of the ⁶⁸Ge/⁶⁸Ga-generator was carried out according to the procedure developed by Eppard et al. [53]. Precursor 11 (5–20 nmol, 5–20 µL 1 mM stock solution) was added to 400 µL 1 M HEPES (pH = 4.5 or 5.5) buffer solution followed by the addition of gallium-68 (ca. 100 MBq).

The reaction solutions were heated at 95 °C, and reaction controls were taken at different time points to investigate reaction kinetics analyzed via radio-TLC. Additional AmOAc/MeOH (1:1) radio-TLCs and radio-HPLC were carried out for the last reaction control to double-check radiochemical conversion (RCC) and purity (RCP).

2.3.2. ¹⁷⁷Lu-Radiolabeling

[¹⁷⁷Lu]LuCl₃ in 0.04 M HCl) was purchased from ITM (Isotope Technologies Munich SE, Munich, Germany). Then, 400 µL 1 M HEPES (pH = 5.5) buffer solution and the respective quantity of precursor (1 mM stock solution) were added to lutetium-177 (50–100 MBq) and heated at 95 °C. Reaction controls were taken at different time points to investigate reaction kinetics analyzed via radio-TLC. Additional AmOAc/MeOH (1:1) radio-TLCs and radio-HPLC were carried out for the last reaction control to double-check radiochemical conversion (RCC) and purity (RCP).

2.3.3. ⁹⁰Y-Radiolabeling

[⁹⁰Y]YCl₃ (in 0.04 M HCl) was purchased from EZAG (Eckert & Ziegler Strahlen- und Medizintechnik AG, Berlin, Germany). Then, 400 µL 1 M HEPES (pH = 4.5) buffer solution and DO3A.Glu.(FAPi)₂ (5 or 10 nmol, 5 or 10 µL of 1 mM stock solution) were added to yttrium-90 (30–60 MBq) and heated at 95 °C. Reaction controls were taken at different time points to investigate reaction kinetics analyzed via radio-TLC.

2.3.4. ²²⁵Ac-Radiolabeling

[²²⁵Ac]AcCl₃ (in 0.04 M HCl) was purchased from ITM (Isotope Technologies Munich SE, Munich, Germany).

Small scale labeling was carried out as follows: ca. 500 kBq [²²⁵Ac]AcCl₃ (in 50 µL 0.04 M HCl) was added to 500 µL 0.1 M sodium ascorbate (pH = 7.0) buffer solution, and then, the respective quantity of precursor 11 (10 or 20 nmol, 10 or 20 µL of 1 mM stock solution) was added and heated at 95 °C for 60 min.

Bigger scale labeling was carried out similarly: 1.6–3.2 MBq [²²⁵Ac]AcCl₃ (in 100 µL 0.04 M HCl) was added to 1000 µL 0.1 M sodium ascorbate (pH = 7.0) buffer solution, and then, the respective quantity of precursor 11 (30–40 nmol/MBq from 1 mM stock solution) was added and heated at 95 °C for 60 min.

Reaction controls were taken at different time points to investigate reaction kinetics. The radio-TLCs were dried with a heating gun and imaged at different time points (1 h, 1 d) after the TLC had been developed. Additional AmOAc/MeOH (1:1) radio-TLCs were developed for the last reaction control.

Subsequent purification using SepPak^® Light C18 cartridge yielded the product. The cartridge was conditioned with 2 mL EtOH and 5 mL H₂O. The reaction solution was pushed through the cartridge. The reaction vial was rinsed with 0.1 M sodium ascorbate, and the step was repeated once. The product was washed with 1 mL H₂O and eluted with 2 mL EtOH/saline (1:1). It was diluted with 8 mL of saline containing 100 mg sodium ascorbate to give the final formulation. The radiochemical yield (>98%) was determined via radio-TLC and high-resolution gamma spectroscopy with HPGe detector. For the gamma spectroscopy, a TLC was developed in standard 0.1 M citrate buffer, cut into two pieces at R_f = 0.2–0.3 and measured separately after one hour for 15 min each. The integral of the ²²¹Fr line (E_γ = 218 keV) of each of the two pieces was considered for the RCC determination.

2.3.5. Complex Stability Measurements

After radiolabeling was carried out (RCP > 95%), the stability in human serum (HS) and phosphate-buffered saline (PBS) was investigated (n = 3) by incubating ca. 10 MBq of the labeled tracer solution in 0.5 mL HS and PBS at 37 °C for 120 min (⁶⁸Ga), for 6 d (⁹⁰Y) and 14 d (¹⁷⁷Lu). The complex stability was determined via radio-TLC with 0.1 M citrate buffer (pH = 4.0) as the mobile phase.

For ²²⁵Ac, 350–400 kBq was incubated in 0.5 mL HS and PBS as well as 200 kBq in 1 mL final formulation solution (“pure”). The complex stability was investigated over 20 days, and the citrate radio TLCs were imaged 1 d after they were developed.

2.3.6. Determination of logD_7.4 (Lipophilicity Measurement)

After radiolabeling was carried out (RCP > 95%), the logD_7.4 value was determined by diluting 10 MBq of the labeled tracer solution to 700 µL with PBS (n = 4). To each, 700 µL of 1-octanol was added and shaken vigorously for 1–2 min (1500 rpm) followed by centrifugation for 1–2 min. Then, 400 µL of the organic and aqueous phase were separated into new Eppendorf tubes. Samples of 3 µL (PBS) or 6 µL (1-octanol) were pipetted onto a TLC plate. Since most of the activity was in the aqueous phase, this was diluted to 700 µL with PBS, and again, 700 µL of 1-octanol was added. The procedure was repeated twice. The TLC plate was imaged (exposure time: 5–10 min), and the integral of each spot (octanol phase: I_O, aqueous PBS phase: I_PBS) was determined. The logD_7.4 value was calculated using Equation (1) where the different volumes of V_O = 6 µL und V_PBS = 3 µL were taken into account.

$$\log D_{7.4}=\log \left(\frac{I_O}{2·I_{PBS}}\right)$$

(1)

Only the values of the second and third extraction were considered in the calculation.

2.4. In Vitro Inhibition Assays (IC₅₀ Measurements)

rhFAP (recombinant human fibroblast activation protein), DPP8 (dipeptidyl peptidase VIII) and DPP9 (dipeptidyl peptidase IX) were expressed and purified as described previously [54,55]. DPP4 (dipeptidyl peptidase IV) was purified from human seminal plasma as described previously [56]. Human recombinant PREP (prolylendopeptidase) was expressed and purified as described before [54].

IC₅₀ measurements for FAP and PREP were carried out as described before [30,55]. Briefly, Z-Gly-Pro-7-amino-4-methylcoumarine (AMC, Bachem) at a final concentration of 50 µM at pH 8.0 in 0.05 M Tris-HCl buffer, 1 mg/mL BSA and 140 mM NaCl was used as the substrate for FAP IC₅₀ measurements. For PREP, N-succinyl-Gly-Pro-AMC (Bachem) at a final concentration of 250 µM at pH 7.4 in 0.1 M K-phosphate, 1 mM EDTA, 1 mM DTT and 1 mg/mL BSA buffer was used.

IC₅₀ measurements for DPP4, DPP8 and DPP9 were performed as published before, using Ala-Pro-pNA as the substrate at the respective concentrations of 25 µM (DPP4), 300 µM (DPP8) and 150 µM (DPP9) at pH 7.4 in 0.05 M HEPES-NaOH buffer with 0.1% Tween-20, 0.1 mg/mL BSA and 150 mM NaCl) [30,57].

All enzymatic activities were determined kinetically for 15 min at 37 °C by measuring initial velocities of AMC release (λex = 380 nm and λem = 465 nm) or pNA release (405 nm) from the substrates mentioned above with an Infinite 200 plate reader from Tecan Group Ltd. (Männedorf, Switzerland). The Magellan software was used to process the data, and data fitting was performed using a non-linear fitting model in Grafit 7.4. All IC₅₀ measurements were carried out in triplicate with at least eight different inhibitor concentrations tested.

2.5. Patient Study

2.5.1. Radiosynthesis

[⁶⁸Ga]Ga-DOTA.SA.FAPi was synthesized as described by Ballal et al. [58]. [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ was synthesized as described by Ballal et al. [39].

[¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ was radiolabeled by adding 6.1 GBq [¹⁷⁷Lu]LuCl₃ (obtained from BRIT (India) in 0.01 M supra pure HCl) in 0.1 M sodium acetate buffer (pH = 5.0) followed by 150 nmol DOTAGA.Glu.(FAPi)₂ 7 (≈25 nmol/GBq). The solution was heated at 95 °C for 30 min followed by purification through Sep-Pak C18 light cartridge and eluted with 1 mL EtOH/H₂O (1:1). Radiochemical quality control was carried out using the instant thin-layer chromatography method with 0.1 M trisodiumcitrate buffer (pH = 4.0) as solvent. The product was administered with a radiochemical purity of 95%. The radiochemical yield was 90%.

2.5.2. Clinical Image Acquisition and Analysis

The patient underwent PET scans with [¹⁸F]F-FDG and [⁶⁸Ga]Ga-DOTA.SA.FAPi mean injected activities of 185 and 222 MBq, respectively. The scans were acquired within a month time interval with a GE Discovery 710* 128 Slice PET/CT Scanner (40 mm detector at 0.35 s rotation speed). The whole-body PET/CT scans were acquired one hour post-injection. An initial scout image was acquired first, followed by a CT scan, and PET acquired at 2 min per bed. The whole-body CT scan parameters included 300–350 mAs, 120 kVp, slice thickness of 5 mm, and pitch 1 was acquired. [¹⁸F]F-FDG and [⁶⁸Ga]Ga-DOTA.SA.FAPi PET/CT scans were loaded simultaneously and co-registered using carina as an anatomical landmark registration technique. The scan interpretations were conducted by two experienced nuclear medicine physicians and reviewed by a third. ROIs were drawn according to the PET Response Criteria in Solid Tumors (PERCIST 1.0). For quantitative assessment of standardized uptake values (SUV). corrected for lean body mass, 3D auto-contour ROI at a 40% threshold of SULpeak technique was used.

Planar whole-body scans after administration of [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ or [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ were performed on a Dual Head Gamma Camera (GE Discovery NM/CT 670) using a high-energy general purpose (HEGP) collimator. Scans were acquired in the lutetium-177 window at a 20% energy window with peaks at 208 and 113 keV. The matrix size for the whole-body scan was 256 × 1024. Anterior and posterior views were acquired by using both detectors.

The clinical study was approved by the Institute Ethics committee at All India Institute of Medical Sciences (IECPG-22/27.02.2020). All patients gave their written informed consent.

3. Results and Discussion

3.1. Organic Synthesis

FAPi-NH₂ 1 was synthesized according to the procedure in the supporting information (cf. Figure S1).

The synthesis of DOTAGA.Glu.(FAPi)₂ 7 and DO3A.Glu.(FAPi)₂ 11 is shown in Figure 2 and was started from FAPi-NH₂ 1 and Boc-Glu-OH 2.

First, 1 was coupled twice to obtain Boc-Glu.(FAPi)₂ 3. Here, HOBt and EDC*HCl with DIPEA in DMF were found to be the best coupling conditions, and 1 needs to be used in slight excess (2.5–3.0 eq). The cleavage of the Boc-protective group can be carried out under very mild conditions with 4 M HCl in 1,4-dioxane (8.6 eq) and acetonitrile as solvent, alternatively using trifluoracetic acid (TFA). The following coupling of the FAPi dimer Glu.(FAPi)₂ 4 with DOTA-tris(tert-butyl ester) 8 and DOTAGA(^tBu)₄ 5 were both carried out with HATU and DIPEA in DMF. For DO3A, only small amounts of product could be found in LC-MS measurements, and no product could be isolated after column chromatography. Therefore, an alternative route was tried out. Compound 8 was first converted into DOTA(^tBu)₃-NHS 9. After coupling with 4 with DIPEA in DMF, the tert-butyl ester groups of the chelator were cleaved and following RP-HPLC purification yielded DO3A.Glu.(FAPi)₂ 11 with 20% over four steps. DOTAGA.Glu.(FAPi)₂ 7 could be obtained with similar yields.

The ^natLu-complexes were obtained by adding 0.1 M LuCl₃ solution (2.0 eq) to 1 M HEPES buffer (pH = 5.5) and the corresponding precursors 7 and 11, respectively. Subsequent RP-HPLC purification gave the Lu-complexed derivatives ^natLu-7 and ^natLu-11.

3.2. Radiosynthesis

3.2.1. [⁶⁸Ga]Ga-DOTAGA.Glu.(FAPi)₂ (⁶⁸Ga-7)

Moon et al. [31] observed that the ⁶⁸Ga-radiolabeling of the dimers DOTA.(SA.FAPi)₂ and DOTAGA.(SA.FAPi)₂ reached better radiochemical conversion (RCC) rates with HEPES buffer compared to ammonium acetate (AmOAc) and sodium acetate (NaOAc). This could also be observed with the new dimers. Labeling of ca. 100 MBq ⁶⁸Ga in 500 µL 1 M AmOAc (pH = 4.5) only gave 57% RCC with 10 nmol DOTAGA.Glu.(FAPi)₂ 7 (cf. Figure S4). Furthermore, 20 nmol was needed to give good yields of >90%. In comparison, 10 nmol 7 and 100 MBq ⁶⁸Ga in 500 µL 1 M HEPES (pH = 4.5) gave >90% after 1.5 min and >95% after 15 min (Figure 3). Similar results were achieved in a fourfold upscale with 400 MBq ⁶⁸Ga. The second TLC system 1 M AmOAc/MeOH (1:1) (RCC = 98.8 ± 0.5%) and Radio-HPLC (RCP = 99.1%, cf. Figure S13) gave confirming results.

The complex stability was determined in human serum (HS) and phosphate-buffered saline (PBS) in triplicate each (n = 3) by incubating ca. 10 MBq of ⁶⁸Ga-7 in 0.5 mL of HS/PBS at 37 °C for 120 min and analyzing via radio-TLC. The complex is stable under these conditions (Figure S5).

3.2.2. [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ (¹⁷⁷Lu-7)

Labeling of 7 with lutetium-177 was carried out in 400 µL 1 M HEPES buffer (pH = 5.5) at 95 °C and gave excellent results with RCC > 99% (1 min) with 2 and 5 nmol 7 and >98% (1 nmol) with just 1 nmol that was given to the buffer solution with 50–100 MBq [¹⁷⁷Lu]LuCl₃ (Figure 4).

In a proportional upscale, this would lead to 10 nmol per GBq lutetium-177. The second radio-TLC system (RCC > 99%) and radio-HPLC (cf. Figure S14: RCP = 99.8%) also showed quantitative labeling without any subsequent purification step to separate free noncomplexed lutetium-177.

The complex stability of ¹⁷⁷Lu-7 was determined similarly to the ⁶⁸Ga-complex ⁶⁸Ga-7, except that the incubation at 37 °C was carried out for 14 days. ¹⁷⁷Lu-7 showed no degradation, and the complex was stable over two half-lives (Figure S6).

3.2.3. [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ (²²⁵Ac-7)

Actinium-225 (t_1/2 = 9.9 d) is part of the neptunium cascade and has no dominant gamma-ray emission lines itself. Therefore, the imaging has to be performed via its daughter nuclides. The gamma lines of francium-221 (t_1/2 = 4.9 min, Eγ = 218 keV) and bismuth-213 (t_1/2 = 45.6 min, Eγ = 440 keV) are the predominant ones that can be used in TLC imaging.

The radio-TLCs were always imaged after one hour and one day. Waiting at least one hour is necessary since the equilibrium between actinium-225 and its daughters is disrupted while running the TLC, and the ²²⁵Ac/²²¹Fr-equilibrium has to be reestablished. At this time point, bismuth-213 also contributed to the TLC imaging, which is why the ²²⁵Ac-RCY/RCC is systematically underestimated at this time point. This was broadly investigated with a statistic model by Kelly et al. [59] where they found that a RCP > 90% after 2 h results in a “true” RCP > 97% after 1 day. After one day, bismuth-213 and all the other daughter nuclides decayed. Imaging at this time point gives the distribution of actinium-225. In gamma spectroscopy the gamma-ray lines of francium-221 and bismuth-213 can be distinguished. The amount of actinium-225 can be calculated via the ²²¹Fr peak; thus, waiting one hour is sufficient in this case.

At first, test labeling experiments were carried out. Therefore, 10 or 20 nmol of 7 were added to a solution of ca. 500 kBq [²²⁵Ac]AcCl₃ and 0.1 M sodium ascorbate (pH = 7.0), which was heated at 95 °C for 60 min (n = 2). For 10 nmol, the RCC was 86.7 ± 3.6% (60 min, 1 d) when imaging after one day. As expected, the RCCs are slightly lower when imaging after one hour, in this case 84.3 ± 4.0% (60 min, 1 h). For 20 nmol, the RCCs were > 90% (15 min) and 91.8 ± 4.7% (60 min, 1 h) and 96.5 ± 1.9% (60 min, 1 d). Exemplary reaction kinetics are shown in Figure S5. It can be seen that at early imaging time points (e.g., 1 h), four spots can be seen, with R_f ≈ 0.5 being free actinium-225 and R_f = 0.0 being the ²²⁵Ac-complex [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ ²²⁵Ac-7. They can also be seen when the exact same TLC is measured again the next day. The other two spots disappear at the radio-TLc after one day. When measuring at additional time points (e.g., 2 h, 3 h etc.), gradually decreasing intensity is observed. This most likely fits the physical half-life of ²¹³Bi and the ability of Bi to form complexes with DOTA conjugates as a trivalent metal. Therefore, R_f > 0.7 is free ²¹³Bi, whereas the spot at R_f = 0.1–0.2 is considered to be [²¹³Bi]Bi-DOTAGA.Glu.(FAPi)₂, also due its gradual increasing intensity during the reaction.

Higher amounts were used for labeling in similar conditions: 1.6–3.2 MBq [²²⁵Ac]AcCl₃ and 30–40 nmol/MBq were heated in 1 mL 0.1 M sodium ascorbate (pH = 7.0) at 95 °C for 60 min (n = 3). Using 40 instead of 30 nmol/MBq did not significantly influence the RCC; hence, the data were merged. The reaction kinetics were analyzed via radio-TLCs (Figure 5) that were imaged one hour after the TLC had been developed, and the exact same TLC was measured again the next day.

Figure 5 shows the underestimation of the imaging after one hour compared to imaging after one day. The maximum RCC of 87.7 ± 3.2% (1 h) and 94.3 ± 2.1% (1 d) is reached after 15 min of heating. The RCC is not quantitative, but increasing to 40 nmol/MBq did not increase RCC. Higher activity concentrations per volume (MBq/mL) might improve the yield, for example by using more activity, less volume or both.

With longer heating times, the RCC decreases again, which might indicate radiolysis. The effect is stronger in the data after one hour. Kelly et al. [59] also found out that the underestimation is not linear and varies depending on the value of RCP/RCC, which might play a role here. In conclusion, the heating should be shortened (15–30 min) in future ²²⁵Ac-labelings of 7. Lower reaction temperatures should also be investigated in future labeling experiments since the solution became slightly yellow after long heating times, which is probably due to the oxidation of ascorbate.

The RCCs were determined with AmOAc (pH = 4.0)/MeOH (1:1) radio-TLC and gamma spectroscopy measurements of a citrate TLC strip match and confirm the results (

Table 1. Comparison of the determined radiochemical conversion rates (RCCs) with the different methods at different time points of the reaction (15 and 60 min: n = 3; after C18 cartridge purification: n = 1). The imaging/measurement was carried out 1 hour (1 h) or 1 day (1 d) after the radio TLC had been developed.

RCC/%	1 h (Citrate)	1 d (Citrate)	1 d (AmOAc/MeOH)	1 h (γ)
after 15 min	87.7 ± 3.2%	94.3 ± 2.1%	-	-
after 60 min	71.3 ± 6.4%	88.9 ± 2.8%	90.8 ± 2.3%	94.8 ± 2.5%
after C18	93.9%	98.1%	97.9%	100%

).

For the complex stability measurements of ²²⁵Ac-7 in HS and PBS, 350–400 kBq of the labeling solution was directly added to HS and PBS (0.5 mL, n = 3) and incubated for 20 days at 37 °C (Figure S9). The complex [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ remains stable in PBS for a couple of days (89.9 ± 0.7% after 2 d), but then, gradual radiolysis occurs (45.7 ± 0.2% after 20 d). In contrast, the stability in HS is much higher despite having the same activity concentration (85.5 ± 1.7% after 20 d). Higher dilution and the addition of sodium ascorbate and ethanol results in a high stability of the pure final formulation (95.4 ± 1.0% after 20 d). Both are known to be scavengers and are able to reduce radiolysis. It seems reasonable to dilute the final product and add scavengers as much as possible, especially for ²²⁵Ac-radiopharmaceuticals due to the high-energy alpha-particles and the high potential for radiolysis.

3.2.4. [⁶⁸Ga]Ga-DO3A.Glu.(FAPi)₂ (⁶⁸Ga-11)

Labeling of DO3A.Glu.(FAPi)₂ 11 with gallium-68 was performed similarly to labeling of 7. Since the generator had much lower activities, cationic post-processing was carried out before labeling [53]. In addition, 1 M HEPES buffer was tested at two different pH values (4.5 and 5.5). Three different precursor amounts were used to label 100 MBq ⁶⁸Ga at each pH value (Figure 6).

Only with 5 nmol of precursor 11 the differences were significant, and pH = 4.5 seems to be better suited than pH = 5.5 (85.9 ± 0.3% vs. 42.4 ± 1.0% after 30 min). For 10 nmol (96.3 ± 2.1% vs. 96.4 ± 0.5%) and 20 nmol (99.1 ± 0.4% vs. 98.7 ± 0.3%), the RCCs were almost identical. This suggests that the pH value of the reaction is flexible to a certain extent. Radio-HPLC (Figure S15) shows RCP = 98.7%.

The complex stability of ⁶⁸Ga-11 was determined analogously to ⁶⁸Ga-7. Almost no radiolysis had taken place, and after 120 min, still > 98% of complexes were intact in both HS and PBS (Figure S10).

3.2.5. [¹⁷⁷Lu]Lu-DO3A.Glu.(FAPi)₂ (¹⁷⁷Lu-11)

[¹⁷⁷Lu]Lu-DO3A.Glu.(FAPi)₂ ¹⁷⁷Lu-11 was synthesized similarly to the DOTAGA derivative ¹⁷⁷Lu-7. When comparing the reaction kinetics in Figure 7 with Figure 4, the labeling kinetics of ¹⁷⁷Lu-11 are somewhat slower than those of ¹⁷⁷Lu-7.

Whereas the RCC is >99% after 1.5 min for 2 nmol 13, it takes 5 min to reach >95% with ¹⁷⁷Lu-11, and even 5 min to obtain RCC > 98% with 5 nmol ¹⁷⁷Lu-11. Nevertheless, ¹⁷⁷Lu-11 can be synthesized in practically quantitative RCCs (>98% with 2 nmol for 30 min; >99% with 5 nmol for 10 min). The second TLC system and radio-HPLC (cf. Figure S16) also gave almost quantitative RCC/RCP.

The complex stability measurements of ¹⁷⁷Lu-11 were performed as described before for ¹⁷⁷Lu-7. Slight decomposition could be observed in PBS (94.7 ± 2.0% intact conjugates after 14 days), whereas ¹⁷⁷Lu-11 seems to be stable in HS, with both having 20 MBq/mL in the beginning (Figure S11).

¹⁷⁷Lu-11 is slightly less stable than ¹⁷⁷Lu-7 since the additional carboxyl group of DOTAGA might be able to take part in the complexation of lutetium-177, which could give it additional stability. Lutetium can form a complex with unconjugated DOTA where all eight donor ligands (4 × N in the macrocyclic + 4 × O⁻ from the acetate arms) are part of the complex [60].

3.2.6. [⁹⁰Y]Y-DO3A.Glu.(FAPi)₂ (⁹⁰Y-11)

DO3A.Glu.(FAPi)₂ 11 was additionally labeled with yttrium-90 in 1 M HEPES buffer (pH = 4.5). For these reaction kinetic studies, 5 and 10 nmol 11 were labeled with 30–60 MBq [⁹⁰Y]YCl₃ in 400 µL buffer at 95 °C. The reaction kinetics were analyzed via radio-TLC and (see Figure 8).

Quantitative RCC > 99% could be achieved with 10 nmol in 15 min. With 5 nmol, the reaction is much slower, but after 45 min, RCC > 99% could also be reached.

Subsequent complex stability studies of ⁹⁰Y-11 were carried out the same way as with ⁶⁸Ga- and ¹⁷⁷Lu-cmplexes. The complex can be considered stable (>98%) in both HS and PBS after more than two half-lives (6 days) at 37 °C (see Figure S12).

3.2.7. Lipophilicity

The lipophilicity was investigated experimentally via the shake-flask method for the ⁶⁸Ga-complexes ⁶⁸Ga-7 and ⁶⁸Ga-11 as well as for the ¹⁷⁷Lu-complexes ¹⁷⁷Lu-7 and ¹⁷⁷Lu-11 with ca. 10 MBq per vial (n = 4) with PBS and 1-octanol. The logD_7.4 values were obtained by determining the integral of each spot on the radio-TLC and calculation with Equation (1).

Table 2. logD_7.4 values of different labeled FAPi-based radiopharmaceuticals. The values for [⁶⁸Ga]Ga-DOTA.SA.FAPi and [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂ are taken from Moon et al. [29] and Moon et al. [31].

Complex	logD7.4
[68Ga]Ga-DOTA.SA.FAPi [29]	−2.68 ± 0.06
[68Ga]Ga-DOTAGA.(SA.FAPi)2 [31]	−2.02 ± 0.06
[68Ga]Ga-DOTAGA.Glu.(FAPi)2 (68Ga-7)	−2.48 ± 0.05
[177Lu]Lu-DOTAGA.Glu.(FAPi)2 (177Lu-7)	−2.77 ± 0.10
[68Ga]Ga-DO3A.Glu.(FAPi)2 (68Ga-11)	−2.08 ± 0.07
[177Lu]Lu-DO3A.Glu.(FAPi)2 (177Lu-11)	−1.77 ± 0.10

gives an overview and compares the logD_7.4 values with those of previous works from our group [29,31].

3.2.8. Comparison with DOTAGA.(SA.FAPi)₂

The monomeric radiotracer [⁶⁸Ga]Ga-DOTA.SA.FAPi showed relatively strong hydrophilic characteristics. This may be one of the reasons that in vivo PET studies indicated a relatively fast renal clearance [58]. In contrast, the dimer [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ predominantly showed biliary excretion over renal excretion [39]. The washout from the hepatobiliary system/liver was slower than that of the monomeric radiotracers (the same for the renal excretion). In particular, [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ remained in the large intestines/colon region for a longer time during excretion. The colon was identified as the critical organ, and the radiation dose to the kidneys was increased compared to the monomer [39,52]. Therefore, we tried to synthesize compounds that are more hydrophilic than DOTAGA.(SA.FAPi)₂ to reduce biliary excretion and thus the radiation dose to the colon.

The DOTAGA complexes ⁶⁸Ga-7 and ¹⁷⁷Lu-7 are definitely more hydrophilic than the first-generation dimer DOTAGA.(SA.FAPi)₂ and the DOTA complexes ⁶⁸Ga-11 and ¹⁷⁷Lu-11. The hydrophilicity of ⁶⁸Ga-7 is comparable to the monomer [⁶⁸Ga]Ga-DOTA.SA.FAPi. Radioligand therapy (RLT) with radiometals such as lutetium-177 is the main objective of these new dimers. [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ ¹⁷⁷Lu-7 shows the highest hydrophilicity of all complexes (logD_7.4 = −2.77 ± 0.10). There is no logD_7.4 value for [¹⁷⁷Lu]Lu-DOTA.SA.FAPi that allows for a direct comparison. Nonetheless, these results are very promising to translate this molecule from bench to bedside.

The molecular design of the FAPi homodimers DOTA.(SA.FAPi)₂ and DOTAGA.(SA.FAPi)₂ may create radiolabeling limitations due to steric hindrance as a result of the chelator being in the center of the molecule. The DOTA chelator only has six donor ligands because it is attached to the two targeting vectors on two binding sites, which only allow for labeling with gallium-68 [31]. The DOTAGA derivative has seven donor ligands for complexation and can be labeled with lutetium-177, while it was cumbersome in the case of actinium-225. The new FAPi dimer DOTAGA.Glu.(FAPi)₂ 7 has eight donor ligands and can be labeled with actinium-225 with high RCC > 90%. ²²⁵Ac-labeling of DO3A.Glu.(FAPi)₂ 11 was just as problematic as with DOTAGA.(SA.FAPi)₂. In general, DOTA-conjugated precursors have been investigated for ²²⁵Ac-labeling quite extensively and allow for efficient labeling when the chelator is in exo-position [61,62,63,64,65,66].

In summary, DOTAGA.Glu.(FAPi)₂ showed superior labeling properties compared to the other dimeric compounds.

3.3. In Vitro Inhibition Assays

High affinity for FAP and low affinity for the very similar proteases PREP and DPPs (DPP4, DPP8 and DPP9) are important requirements for FAPi-based radiopharmaceuticals to be promising candidates for subsequent in vivo studies.

The IC₅₀ values were determined in in vitro studies.

Table 3. IC₅₀ value and selectivity index (SI) of different FAP inhibitors: DOTAGA.Glu.(FAPi)₂ 7, [^natLu]Lu-DOTAGA.Glu.(FAPi)₂ ^natLu-7, DO3A.Glu.(FAPi)₂ 11 as well as DOTAGA.(SA.FAPi)₂, [^natLu]Lu-DOTAGA.(SA.FAPi)₂ and the initial FAP inhibitor UAMC-1110 from previous works (Jansen et al. [24] and Moon et al. [31]).

Compound	DOTAGA.Glu.(FAPi)2 (7)	[natLu]Lu-DOTAGA.Glu.(FAPi)2 (natLu-7)	DO3A.Glu.(FAPi)2 (11)	DOTAGA.(SA.FAPi)2 [31]	[natLu]Lu- DOTAGA.(SA.FAPi)2 [31]	UAMC-1110 [24,31]
IC50(FAP)/nM	0.26 ± 0.04	0.33 ± 0.02	0.60 ± 0.04	0.92 ± 0.06	1.54 ± 0.15	0.43 ± 0.02
IC50(PREP)/µM	0.59 ± 0.10	0.43 ± 0.16	1.00 ± 0.14	0.39 ± 0.02	0.56 ± 0.04	1.80 ± 0.01
IC50(DPP4)/µM	1.19 ± 0.08	0.65 ± 0.04	0.54 ± 0.06	0.40 ± 0.07	0.63 ± 0.07	>10
IC50(DPP8)/µM	0.029 ± 0.004	0.22 ± 0.02	1.03 ± 0.18	0.42 ± 0.04	0.41 ± 0.03	>10
IC50(DPP9)/µM	0.083 ± 0.0015	0.19 ± 0.01	0.95 ± 0.11	0.16 ± 0.02	0.18 ± 0.02	4.70 ± 0.40
SI(PREP/FAP)	2269	1292	1667	424	364	4186
SI(DPP4/FAP)	4577	1967	900	435	409	23,256
SI(DPP8/FAP)	112	661	1717	456	266	23,256
SI(DPP9/FAP)	319	557	1583	174	117	10,930

summarizes the results of the IC₅₀ measurements. The selectivity index (SI) is the ratio of the IC₅₀ value of the respective protease (PREP, DPP4, DPP8 and DPP9) to the IC₅₀ value of FAP.

Compounds 7, ^natLu-7 and 11 show very high affinity for FAP, as reflected in the subnanomolar IC₅₀ values. The affinities of 7 and ^natLu-7 are very similar, and the different selectivity indexes do not differ substantially as well. This supports previous results where the IC₅₀ values of the precursor and its respective metal complex (^natGa or ^natLu) do not differ much [29,30,31], as can be seen with DOTAGA.(SA.FAPi)₂ and [^natLu]Lu-DOTAGA.(SA.FAPi)₂ for example (see Table 3) [31]. The affinity and selectivity for all new compounds were better than for DOTAGA.(SA.FAPi)₂. This also includes the comparison of the Lu-complex ^natLu-7 with [^natLu]Lu-DOTAGA.(SA.FAPi)₂.

Although selectivity indexes (SIs) are lower compared to UAMC-1110, high selectivity for FAP over PREP and DPP4 was measured, which are most relevant among the related proteases. In contrast to FAP, they are ubiquitously expressed in healthy tissue. A high affinity for PREP or DPP4 would lead to worsened tumor selectivity and a diminished tumor-to-background ratio in PET/SPECT scans and/or RNT. Overall, these results strongly suggest that compounds 7 and 11 might also have superior properties in vivo compared to DOTAGA.(SA.FAPi)₂ and that especially FAPi dimer 7 is suitable for further investigations.

The IC₅₀(FAP) values reported for BiOncoFAP-DOTAGA and [^natLu]Lu-BiOncoFAP-DOTAGA were 0.17 and 0.19 nM, respectively [50]. These values seem to be equal to 7 and ^natLu-7, although it has to be noted that the assay conditions were different (substrate/enzyme/buffer concentration, temperature, volume etc.). For the dimer [⁶⁸Ga]Ga-DOTA-2P(FAPI)₂, the assay setup differed drastically, as a competitive assay of the radiolabeled agent against FAPI-46 was carried out and resulted in an IC₅₀ = 3.68 ± 1.82 nM [48]. The different assay conditions, especially of Zhang et al. [48], limit the comparability of the different IC₅₀ values. On top, no IC₅₀ values for the related proteases and therefore no selectivity indexes were reported by Zhao et al. [48] or by Galbiati et al. [50].

3.4. Patient Study (Medullary Thyroid Cancer)

A 40-year-old male was diagnosed with medullary thyroid cancer in 2016. The patient underwent total thyroidectomy and bilateral radical neck dissection in prior treatments.

[¹⁸F]F-FDG and [⁶⁸Ga]Ga-DOTA.SA.FAPi PET/CT scans were carried out. [¹⁸F]F-FDG PET/CT (see Figure 9a) showed an FDG-avid soft tissue lesion noted measuring 3 × 3.5 cm in the thyroid bed region (SULpeak: 3.4), avid bilateral level VI, bilateral supraclavicular lymph nodes noted, sub-centimetric tracer avid level V lymph nodes, superior mediastinal/bilateral upper paratracheal, and bilateral paraoesophageal lymph nodes. Few hypodense lesions were noted in the right lobe of the liver involving segment V with low [¹⁸F]F-FDG uptake of 1.75. Diffuse physiological FDG distribution was seen in the axial and appendicular skeleton. Unlike [¹⁸F]F-FDG, [⁶⁸Ga]-DOTA.SA.FAPi showed superior uptake in all the lesions (soft tissue neck lesion: SULpeak 17.98) corresponding to [¹⁸F]F-FDG PET/CT scan and better delianiation in the liver lesions (SULpeak:10.5) on MIP itself (see Figure 9b). The patient was administered two cycles of [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂. Figure 9c shows whole-body scans at 24 and 48 h post-injection. Despite uptake in the lesions (neck nodes and mass), huge amounts of radiotracer uptake were observed in the liver (24 h p.i.) and colon (48 h p.i.), causing high radiation burden to the colon and liver. For the third cycle of treatment, the new FAPi dimer [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ ¹⁷⁷Lu-7 was considered.

The patient was administered 5.5 GBq of [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ ¹⁷⁷Lu-7, which was previously synthesized according to the procedure described in Methods and Materials (Section 2.5.1). Furthermore, 24 h p.i. high uptake in the neck and mediastinal metastases was comparable to the first-generation dimer. In contrast, only negligible radiotracer uptake in the liver and colon was observed. This suggests a different excretion pattern, possibly a higher proportion of renal instead of hepatobiliary excretion (improved pharmacodynamic profile) or a faster washout (improved pharmacokinetic profile), or both, which results in a significantly reduced radiation dose to the critical healthy organs liver and colon.

While this is a first proof-of-concept investigation and not of quantitative dimension, the results support the assumption that the new ¹⁷⁷Lu-labeled FAPi homodimer ¹⁷⁷Lu-7 has favorable pharmacologic properties compared to [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂. More detailed studies regarding biodistribution as well as safety and efficacy are currently ongoing, which are needed to better understand the pharmacokinetic and pharmacodynamic behavior of [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ ¹⁷⁷Lu-7 in systematic studies.

4. Conclusions

DOTAGA.Glu.(FAPi)₂ and DO3A.Glu.(FAPi)₂ represent the second generation of our homodimeric FAPi compounds. They were synthesized with glutamic acid as a central linker unit and successfully labeled with different trivalent radiometals. Both precursors show excellent labeling properties with ⁶⁸Ga and ¹⁷⁷Lu. In contrast to DO3A.Glu.(FAPi)₂ and the first-generation dimer DOTAGA.(SA.FAPi)₂, DOTAGA.Glu.(FAPi)₂ could also be labeled successfully with ²²⁵Ac in high yields. All complexes showed high stability and hydrophilicity. Beyond that, IC₅₀ values were determined to confirm the very high affinity for fibroblast activation protein alpha (FAP) and high selectivity for FAP over prolyl endopeptidase (PREP) and the dipeptidyl peptidase (DPP4). The in vitro studies showed the best results for DOTAGA.Glu.(FAPi)₂, but the results for DO3A.Glu.(FAPi)₂ were also better than for DOTAGA.(SA.FAPi)₂. It is also noteworthy that the respective FAP affinity of the new homodimeric compounds is higher than for the initial FAP inhibitor UAMC-1110 itself, which was measured in the same assay. Similar FAP affinities were reported for BiOncoFAP-DOTAGA and its ^natLu-complex by Galbiati et al. [50], but no selectivity data were reported. The comparison of the IC₅₀ values is difficult and should be cautiously interpreted, as the inhibition assays are different from each other (especially regarding Zhao et al. [48]).

DOTAGA.Glu.(FAPi)₂ proved to be the most promising candidate for further in vivo studies that are currently ongoing. The compound seems especially suited for therapeutic applications with ¹⁷⁷Lu, ⁹⁰Y and ²²⁵Ac. Especially, FAP-targeted alpha-particle therapy (FAP-TAT) with ²²⁵Ac-radiopharmaceuticals is a new treatment option that could potentially be more efficacious than with beta-emitting therapeutics since stromal cells, similar to how CAFs are reported to be more resistant to radiation [67,68,69]. Therefore, particles with higher energy and linear energy transfer such as alpha particles might be needed to address the specifics of the tumor stroma. The stroma can build a physical barrier around the tumor that is hard to penetrate for some agents (e.g., chemotherapeutics), which results in poor efficacy [5,70,71]. The destruction of tumor stroma with alpha-emitters in combination with other non-radioactive agents (e.g., chemotherapeutics, targeted drug delivery systems, CAR T-cell therapy etc.) might also be a promising therapy approach, and [²²⁵Ac]Ac-DOTAGA.Glu.(FAPi)₂ might be an interesting candidate for FAP-TAT in the future.

A first proof-of-concept patient study with [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ showed promising results. The pharmacological properties are superior to those of [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂. with a high radiotracer uptake and long tumor retention in lesions. At the same time, the excretion kinetics are faster, and minimized uptake in non-target tissues, especially the colon as the critical organ for [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂, was observed. Improved pharmacologic properties are essential to ensure a safe therapy without adverse side effects. Further studies are currently ongoing and will investigate biodistribution, efficacy and safety in more detail.