[99mTc]Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience

Purpose The clinical success non-invasive imaging of CXCR4 expression using [68 Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of 99mTc-labeled cyclic pentapeptides based on the PentixaFor scaffold. Methods Six mas3-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa3 linkers (L1–L6) as well as the corresponding HYNIC- and N4-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC50 and IC50inv) were carried out using Jurkat T cell lymphoma cells and [125I]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [99mTc]Tc-N4-L6-CPCR4 ([99mTc]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [99mTc]Tc-N4-L6-CPCR4 SPECT/planar imaging with individual dosimetry. Results Of the six mas3-conjugated peptides, mas3-L6-CPCR4 (mas3-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC50 = 5.0 ± 1.3 nM). Conjugation with N4 (N4-L6-CPCR4) further improved hCXCR4 affinity to 0.6 ± 0.1 nM. [99mTc]Tc-N4-L6-CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 ± 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [99mTc]Tc-N4-L6-CPCR4 (termed [99mTc]Tc-PentixaTec) was selected for first-in-human application. [99mTc]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1–3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging. Conclusion The successive optimization of the amino acid composition of the linker structure and the N-terminal 99mTc-labeling strategies (mas3 vs HYNIC vs N4) has provided [99mTc]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00259-023-06395-x.

In all of these indications, [ 68 Ga]Ga-Pentixafor was shown to allow for sensitive and high-contrast positron emission tomography (PET).To date, it is the only CXCR4-targeted imaging agent that has achieved broad clinical applicability so far [16][17][18].However, given the continuously expanding field of clinical indications for non-invasive in vivo CXCR4 visualization and quantification, development of a ligand amenable to radiolabeling with gamma emitters (such as 99m Tc) for use in conventional scintigraphy or single photon emission computed tomography (SPECT) with its lower costs and general availability would be highly desirable.
Our own experience with PentixaFor-based radioligands, however, has consistently demonstrated that even minor modifications of the peptide scaffold (either directly at the D-Orn side chain or at the far end of the D-Orn-AMBS-moiety) may lead to substantial losses in CXCR4 binding affinity [23][24][25].Thus, implementation of a Tc-labeling strategy based on the PentixaFor-backbone represents a challenge, since the required structural modifications to accommodate 99m Tc-chelation are usually quite extensive [26].In a previous study on PentixaForand PentixaTher-based DOTA-conjugated tracers, replacement of the AMBS-by a Abz-a-r-linker had emerged as a powerful tool to improve ligand affinity and internalization.Furthermore, the flexibility of the tracer scaffold towards structural modifications at the far end of the linker was substantially improved [24].We therefore selected the CPCR4-Abz-ar scaffold as a starting point for the development of novel 99m Tc-labeled analogs with further optimized linkers.Upon selection of the most promising peptide-linker construct using mas 3 -based labeling chemistry, other 99m Tc-labeling methods (HYNIC, N 4 ) were also implemented, and a comparative indepth preclinical characterization was performed.Based on the particularly promising in vitro and in vivo characteristics of [ 99m Tc]Tc-N 4 -dap-r-a-Abz-CPCR4 ([ 99m Tc]Tc-N 4 -L6-CPCR4, [ 99m Tc]Tc-PentixaTec), we also report its first in human application in four patients with hematological malignancies.

Precursor synthesis
All compounds in this study were synthesized by combining solid phase peptide synthesis (SPPS) using a standard Fmocprotocol and solution phase fragment condensation strategies.Briefly, the pentapeptide backbone CPCR4 was synthesized according to a literature protocol [27] and then functionalized with the corresponding linkers (L1-L6) and respective Tcchelators mas 3 , HYNIC and N 4 as described in detail in the supplementary information.

Radiolabeling
Labeling with 99m Tc was carried out using kit-like lyophilized reaction vials (see supplementary material), based on established labeling protocols for mas 3 - [28], HYNIC- [29], and N 4 -conjugated peptide tracers [30] with minor modifications.Details are provided in the supplementary material.

Determination of lipophilicity (logD)
The lipophilicity (logD, partition coefficient between n-octanol and PBS, pH 7.4) of all 99m Tc-labeled compounds in this study was determined using a modified shake-flask method as described [31].

Determination of inv-IC 50 (hCXCR4)
For the determination of the "inverse IC 50 " (inv-IC 50 ) of the 99m Tc-labeled compounds in this study, the same experimental protocol as used for the IC 50 -determination was employed.However, the respective 99m Tc-labeled peptide of interest was used as radioligand (0.2 nM), and unlabeled FC-131 was used as a standard competitor.Both IC 50 values and inv-IC 50 values were calculated using GraphPad Prism 6 (GraphPad Software Inc., San Diego, USA).

Dual tracer internalization studies
Dual tracer internalization studies using [ 125 I]FC-131 as internal reference were conducted in hCXCR4 expressing Chem-1 cells in analogy to a previously established protocol [34].A detailed assay protocol is provided in the supplementary material section.

In vivo evaluation
All animal experiments were conducted in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals.

Biodistribution studies
Female CB17/SCID mice bearing subcutaneous Jurkat human T cell lymphoma xenografts (for more detailed information, see supplemental material) were injected intravenously with the respective 99m Tc-labeled tracer (5-20 MBq, 0.1-0.2nmol/mouse) under isofluorane anesthesia and were sacrificed at 1 h post-injection (p.i.).The organs of interest were dissected, and the activity concentration in weighed tissue samples was quantified using a WIZARD 2 ® 2480 automatic γ-Counter from Perkin Elmer (Waltham MA, USA).Biodistribution data are given in %iD/g and represent means ± SD (groups of n = 4-5 animals).Statistical analysis (t-test) was performed using GraphPad Prism.

Small animal SPECT/CT imaging
SPECT/CT imaging was performed on a VECTor 4 smallanimal SPECT/PET/OI/CT scanner (MILabs BV, Utrecht, The Netherlands).Static images were acquired with 45-min acquisition time using the HE-GP-RM collimator and a stepwise multi-planar bed movement.All images were reconstructed using the MILabs Rec software (version 10.02) and a pixel-based Similarity-Regulated Ordered Subsets Expectation Maximization (SROSEM) algorithm with a window-based scatter correction (20% below and 20% above the photopeak, respectively; voxel size CT: 80 µm, voxel size SPECT: 0.8 mm, 1.6 mm (FWHM) Gaussian blurring post-processing filter, with calibration factor in kBq/mL and decay correction, no attenuation correction).Image analysis was carried out using PMOD 4.0 (PMOD TECHNOLO-GIES LLC, Fällanden, Switzerland).

Patients
The use of [ 99m Tc]Tc-PentixaTec was in compliance with the German Medicinal Products Act, AMG §13 No 2b and reported to the regulatory office (Regierung von Oberbayern).The analysis of patient data was approved by the Ethics Committee of Ludwig-Maximilians Universität München (permit 22-0850).
Five patients (age, 48-72 y, median 66 y) with a history of hematological malignancy underwent imaging after intravenous injection of a median of 558 MBq (range, 417-601) [ 99m Tc]Tc-PentixaTec.One patient suffered from marginal zone lymphoma, one from B-NHL, and the remaining three individuals had a history of multiple myeloma.Safety was assessed by monitoring adverse events after administration of [ 99m Tc]PentixaTec.

Human [ 99m Tc]Tc-PentixaTec gamma camera imaging
[ 99m Tc]Tc-PentixaTec scans were performed using a dual head gamma camera (GE Discovery NM/CT 670 Pro, Milwaukee, WI, USA).In the four patients undergoing dosimetry, planar dynamic whole body scans were performed 5 min, 30 min, and 1 h with a bed speed of 30 cm/min; 2 h, 3 h, and 5 h with a speed of 12 cm/min; and 24 h with a speed of 5 cm/min after tracer injection.SPECT/CT imaging was performed following the 3-h planar imaging with 60 views and a frame duration of 8 s.Attenuation maps were generated on the basis of low-dose CT.

Blood clearance
Venous blood samples (2.7 mL) were drawn directly before each planar scan.A cross-calibrated well-counter (Digibase, Ametek, Oak Ridge, TN, USA) was used to measure activity in the blood samples, resulting in the average activity per mL blood.Plasma half-life was calculated using NUKFIT software [35].

Radiation dosimetry and biodistribution
Radiation dosimetry calculations were performed using the RADAR (Radiation Dose Assessment Resource) [36] method as implemented in the OLINDA/EXM software and according to the recommendations of the Medical Internal Radiation Dose (MIRD) committee [37].The individual absorbed organ doses (ODs) and the effective doses (EDs) were corrected to consider the current tissue weighting factors of ICRP 103 [38].For a detailed description of image analysis for dosimetry, please refer to the supplementary materials section.

Identification of a suitable linker unit
In a first step, the effect of direct mas 3 -functionalization of the r-a-Abz-CPCR4 backbone on CXCR4 affinity was investigated.Compared to the corresponding nat Ga/ nat Lu-complexes of DOTA-r-a-Abz-CPCR4, which had shown CXCR4 affinities of 0.4 ± 0.1 and 1.5 ± 0.1 nM, respectively [24], the affinity of mas 3 -r-a-Abz-CPCR4 (mas 3 -L1-CPCR4), despite being in the same range as the clinically used reference ligands nat Ga-PentixaFor and nat Lu-PentixaTher (Table 1), was substantially lower.To investigate if the interaction with the CXCR4 binding pocket may be improved by a linker extension, additional neutral (D-Ala = a), aromatic (D-Phe = f), or cationic (D-Arg = r, D-His = h, D-Dap = dap) amino acids were introduced (Table 1).Of all modifications, only small cationic side chains (h and dap in L5 and L6, respectively) were tolerated without compromising CXCR4affinity.The fourfold increased affinity of mas 3 -L6-CPCR4 compared to mas 3 -L1-CPCR4 prompted its further preclinical evaluation.Furthermore, the L6-CPCR4 construct was chosen for further functionalization with HYNIC and N 4 , allowing a side-by-side comparison of alternative 99m Tclabeling strategies.

Synthesis of L6-CPCR4-based chelator conjugates
Synthetically, all peptides (Tables 1 and 2) were accessible via a combined solid phase peptide synthesis/solution phase fragment condensation strategy (for details, see supplementary material).Since the conjugation of the complete Abz-a-r-Xmas 3 constructs to the CPCR4 backbone (as employed for peptides mas 3 -L3/L4/L5-CPCR4) was found to be inefficient, two successive coupling steps were employed for the synthesis of all other peptides.For the peptides mas 3 /HYNIC/N 4 -L6-CPCR4, this involved attachment of the Fmoc-protected Abz-a-r(Pbf)-dap(Boc)-linker (L6) to the peptide core as a first step, and, after Fmoc deprotection and purification of the CPCR4-L6-scaffold, functionalization with the corresponding technetium-chelator, followed by final cleavage of all acidlabile protecting groups.In the case of HYNIC-L6-CPCR4, the formation of trifluoroacetyl-HYNIC as a side product during the last deprotection step limited the yield of the isolated product to 3.4% based on L6-CPCR4.During the synthesis of N 4 -L6-CPCR4, pre-activation of (Boc) 4 N 4 -COOH with HOAt/HATU and the use of 2,4,6-collidine as a base was found to successfully prevent the previously observed elimination of Boc-protected 1,2-ethylenediamine from the (Boc) 4 N 4 moiety during the condensation reaction.N 4 -L6-CPCR4 was thus obtained in reasonable yields of 12-17% based on L6-CPCR4 after RP-HPLC purification.

Radiolabeling
Both [ 99m Tc]Tc-mas 3 -L6-CPCR4 and [ 99m Tc]Tc-N 4 -L6-CPCR4 were consistently obtained in > 95% radiochemical yield and radiochemical purity, respectively, using prefabricated, lyophilized radiolabeling kits.For in vitro and in vivo applications, the radiolabeling products were thus only diluted with saline or PBS and sterile filtered without the requirement for further purification.In contrast, 99m Tc-labeling yields for [ 99m Tc]Tc-HYNIC-L6-CPCR4 remained below 50% (n = 7), necessitating cartridge purification before further use.Interestingly, radiolabeling yields for the HYNIC-containing reference compound [ 99m Tc] Tc-HYNIC-CPCR4 ([ 99m Tc]Tc-CXCR4-L [21,22]) were always > 85%, suggesting a potential influence of the adjacent amino acid side chains of the linker unit in [ 99m Tc] Tc-HYNIC-L6-CPCR4 on the labeling efficiency.Cationpi interactions between the cationic D-Dap-and/or D-Argside chain and the aromatic system in HYNIC may lead to a decreased electron donating capacity of the HYNIC moiety and thus contribute to the observed reduction in 99m Tc-labeling efficiency [39].
For this reason, we directly determined the CXCR4-affinity of the respective 99m Tc-labeled peptides (and of [ 177 Lu]Lu-PentixaTher as reference) using an inverse IC 50 setup (inv-IC 50 ).Here, the competition binding study is performed using a fixed concentration of the respective radioligand of interest (0.2 nM) and FC-131 as standard competitor.Under these conditions, a higher absolute inv-IC 50 corresponds to a higher CXCR4 affinity of the radioligand, since more unlabeled FC-131 is needed to replace receptor-bound tracer.
In accordance with its pronounced hydrophilicity (Table 2), [ 99m Tc]Tc-HYNIC-L6-CPCR4 showed the most rapid overall background clearance, with the liver, the kidneys, and the tumor being the only organs with an activity accumulation above background level (p < 0.0001).This is also reflected by the excellent imaging contrast observed in small animal [ 99m Tc]Tc-HYNIC-L6-CPCR4 SPECT/CT in a Jurkat tumor bearing mouse (Fig. 2).In contrast, for the most lipophilic compound in this series, [ 99m Tc]Tc-mas 3 -L6-CPCR4, with its borderline logD of − 1.54, increased background activity levels in all organs were observed (Table 3, Fig. 2).Although most of the tracer is renally excreted, the almost threefold higher intestinal activity uptake of [ 99m Tc]Tc-mas 3 -L6-CPCR4 compared to [ 99m Tc] Tc-HYNIC-L6-CPCR4 (p < 0.0001) and [ 99m Tc]Tc-N 4 -L6-CPCR4 (p < 0.0001) hints towards a certain degree of hepatobiliary clearance, which generally is deemed an unfavorable feature for high-contrast imaging, especially at later time points.Of the compounds investigated, [ 99m Tc]Tcmas 3 -L6-CPCR4 shows the lowest tumor uptake (Table 3, Fig. 2), which is in line with its lower CXCR4 affinity compared to the other two tracers in this study.
In accordance with its particularly high CXCR4 affinity and internalization efficiency, [ 99m Tc]Tc-N 4 -L6-CPCR4 shows enhanced tumor accumulation compared to its mas 3and HYNIC-analogs.However, this difference is not as marked as anticipated (p = 0.002 and p = 0.5, respectively).Conversely, all tissues known to be mCXCR4-positive, i.e., lung, liver, and spleen [43], show notable [ 99m Tc]N 4 -L6-CPCR4 uptake.We had previously encountered the same effect for [ 177 Lu]Lu-DOTA-r-a-Abz-iodoCPCR4 in direct comparison with [ 177 Lu]Lu-PentixaTher at 1 h p.i. [24].The unexpectedly low tumor accumulation of the [ 177 Lu] Lu-DOTA-r-a-Abz-analog (based on its in vitro characteristics) was attributed to a "sink effect" in mCXCR4-positive organs, especially the liver, due to the > 11-fold higher mCXCR4 affinity of [ 177 Lu]Lu-DOTA-r-a-Abz-iodoCPCR4 compared to [ 177 Lu]Lu-PentixaTher.To confirm if this is also the case for [ 99m Tc]Tc-N 4 -L6-CPCR4, we additionally determined its mCXCR4 affinity in HEK cells stably transduced with mCXCR4, using [ 125 I] CPCR4.3 as radioligand.Compared to previous experiments using mCXCR4 overexpressing Eµ-myc 1080 mouse lymphoma cells [24,33], the absolute IC 50 value for the reference nat Lu-PentixaTher in the new experimental setup was fivefold higher (IC 50 = 2568 ± 114 nM vs 567 ± 62 nM, respectively).With an IC 50 of 66.4 ± 16.3 nM, N 4 -L6-CPCR4 has thus an almost 40-fold higher mCXCR4 affinity than nat Lu-PentixaTher, which is even higher than that of the [ 177 Lu]Lu-DOTA-r-a-Abzligand cited above [24].This corroborates the assumption that a significant proportion of [ 99m Tc]Tc-N 4 -L6-CPCR4 in the lung, liver, and spleen is mCXCR4 specific, as has also been demonstrated the a blocking experiment (coinjection of 50 µg AMD3100) shown in Fig. 2. Thus, importantly, the observed high liver uptake of [ 99m Tc]Tc-N 4 -L6-CPCR4 in mice will most probably not be predictive for the human situation, because hCXCR4 is not expressed in the normal liver [44].
Despite this non-negligible on-target-off-site accumulation of [ 99m Tc]Tc-N 4 -L6-CPCR4, however, its tumor-tobackground ratios for all non-target organs are comparable to those obtained for the more hydrophilic analog [ 99m Tc] Tc-HYNIC-L6-CPCR4 (Supplementary Fig. 1), and the observed minor differences are statistically not significant.Thus, since [ 99m Tc]Tc-N 4 -L6-CPCR4 showed the highest CXCR4 affinity, internalization, and tumor accumulation of the novel 99m Tc-labeled CPCR4-analogs in this study and was reliably obtained in high radiochemical yields using a kit-like procedure, [ 99m Tc]Tc-N 4 -L6-CPCR4 (termed [ 99m Tc]Tc-PentixaTec for the remainder of the manuscript) was selected as lead compound for clinical translation.

In vitro and in vivo stability of [ 99m Tc]Tc-PentixaTec
In preparation for the early translation to the human setting, in vitro and in vivo stability of [ 99m Tc]Tc-PentixaTec in various media was assessed (see Supplementary Fig. 2).Only when diluted with saline and under challenging conditions (citrate and DTPA), progressive decomposition of the radiopharmaceutical was observed.In contrast, [ 99m Tc] Tc-PentixaTec was found to be fully stable in all physiological media (whole blood, serum, human serum albumin (2.5 and 5%)) over an extended period of time (13 h).In the original product formulation, the percentage of intact [ 99m Tc]Tc-PentixaTec slowly decreased from 94.5 to 90.6% within 2.5 h, suggesting that the tracer should ideally be administered within 2 h after the end of synthesis to ensure maximum radiochemical purity of the tracer.

Proof-of-concept clinical [ 99m Tc]Tc-PentixaTec SPECT/CT imaging
In a next step, CXCR4-directed scintigraphy with [ 99m Tc] Tc-PentixaTec was performed in five patients with a history of hematologic disease.Out of those, four individuals underwent dedicated dosimetry.Imaging was well tolerated and no adverse events were recorded.In all patients, [ 99m Tc]Tc-PentixaTec was rapidly cleared from the blood pool with a median plasma half-life of 19 min (range 15-25 min, see Supplementary Fig. 3) and exhibited a very favorable in vivo distribution with no relevant background accumulation.Of note, as compared to [ 99m Tc]Tc-CPCR4-L, [ 99m Tc]Tc-PentixaTec demonstrated no significant hepatobiliary excretion.An example of clearance kinetics and normal biodistribution of [ 99m Tc]Tc-Pen-tixaTec over 24 h is depicted in Fig. 3. Figure 4 provides an illustrative comparison between [ 18 F]FDG-PET/CT and [ 99m Tc]Tc-PentixaTec scintigraphy/SPECT/CT in a patient with B cell non-Hodgkin's lymphoma who underwent chemokine receptor imaging for assessment of potential CXCR4-directed radioligand therapy.

[ 99m Tc]Tc-PentixaTec patient dosimetry
Table 4 summarizes the calculated absorbed organ doses for four patients (the remaining subject with B-NHL did not undergo dedicated dosimetry).The spleen as a pool for CXCR4 expressing blood cells received the highest absorbed dose with a median value of 1.95E − 02 mGy/MBq (range, 1.61E − 02-2.38E− 02), followed by the kidneys (median Injection of a standard activity of 500 MBq [ 99m Tc]Tc-PentixaTec resulted in a median effective dose of 2.7 mSv (range, 2.1-3.4).Therefore, [ 99m Tc]Tc-PentixaTec compares favorably to established PET agents such as [ 68 Ga] Ga-PentixaFor or [ 68 Ga]Ga-NOTA-NFB for which imaging with a standard activity of 150 MBq has been reported to result in effective doses of 2.3 mSv [45] and 3.8 mSv [46], respectively.

Conclusion
In this study, both the role of the structural composition of the Abz-a-r-(aa 3 ) linker, connecting the CPCR4 peptide core and the radiolabel, and the influence of the radiolabeling strategy (mas 3 vs HYNIC vs N 4 ) on the overall in vitro and in vivo characteristics of a given CPCR4-linker-construct (L6-CPCR4) were investigated in detail.Our results demonstrate that positioning a small cationic amino acid at the far end of the linker (aa 3 = dap, L6) greatly improves CXCR4 affinity and the flexibility towards N-terminal structural modifications (different chelators) in CPCR4-Abz-a-r-aa 3 -based peptides.Of all compounds investigated, [ 99m Tc]Tc-N 4 -L6-CPCR4 ([ 99m Tc]Tc-PentixaTec) showed the most promising overall in vitro and in vivo characteristics, combining a robust, clinically translatable radiolabeling chemistry, high in vitro stability, a suitable lipophilicity, excellent CXCR4 targeting and internalization, both in vitro and in vivo, and an appropriate pharmacokinetic profile, allowing high-contrast visualization of CXCR4 expressing tissues at early time points.In a first proof-of-concept human application in a group of five patients with hematological cancers, [ 99m Tc] Tc-PentixaTec was well tolerated, allowed high-contrast delineation of CXCR4 expressing tumors, and exhibited a favorable overall biodistribution and dosimetry profile.Further clinical research to consolidate the value of [ 99m Tc]Tc-PentixaTec SPECT/planar imaging as an alternative modality to CXCR4-targeted PET is therefore highly warranted.

Fig. 2
Fig. 2 Maximum-intensity projection images obtained from static SPECT/CT imaging of Jurkat xenograft-bearing CB17 SCID mice at 1 h p.i.; solid arrows indicate tumor, dashed arrows indicate the liver, and pointed arrows indicated the kidneys

Fig. 3 Fig. 4
Fig. 3 Biodistribution and clearance kinetics of [ 99m Tc]Tc-PentixaTec in a patient with a history of multiple myeloma and a suspicious finding in the right tibia in [ 18 F]FDG PET/CT.CXCR4-directed whole-

Table 1
CXCR4 affinities of the novel mas 3 -conjugated ligands compared to selected reference compounds

Table 2
[21,22]ffinities, internalization (in % of the reference ([ 125 I]FC-131)), and lipophilicities (logD) of the novel ligands and selected reference compounds *Specific internalization (total internalized activity corrected by internalization in the presence of 10 µM AMD3100) of the reference compound [ 125 I]FC-131 was determined in the respective dual tracer experiment and used for data normalization # Name in the literature[21,22]: CXCR4-L