Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.

Lessons learned in application driven imaging agent design for image-guided surgery.

To meet the growing demand for intraoperative molecular imaging, the development of compatible imaging agents plays a crucial role. Given the unique requirements of surgical applications compared to diagnostics and therapy, maximizing translational potential necessitates distinctive imaging agent designs. For effective surgical guidance, exogenous signatures are essential and are achievable through a diverse range of imaging labels such as (radio)isotopes, fluorescent dyes, or combinations thereof. To achieve optimal in vivo utility a balanced molecular design of the tracer as a whole is required, which ensures a harmonious effect of the imaging label with the affinity and specificity (e.g., pharmacokinetics) of a pharmacophore/targeting moiety. This review outlines common design strategies and the effects of refinements in the molecular imaging agent design on the agent's pharmacological profile. This includes the optimization of affinity, pharmacokinetics (including serum binding and target mediated background), biological clearance route, the achievable signal intensity, and the effect of dosing hereon.

[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.

The role of fluorescent and hybrid tracers in radioguided surgery in urogenital malignancies.

The increasing availability of new imaging technologies and tracers has enhanced the application of nuclear molecular imaging in urogenital interventions. In this context, preoperative nuclear imaging and radioactivity-based intraoperative surgical guidance have become important tools for the identification and anatomical allocation of tumor lesions and/or suspected lymph nodes. Fluorescence guidance can provide visual identification of the preoperatively defined lesions during surgery. However, the added value of fluorescence guidance is still mostly unknown. This review provides an overview of the role of fluorescence imaging in radioguided surgery in urogenital malignancies. The sentinel node (SN) biopsy procedure using hybrid tracers (radioactive and fluorescent component) serves as a prominent example for in-depth evaluation of the complementary value of radio- and fluorescence guidance. The first large patient cohort and long-term follow-up studies show: 1) improvement in the SN identification rate compared to blue dye; 2) improved detection of cancer-positive SNs; and 3) hints towards a positive effect on (biochemical) recurrence rates compared to extended lymph node dissection. The hybrid tracer approach also highlights the necessity of a preoperative roadmap in preventing incomplete resection. Recent developments focus on receptor-targeted approaches that allow intraoperative identification of tumor tissue. Here radioguidance is still leading, but fluorescent and hybrid tracers are also finding their way into the clinic. Emerging multiwavelength approaches that allow concomitant visualization of different anatomical features within the surgical field may provide the next step towards even more refined procedures.

Image-Guided Surgery: Are We Getting the Most Out of Small-Molecule Prostate-Specific-Membrane-Antigen-Targeted Tracers?

Expressed on virtually all prostate cancers and their metastases, the transmembrane protein prostate-specific membrane antigen (PSMA) provides a valuable target for the imaging of prostate cancer. Not only does PSMA provide a target for noninvasive diagnostic imaging, e.g., PSMA-positron emission tomography (PSMA-PET), it can also be used to guide surgical resections of PSMA-positive lesions. The latter characteristic has led to the development of a plethora of PSMA-targeted tracers, i.e., radiolabeled, fluorescent, or hybrid. With image-guided surgery applications in mind, this review discusses these compounds based on clinical need. Here, the focus is on the chemical aspects (e.g., imaging label, spacer moiety, and targeting vector) and their impact on in vitro and in vivo tracer characteristics (e.g., affinity, tumor uptake, and clearance pattern).

Chemokine receptor - Directed imaging and therapy.

The C-X-C chemokine receptor 4 (CXCR4) and its natural ligand CXCL12 are key factors in the process of cell migration, homing of hematopoietic stem cells to the bone marrow, and represent important mediators of angiogenesis and cell proliferation. The CXCR4/CXCL12 interplay can be disrupted by CXCR4 antagonists such as Plerixafor which are already in daily clinical use, i.e. for mobilization and subsequent harvesting of hematopoietic progenitor cells and stem cell transplantation. In a pathological condition, involvement in the process of metastasis and homing of cancer cells to a protective niche has been described, making CXCR4 an attractive target for imaging and treatment of malignant diseases. Recently, radiolabeled analogs of CXCR4 antagonists (e.g., [68Ga]Pentixafor) have been introduced which can be used for non-invasive imaging of CXCR4 expression in animal models and humans using positron emission tomography. In addition, beta emitter-labeled antagonists (i.e., [177Lu]/[90Y]Pentixather) have been used in small patient cohorts for treatment of hematological neoplasms such as lymphoma, multiple myeloma and acute myeloid leukemia. This review reports on current imaging protocols for CXCR4-directed positron emission tomography in preclinical models and in humans. Furthermore, a theranostic approach using beta emitter-labeled antagonists is highlighted. Molecular imaging of the CXCR4/CXCL12 axis can contribute to further understand the process of metastatic spread and the intra-/interindividual heterogeneity of tumors. In addition, CXCR4 directed imaging allows tracking of activated, CXCR4+ immune cells. This allows for watching inflammatory processes, thus contributing to enlighten the role of the immune system in a variety of cardiovascular and neurological diseases.

Value of 111 In-prostate-specific membrane antigen (PSMA)-radioguided surgery for salvage lymphadenectomy in recurrent prostate cancer: correlation with histopathology and clinical follow-up.

OBJECTIVES: To evaluate the use of 111 In-labelled prostate-specific membrane antigen (PSMA)-I&T-based radioguided surgery (111 In-PSMA-RGS) for salvage surgery in recurrent prostate cancer (PCa) using comparison of intra-operative gamma probe measurements with histopathological results of dissected specimens. In addition, to determine the success of 111 In-PSMA-RGS with regard to postoperative prostate-specific antigen (PSA) responses, PCa-specific treatment-free survival rates and postoperative complication rates. PATIENTS AND METHODS: A total of 31 consecutive patients with localized recurrent PCa undergoing salvage surgery with PSMA-targeted radioguided surgery using a 111 In-labelled PSMA ligand between April 2014 and July 2015 were retrospectively included in this study. The preoperative (interquartile range; range) median PSA level was 1.3 (0.57-2.53 ng/mL; 0.2-13.9 ng/mL). Results of ex vivo radioactivity rating (positive vs negative) of resected tissue specimens were compared with findings of postoperative histological analysis. Best PSA response without additional treatment was determined after 111 In-PSMA-RGS, and salvage-surgery-related postoperative complications and PCa-specific additional treatments were recorded. RESULTS: In 30/31 patients, 111 In-PSMA-RGS allowed intra-operative identification of metastatic lesions. In total, 145 surgical specimens were removed and 51 showed metastatic involvement at histological analysis. According to 111 In-PSMA-RGS ex vivo measurements, 48 specimens were correctly classified as metastatic and 87 as cancer-free, four were false-negative and six were false-positive compared with histological evaluation. Follow-up information was available for 30/31 patients. PSA declines of >50% and >90% were observed in 23/30 patients and in 16/30 patients, respectively. In 18/30 patients, a PSA decline to <0.2 ng/mL was observed. In 10/30 patients further PCa-specific treatment was given after a median (range) of 125 (48-454) days post-111 In-PSMA-RGS. The remaining 20 patients remained treatment-free at a median (range) follow-up of 337 (81-591) days. Of 30 patients, 10 presented with surgery-related complications (Clavien-Dindo grade 1, n = 6, Clavien-Dindo grade 3b, n = 4). CONCLUSION: 111 In-PSMA-RGS proved to be of high value for intra-operative detection of even small metastatic lesions in patients with PCa scheduled for salvage lymphadenectomy. It allows the exact localization and resection of metastatic tissue during 111 In-PSMA-RGS and is therefore anticipated to have a beneficial influence on further disease progression; however, identification of suitable patients on the basis of PSMA-positron-emission tomography imaging as well as clinical variables is essential for satisfactory results to be obtained.

Systemic Radioligand Therapy with (177)Lu Labeled Prostate Specific Membrane Antigen Ligand for Imaging and Therapy in Patients with Metastatic Castration Resistant Prostate Cancer.

PURPOSE: We report our initial clinical experience with ß -emitting (177)Lu-PSMA-I&T ((177)Lu labeled prostate specific membrane antigen ligand for imaging and therapy) for systemic treatment of metastatic castration resistant prostate cancer. MATERIALS AND METHODS: Patients with metastatic castration resistant prostate cancer who experienced treatment failure with chemotherapy and novel androgen receptor targeted therapy were treated for 8 weeks with up to 4 cycles of (177)Lu-PSMA-I&T. We report safety data, the antitumor response with prostate specific antigen decreases and the radiographic tumor response as well as the clinical outcome with changes in ECOG (Eastern Cooperative Oncology Group) performance status and pain severity. RESULTS: The first 3 patients were treated with a lower activity of 3.7 GBq in cycle 1. Due to a favorable safety profile the activity was increased to 7.4 GBq in 19 subsequent patients who completed a total of 40 cycles. With the higher activity no grade 3/4 toxicities were observed. The main nonhematological and hematological grade 1/2 toxicities were dry mouth in 7 patients (37%), anemia in 6 (32%) and thrombopenia in 5 (25%). The proportion of patients who achieved a maximum prostate specific antigen decrease of 30% or greater, 50% or greater and 90% or greater was 56%, 33% and 11%, respectively. Combined assessment of bone and soft tissue metastases showed complete remission in 5% of patients, stable disease in 63% and progressive disease in 32%. ECOG performance status improved or was stable in 74% of patients. Of men with bone pain 58% achieved complete resolution or reduced pain. CONCLUSIONS: Radioligand therapy with (177)Lu-PSMA-I&T appears to be safe and active in heavily pretreated patients with metastatic castration resistant prostate cancer.

Targeted positron emission tomography imaging of CXCR4 expression in patients with acute myeloid leukemia.

Acute myeloid leukemia originates from leukemia-initiating cells that reside in the protective bone marrow niche. CXCR4/CXCL12 interaction is crucially involved in recruitment and retention of leukemia-initiating cells within this niche. Various drugs targeting this pathway have entered clinical trials. To evaluate CXCR4 imaging in acute myeloid leukemia, we first tested CXCR4 expression in patient-derived primary blasts. Flow cytometry revealed that high blast counts in patients with acute myeloid leukemia correlate with high CXCR4 expression. The wide range of CXCR4 surface expression in patients was reflected in cell lines of acute myeloid leukemia. Next, we evaluated the CXCR4-specific peptide Pentixafor by positron emission tomography imaging in mice harboring CXCR4 positive and CXCR4 negative leukemia xenografts, and in 10 patients with active disease. [(68)Ga]Pentixafor-positron emission tomography showed specific measurable disease in murine CXCR4 positive xenografts, but not when CXCR4 was knocked out with CRISPR/Cas9 gene editing. Five of 10 patients showed tracer uptake correlating well with leukemia infiltration assessed by magnetic resonance imaging. The mean maximal standard uptake value was significantly higher in visually CXCR4 positive patients compared to CXCR4 negative patients. In summary, in vivo molecular CXCR4 imaging by means of positron emission tomography is feasible in acute myeloid leukemia. These data provide a framework for future diagnostic and theranostic approaches targeting the CXCR4/CXCL12-defined leukemia-initiating cell niche.

Twins in spirit part II: DOTATATE and high-affinity DOTATATE--the clinical experience.

PURPOSE: Over recent decades interest in diagnosis and treatment of neuroendocrine tumours (NET) has steadily grown. The basis for diagnosis and therapy of NET with radiolabelled somatostatin (hsst) analogues is the variable overexpression of hsst receptors (hsst1-5 receptors). We hypothesized that radiometal derivatives of DOTA-iodo-Tyr(3)-octreotide analogues might be excellent candidates for somatostatin receptor imaging. We therefore explored the diagnostic potential of (68)Ga-DOTA-iodo-Tyr(3)-octreotate [(68)Ga-DOTA,3-iodo-Tyr(3),Thr(8)]octreotide ((68)Ga-HA-DOTATATE; HA, high-affinity) compared to the established (68)Ga-DOTA-Tyr(3)-octreotate ((68)Ga-DOTATATE) in vivo. METHODS: The study included 23 patients with known somatostatin receptor-positive metastases from NETs, thyroid cancer or glomus tumours who were investigated with both (68)Ga-HA-DOTATATE and (68)Ga-DOTATATE. A patient-based and a lesion-based comparative analysis was carried out of normal tissue distribution and lesion detectability in a qualitative and a semiquantitative manner. RESULTS: (68)Ga-HA-DOTATATE and (68)Ga-DOTATATE showed comparable uptake in the liver (SUVmean 8.9 ± 2.2 vs. 9.3 ± 2.5, n.s.), renal cortex (SUVmean 13.3 ± 3.9 vs. 14.5 ± 3.7, n.s.) and spleen (SUVmean 24.0 ± 6.7 vs. 22.9 ± 7.3, n.s.). A somewhat higher pituitary uptake was found with (68)Ga-HA-DOTATATE (SUVmean 6.3 ± 1.8 vs. 5.4 ± 2.1, p < 0.05). On a lesion-by-lesion basis a total of 344 lesions were detected. (68)Ga-HA-DOTATATE demonstrated 328 lesions (95.3% of total lesions seen), and (68)Ga-DOTATATE demonstrated 332 lesions (96.4%). The mean SUVmax of all lesions was not significantly different between (68)Ga-HA-DOTATATE and (68)Ga-DOTATATE (17.8 ± 11.4 vs. 16.7 ± 10.7, n.s.). CONCLUSION: Our analysis demonstrated very good concordance between (68)Ga-HA-DOTATATE and (68)Ga-DOTATATE PET data. As the availability and use of (68)Ga-HA-DOTATATE is not governed by patent restrictions it may be an attractive alternative to other (68)Ga-labelled hsst analogues.

Validation of a size exclusion method for concomitant purification and formulation of peptide radiopharmaceuticals.

BACKGROUND: Both in clinical routine and in preclinical research, the established standard procedure for the final purification of radiometal-labeled peptide radiopharmaceuticals is cartridge-based reversed-phase (RP) solid phase extraction (SPE). It allows the rapid and quantitative separation of the radiolabeled peptide from hydrophilic impurities and easy integration into automated synthesis procedures. However, product elution from RP cartridges necessitates the use of organic solvents and product recovery is sometimes limited. Thus, an alternative purification method based on commercially available size exclusion cartridges was investigated. RESULTS: Since most peptide radiopharmaceuticals have a molecular weight > 1 kDa, Sephadex G10 cartridges with a molecular size cut-off of 700 Da were used for the final purification of a broad palette of 68Ga-, 64Cu- and 99mTc-labeled experimental peptide radiotracers as well as the clinically relevant ligand PSMA-617. Results (radiochemical purity (RCP, determined by ITLC), recovery from the solid support) were compared to the respective standard RP-SPE method. Generally, retention of unreacted 68Ga, 64Cu and 99mTc salts on the G10 cartridges was quantitative up to the specified elution volume (1.2 mL) for 68Ga and 99mTc and 99.6% for 64Cu. Even at increased elution volumes of 1.5-2 mL, RCPs of the eluted 68Ga- and 99mTc -radiopeptides were > 99%. For all peptides with a molecular weight ≥ 2 kDa, product recovery from the G10 cartridges was consistently > 85% upon respective adjustment of the elution volume. Product recovery was lowest for [68Ga]Ga-PSMA-617 (67%, 1.2 mL to 84%, 2 mL). The pH of the final product solution was found to be volume-dependent (1.2 mL: pH 6.3; 1.5 mL: pH 5.9; 2 mL: pH 5.5). Notably, the G10 cartridges were reused up to 20 times without compromising performance, and implementation of the method in an automated radiosynthesis procedure was successful. CONCLUSIONS: Overall, size exclusion purification yielded all peptide radiopharmaceuticals in excellent radiochemical purities (> 99%) in saline within 10-12 min. Although product recovery is marginally inferior to classical SPE purifications, this method has the advantage of completely avoiding organic solvents and representing a cost-effective, easy-to-implement purification approach for automated radiotracer synthesis.

Site-selective template-directed synthesis of antibody Fc conjugates with concomitant ligand release.

Template-directed methods are emerging as some of the most effective means to conjugate payloads at selective sites of monoclonal antibodies (mAbs). We have previously reported a method based on an engineered Fc-III reactive peptide to conjugate a radionuclide chelator to K317 of antibodies with the concomitant release of the Fc-III peptide ligand. Here, our method was redesigned to target two lysines proximal to the Fc-III binding site, K248 and K439. Using energy minimization predictions and a semi-combinatorial synthesis approach, we sampled multiple Fc-III amino acid substituents of A3, H5, L6 and E8, which were then converted into Fc-III reactive conjugates. Middle-down MS/MS subunit analysis of the resulting trastuzumab conjugates revealed that K248 and K439 can be selectively targeted using the Fc-III reactive variants L6Dap, L6Orn, L6Y and A3K or A3hK, respectively. Across all variants tested, L6Orn-carbonate appeared to be the best candidate, yielding a degree and yield of conjugation of almost 2 and 100% for a broad array of payloads including radionuclide chelators, fluorescent dyes, click-chemistry reagents, pre-targeted imaging reagents, and some cytotoxic small molecules. Furthermore, L6Orn carbonate appeared to yield similar conjugation results across multiple IgG subtypes. In vivo proof of concept was achieved by conjugation of NODAGA to the PD1/PD-L1 immune checkpoint inhibitor antibody atezolizumab, followed by PET imaging of PD-L1 expression in mice bearing PD-L1 expressing tumor xenograft using radiolabeled [64Cu]Cu-atezolizumab.

Orthogonally protected artificial amino acid as tripod ligand for automated peptide synthesis and labeling with [(99m)Tc(OH(2))(3)(CO)(3)](+).

1,2-Diamino-propionic acid (Dap) is a very strong chelator for the [(99m)Tc(CO)(3)](+) core, yielding small and hydrophilic complexes. We prepared the lysine based Dap derivative l-Lys(Dap) in which the ε-NH(2) group was replaced by the tripod through conjugation to its α-carbon. The synthetic strategy produced an orthogonally protected bifunctional chelator (BFC). The -NH(2) group of the α-amino acid portion is Fmoc- and the -NH(2) of Dap are Boc-protected. Fmoc-l-Lys(Dap(Boc)) was either conjugated to the N- and C-terminus of bombesin BBN(7-14) or integrated into the sequence using solid-phase peptide synthesis (SPPS). We also replaced the native lysine in a cyclic RGD peptide with l-Lys(Dap). For all peptides, quantitative labeling with the [(99m)Tc(CO)(3)](+) core at a 10 µM concentration in PBS buffer (pH = 7.4) was achieved. For comparison, the rhenium homologues were prepared from [Re(OH(2))(3)(CO)(3)](+) and Lys(Dap)-BBN(7-14) or cyclo-(RGDyK(Dap)), respectively. Determination of integrin receptor binding showed low to medium nanomolar affinities for various receptor subtypes. The IC(50) of cyclo-(RGDyK(Dap[Re(CO)(3)])) for α(v)ß(3) is 7.1 nM as compared to 3.1 nM for nonligated RGD derivative. Biodistribution studies in M21 melanoma bearing nude mice showed reasonable α(v)ß(3)-integrin specific tumor uptake. Altogether, orthogonally protected l-Lys(Dap) represents a highly versatile building block for integration in any peptide sequence. Lys(Dap)-precursors allow high-yield (99m)Tc-labeling with [(99m)Tc(OH(2))(3)(CO)(3)](+), forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with excellent in vivo characteristics.

Influence of corticosteroid treatment on CXCR4 expression in DLBCL.

BACKGROUND: CXCR4-targeted radioligand therapy (RLT) with [177Lu]Lu/[90Y]Y-PentixaTher has recently evolved as a promising therapeutic option for patients with advanced hematological cancers. Given their advanced disease stage, most patients scheduled for PentixaTher RLT require concomitant or bridging chemotherapy to prevent intermittent tumor progression. These (mostly combination) therapies may cause significant downregulation of tumoral CXCR4 expression, challenging the applicability of PentixaTher RLT. This study therefore aimed at investigating the influence of corticosteroids, a central component of these chemotherapies, on CXCR4 regulation in diffuse large B cell lymphoma (DLBCL). METHODS: Different DLBCL cell lines (Daudi, OCI-LY1, SUDHL-4, -5-, -6 and -8) as well as the human T-cell lymphoma cell line Jurkat were incubated with Dexamethasone (Dex; 0.5 and 5 µM, respectively) and Prednisolone (Pred; 5 and 50 µM, respectively) for different time points (2 h, 24 h). Treatment-induced modulation of cellular CXCR4 surface expression was assessed via flow cytometry (FC) and compared to untreated cells. A radioligand binding assay with [125I]CPCR4.3 was performed in parallel using the same cells. To quantify potential corticosteroid treatment effects on tumoral CXCR4 expression in vivo, OCI-LY1 bearing NSG mice were injected 50 µg Dex/mouse i.p. (daily for 6 days). Then, a biodistribution study (1 h p.i.) using [68Ga]PentixaTher was performed, and tracer biodistribution in treated (n = 5) vs untreated mice (n = 5) was compared. RESULTS: In the in vitro experiments, a strongly cell line-dependent upregulation of CXCR4 was observed for both Dex and Pred treatment, with negligible differences between the high and low dose. While in Jurkat, Daudi and SUDHL-8 cells, CXCR4 expression remained unchanged, a 1.5- to 3.5-fold increase in CXCR4 cell surface expression was observed for SUDHL-5 < SUDHL-4 /-6 < OCI-LY1 via FC compared to untreated cells. This increase in CXCR4 expression was also reflected in correspondingly enhanced [125I]CPCR4.3 accumulation in treated cells, with a linear correlation between FC and radioligand binding data. In vivo, Dex treatment led to a general increase of [68Ga]PentixaTher uptake in all organs compared to untreated animals, as a result of a higher tracer concentration in blood. However, we observed an overproportionally enhanced [68Ga]PentixaTher uptake in the OCI-LY1 tumors in treated (21.0 ± 5.5%iD/g) vs untreated (9.2 ± 2.8%iD/g) mice, resulting in higher tumor-to-background ratios in the treatment group. CONCLUSION: Overall, corticosteroid treatment (Dex/Pred) consistently induced an upregulation of CXCR4 expression DBLCL cells in vitro, albeit in a very cell line-dependent manner. For the cell line with the most pronounced Dex-induced CXCR4 upregulation, OCI-LY1, the in vitro findings were corroborated by an in vivo biodistribution study. This confirms that at least the corticosteroid component of stabilizing chemotherapy regimens in DLBCL patients prior to [177Lu]Lu-PentixaTher RLT does not lead to downregulation of the molecular target CXCR4 and may even have a beneficiary effect. However, further studies are needed to investigate if and to what extent the other commonly used chemotherapeutic agents affect CXCR4 expression on DLBCL to ensure the choice of an appropriate treatment regimen prior to [177Lu]Lu/[90Y]Y-PentixaTher RLT.

Heterogeneity of prostate-specific membrane antigen (PSMA) and PSMA-ligand uptake detection combining autoradiography and postoperative pathology in primary prostate cancer.

BACKGROUND: Targeting prostate-specific membrane antigen (PSMA) has been highly successful for imaging and treatment of prostate cancer. However, heterogeneity in immunohistochemistry indicates limitations in the effect of imaging and radionuclide therapy of multifocal disease. 99mTc-PSMA-I&S is a γ-emitting probe, which can be used for intraoperative lesion detection and postsurgical autoradiography (ARG). We aimed to study its intraprostatic distribution and compared it with (immuno)-histopathology. RESULTS: Seventeen patients who underwent RGS between 11/2018 and 01/2020 with a total of 4660 grids were included in the preliminary analysis. Marked intratumor and intra-patient heterogeneity of PSMA expression was detected, and PSMA negative foci were observed in all samples (100%). Heterogeneous intra-patient PSMA-ligand uptake was observed, and no significant correlation was present between the degree of heterogeneity of PSMA expression and PSMA-ligand uptake. Higher PSMA-ligand uptake was observed in GS ≥ 8 than GS < 8 (p < 0.001). The appearance of Gleason Pattern (GP) 4 was strongly associated with higher uptake (coefficient: 0.43, p < 0.001), while GP 5 also affected the uptake (coefficient: 0.07, p < 0.001). CONCLUSION: PSMA expression and PSMA-ligand uptake show marked heterogeneity. Prostate carcinoma with GP 4 showed significantly higher uptake compared with non-neoplastic prostate tissue. Our analyses extend the scope of applications of radiolabeled PSMA-ligands to ARG for identifying high-grade disease and using its signal as a noninvasive biomarker in prostate cancer.

CXCR4 peptide-based fluorescence endoscopy in a mouse model of Barrett's esophagus.

BACKGROUND: Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett's esophagus. METHODS: Six L2-IL1B mice with advanced stage of disease (12-16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12-14 months old)  by a novel imaging system with two L2-IL1B mice used as negative controls. RESULTS: Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells  by confocal microscopy further confirmed the imaging results. CONCLUSIONS: This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett's esophagus. Further investigations are needed to assess its use in the clinical setting.

In Vivo Targeting of CXCR4-New Horizons.

Given its pre-eminent role in the context of tumor cell growth as well as metastasis, the C-X-C motif chemokine receptor 4 (CXCR4) has attracted a lot of interest in the field of nuclear oncology, and clinical evidence on the high potential of CXCR4-targeted theranostics is constantly accumulating. Additionally, since CXCR4 also represents a key player in the orchestration of inflammatory responses to inflammatory stimuli, based on its expression on a variety of pro- and anti-inflammatory immune cells (e.g., macrophages and T-cells), CXCR4-targeted inflammation imaging has recently gained considerable attention. Therefore, after briefly summarizing the current clinical status quo of CXCR4-targeted theranostics in cancer, this review primarily focuses on imaging of a broad spectrum of inflammatory diseases via the quantification of tissue infiltration with CXCR4-expressing immune cells. An up-to-date overview of the ongoing preclinical and clinical efforts to visualize inflammation and its resolution over time is provided, and the predictive value of the CXCR4-associated imaging signal for disease outcome is discussed. Since the sensitivity and specificity of CXCR4-targeted immune cell imaging greatly relies on the availability of suitable, tailored imaging probes, recent developments in the field of CXCR4-targeted imaging agents for various applications are also addressed.

At the Bench: Pre-clinical evidence for multiple functions of CXCR4 in cancer.

Signaling through chemokine receptor, C-X-C chemokine receptor type 4 (CXCR4) regulates essential processes in normal physiology, including embryogenesis, tissue repair, angiogenesis, and trafficking of immune cells. Tumors co-opt many of these fundamental processes to directly stimulate proliferation, invasion, and metastasis of cancer cells. CXCR4 signaling contributes to critical functions of stromal cells in cancer, including angiogenesis and multiple cell types in the tumor immune environment. Studies in animal models of several different types of cancers consistently demonstrate essential functions of CXCR4 in tumor initiation, local invasion, and metastasis to lymph nodes and distant organs. Data from animal models support clinical observations showing that integrated effects of CXCR4 on cancer and stromal cells correlate with metastasis and overall poor prognosis in >20 different human malignancies. Small molecules, Abs, and peptidic agents have shown anticancer efficacy in animal models, sparking ongoing efforts at clinical translation for cancer therapy. Investigators also are developing companion CXCR4-targeted imaging agents with potential to stratify patients for CXCR4-targeted therapy and monitor treatment efficacy. Here, pre-clinical studies demonstrating functions of CXCR4 in cancer are reviewed.

Novel Peptide-Based PET Probe for Non-invasive Imaging of C-X-C Chemokine Receptor Type 4 (CXCR4) in Tumors.

The recently reported CXCR4 antagonist 3 (Ac-Arg-Ala-[DCys-Arg-2Nal-His-Pen]-CO2H) was investigated as a molecular scaffold for a CXCR4-targeted positron emission tomography (PET) tracer. Toward this end, 3 was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononanetriacetic acid (NOTA). On the basis of convincing affinity data, both tracers, [68Ga]NOTA analogue ([68Ga]-5) and [68Ga]DOTA analogue ([68Ga]-4), were evaluated for PET imaging in "in vivo" models of CHO-hCXCR4 and Daudi lymphoma cells. PET imaging and biodistribution studies revealed higher CXCR4-specific tumor uptake and high tumor/background ratios for the [68Ga]NOTA analogue ([68Ga]-5) than for the [68Ga]DOTA analogue ([68Ga]-4) in both in vivo models. Moreover, [68Ga]-4 and [68Ga]-5 displayed rapid clearance and very low levels of accumulation in all nontarget tissues but the kidney. Although the high tumor/background ratios observed in the mouse xenograft model could partially derive from the hCXCR4 selectivity of [68Ga]-5, our results encourage its translation into a clinical context as a novel peptide-based tracer for imaging of CXCR4-overexpressing tumors.