Mark Bartholomä

High-Resolution PET Imaging with Therapeutic Antibody-based PD-1/PD-L1 Checkpoint Tracers.

Checkpoint-blocking antibodies like those targeting the PD-1/PD-L1 pathway have revolutionized oncology. We developed radiotracers based on therapeutic checkpoint-blocking antibodies permitting sensitive and high-resolution PET imaging of both PD-1 and PD-L1 in immunocompetent mice. ImmunoPET of naive mice revealed similar overall expression patterns for PD-1 and PD-L1 in secondary lymphoid organs (spleen and lymph nodes). Interestingly, PD-L1 was also detected in brown adipose tissue (BAT), confirming the notion that BAT is immunologically relevant. Under pathophysiological conditions, strong expression of the receptor/ligand pair was also found in non-lymphoid tissues. Both were specifically detected in malignant tumors. PD-1 was readily detected after combined immunoradiotherapy causing massive tumor infiltration by PD-1+ lymphocytes. PD-L1 tracer uptake was reduced in PD-L1 knockout tumors. Moreover, monitoring the expression changes of PD-L1 in response to its main inducer, the effector T cell cytokine IFN-γ, revealed robust upregulation in the lung. This suggests that T cell responses in the lung, a vital organ continuously exposed to a variety of antigens, are strongly restrained by the PD-1 checkpoint. In turn, this could explain the association of PD-1 checkpoint inhibition with potentially fatal immune-mediated pneumonitis and partially also its efficacy in lung cancer.

Synthesis, 64Cu-labeling and PET imaging of 1,4,7-triazacyclononane derived chelators with pendant azaheterocyclic arms

The TACN derived chelators NOTI, NOTI-Me and NOTThia were synthesized in a facile, single reaction step. The new chelators were readily labeled with 64Cu under mild conditions over a wide pH range. The corresponding 64Cu complexes are of high stability in vitro as determined by Cu2+ exchange experiments, acid decomplexation and serum stability studies. Single crystal X-ray analysis showed that the six-coordinate copper complexes exhibited a distorted prismatic CuN3N′3 coordination geometry similar to Cu(NOTA). Biological testing by small animal PET imaging revealed retention of 64Cu complexes in the kidneys and, to a lesser extent, in the liver. Altogether, the results presented support the development of bifunctional derivatives for conjugation to targeting vectors and further testing in vivo.

Oncogenic JAK2V617F causes PD-L1 expression, mediating immune escape in myeloproliferative neoplasms

Oncogenic JAK mutation sensitizes myeloproliferative neoplasms to immune checkpoint inhibition. Cancers JAK up an immune checkpoint Myeloproliferative neoplasms, a group of hematologic cancers, are associated with mutations activating the JAK2 oncogene. JAK2 is located on chromosome 9, in the vicinity of the immunosuppressive PD-L1 gene, and Prestipino et al. discovered that myeloproliferative cancers with overactive JAK2 generally have increased PD-L1 as well. Although PD-L1 helps cancers evade the immune system, immune checkpoint inhibitors developed in recent years provide a way to block its function and turn PD-L1 expression into a therapeutic vulnerability for the tumors, as the authors demonstrate in this study. Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Diagnosis of recurrent prostate cancer with PET/CT imaging using the gastrin-releasing peptide receptor antagonist 68Ga-RM2: Preliminary results in patients with negative or inconclusive [18F]Fluoroethylcholine-PET/CT

Purpose/background[18F]fluoroethylcholine (18FECH) has been shown to be a valuable PET-tracer in recurrent prostate cancer (PCa), but still has limited accuracy. RM2 is a gastrin-releasing peptide receptor (GRPr) antagonist that binds to GRPr on PCa cells. Recent studies suggest that GRPr imaging with PET/CT is a promising technique for staging and restaging of PCa. We explore the value of GRPr-PET using the 68Ga-labeled GRPr antagonist RM2 in a selected population of patients with biochemically recurrent PCa and a negative/inconclusive 18FECH-PET/CT.Material and methodsIn this retrospective study 16 men with biochemical PCa relapse and negative (nxa0=xa014) or inconclusive (nxa0=xa02) 18FECH-PET/CT underwent whole-body 68Ga-RM2-PET/CT. Mean time from 18FECH-PET/CT to 68Ga-RM2-PET/CT was 6.1xa0±xa06.8xa0months. Primary therapies in these patients were radical prostatectomy (nxa0=xa013; 81.3%) or radiotherapy (nxa0=xa03; 18.7%). 14/16 patients (87.5%) had already undergone salvage therapies because of biochemical relapse prior to 68Ga-RM2-PET/CT imaging. Meanxa0±xa0SD PSA at 68Ga-RM2-PET/CT was 19.4xa0±xa053.5xa0ng/ml (range 1.06–226.4xa0ng/ml).Results68Ga-RM2-PET/CT showed at least one region with focal pathological uptake in 10/16 patients (62.5%), being suggestive of local relapse (nxa0=xa04), lymph node metastases (LNM; nxa0=xa04), bone metastases (nxa0=xa01) and lung metastasis with hilar LNM (nxa0=xa01). Seven of ten positive 68Ga-RM2 scans were positively confirmed by surgical resection and histology of the lesions (nxa0=xa02), by response to site-directed therapies (nxa0=xa02) or by further imaging (nxa0=xa03). Patients with a positive 68Ga-RM2-scan showed a significantly higher median PSA (6.8xa0ng/ml, IQR 10.2xa0ng/ml) value than those with a negative scan (1.5xa0ng/ml, IQR 3.1xa0ng/ml; pxa0=xa00.016). Gleason scores or concomitant antihormonal therapy had no apparent impact on the detection of recurrent disease.ConclusionEven in this highly selected population of patients with known biochemical recurrence but negative or inconclusive 18FECH-PET/CT, a 68Ga-RM2-PET/CT was helpful to localize PCa recurrence in the majority of the cases. Thus, 68Ga-RM2-PET/CT deserves further investigation as a promising imaging modality for imaging PCa recurrence.

Gastrin-releasing Peptide Receptor Imaging in Breast Cancer Using the Receptor Antagonist 68Ga-RM2 And PET

Introduction: The gastrin-releasing peptide receptor (GRPR) is overexpressed in breast cancer. The present study evaluates GRPR imaging as a novel imaging modality in breast cancer by employing positron emission tomography (PET) and the GRPR antagonist 68Ga-RM2. Methods: Fifteen female patients with biopsy confirmed primary breast carcinoma (3 bilateral tumors; median clinical stage IIB) underwent 68Ga-RM2-PET/CT for pretreatment staging. In vivo tumor uptake of 68Ga-RM2 was correlated with estrogen (ER) and progesterone (PR) receptor expression, HER2/neu status and MIB-1 proliferation index in breast core biopsy specimens. Results: 13/18 tumors demonstrated strongly increased 68Ga-RM2 uptake compared to normal breast tissue (defined as PET-positive). All PET-positive primary tumors were ER- and PR-positive (13/13) in contrast to only 1/5 PET-negative tumors. Mean SUVMAX of ER-positive tumors was 10.6±6.0 compared to 2.3±1.0 in ER-negative tumors (p=0.016). In a multivariate analysis including ER, PR, HER2/neu and MIB-1, only ER expression predicted 68Ga-RM2 uptake (model: r2=0.55, p=0.025). Normal breast tissue showed inter- and intraindividually variable, moderate GRPR binding (SUVMAX 2.3±1.0), while physiological uptake of other organs was considerably less except pancreas. Of note, 68Ga-RM2-PET/CT detected internal mammary lymph nodes with high 68Ga-RM2 uptake (n=8), a contralateral axillary lymph node metastasis (verified by biopsy) and bone metastases (n=1; not detected by bone scan and CT). Conclusion: Our study demonstrates that 68Ga-RM2-PET/CT is a promising imaging method in ER-positive breast cancer. In vivo GRPR binding assessed by 68Ga-RM2-PET/CT correlated with ER expression in primary tumors of untreated patients.

In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector

INTRODUCTIONnWe recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for complexation of the PET nuclides gallium-68 and copper-64. The main objective of this study was to evaluate the stability and pharmacokinetics of 68Ga- and 64Cu-complexes of the bifunctional chelator NODIA-Me 1 covalently bound to a PSMA targeting vector in vivo.nnnMETHODSnNODIA-Me 1 was conjugated to the PSMA targeting Glu-NH-CO-NH-Lys moiety to give the bioconjugate NODIA-Me-NaI-Ahx-PSMA 4. The stability of [68Ga]4 and [64Cu]4 was assessed in vitro by serum stability studies. The PSMA binding affinity was determined in competitive cell experiments in LNCaP cells using 68Ga-PSMA-HBED-CC as radioligand. The stability and pharmacokinetics of [68Ga]4 and [64Cu]4 was evaluated by PET imaging and ex vivo biodistribution studies in mice bearing subcutaneous LNCaP tumors.nnnRESULTSnIn human serum, [68Ga]4 and [64Cu]4 remained intact to 85% (3u202fh) and 92% (24u202fh), respectively. Nature of the metal chelate influenced PSMA binding affinity with IC50 of 233u202f±u202f10u202fnM for uncomplexed 4, 681u202f±u202f7u202fnM for Cu-4 and 176u202f±u202f10u202fnM for Ga-4. In animal studies, [68Ga]4 and [64Cu]4 revealed low uptake (≤1% IA g-1) in the majority of organs. Kidney uptake at 1u202fh p.i. was 6.28u202f±u202f0.92% IA g-1 and 4.96u202f±u202f0.79% IA g-1 and specific tumor uptake was 1.33u202f±u202f0.46% IA g-1 and 2.15u202f±u202f0.38% IA g-1 for [68Ga]4 and [64Cu]4, respectively.nnnCONCLUSIONnThe bifunctional chelator NODIA-Me 1 was successfully conjugated to a PSMA targeting moiety. In small-animal PET imaging and ex vivo biodistribution studies, 68Ga- and 64Cu-labelled conjugates specifically delineated PSMA-positive LNCaP tumors and exhibited rapid renal clearance from non-target tissues with no significant demetallation/transchelation in vivo. The results support further development of this novel chelating platform for production of 68Ga- and 64Cu-labelled radiopharmaceuticals.

Preparation and preclinical evaluation of a 68 Ga-labelled c(RGDfK) conjugate comprising the bifunctional chelator NODIA-Me

BackgroundWe recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three, five-membered azaheterocyclic arms for the development of 68Ga- and 64Cu-based radiopharmaceuticals. Here, a 68Ga-labelled conjugate comprising the bifunctional chelator NODIA-Me in combination with the αvß3-targeting peptide c(RGDfK) has been synthesized and characterized. The primary aim was to evaluate further the potential of our NODIA-Me chelating system for the development of 68Ga-labelled radiotracers.ResultsThe BFC NODIA-Me was conjugated to c(RGDfK) by standard peptide chemistry to obtain the final bioconjugate NODIA-Me-c(RGDfK) 3 in 72% yield. Labelling with [68Ga]GaCl3 was accomplished in a fully automated, cGMP compliant process to give [68Ga]3 in high radiochemical yield (98%) and moderate specific activity (~ 8 MBq nmol− 1). Incorporation of the Ga-NODIA-Me chelate to c(RGDfK) 2 had only minimal influence on the affinity to integrin αvß3 (IC50 values [natGa]3 = 205.1 ± 1.4 nM, c(RGDfK) 2 = 159.5 ± 1.3 nM) as determined in competitive cell binding experiments in U-87 MG cell line. In small-animal PET imaging and ex vivo biodistribution studies, the radiotracer [68Ga]3 showed low uptake in non-target organs and specific tumor uptake in U-87 MG tumors.ConclusionThe results suggest that the bifunctional chelator NODIA-Me is an interesting alternative to existing ligands for the development of 68Ga-labelled radiopharmaceuticals.

Radioimmunotherapy of solid tumors: Approaches on the verge of clinical application

While radioimmunotherapy (RIT) for the treatment of hematological malignancies such as indolent B-cell lymphoma has proven quite successful, clinical results of RIT in solid tumors have only been moderate in the past. The reasons were manifold and can be mostly attributed to the different biological properties of solid tumors vs hematological cancers. Furthermore, the slow clearance of the radiolabelled antibody prevents the use of radiation doses necessary to achieve clinical responses. The long biological half-life of radioimmunoconjugates results in high background levels and is the main reason for radiation related toxicities. In recent years, researchers and clinicians have developed solutions for the successful application of RIT for the treatment of solid tumors. These include compartmental route of administration, neoadjuvant therapies, and pretargeting approaches. In this review, recent developments in RIT for the treatment of solid tumors that address these restrictions as well as future perspectives will be highlighted from a clinical perspective.

Performance of 111In-labelled PSMA ligand in patients with nodal metastatic prostate cancer: correlation between tracer uptake and histopathology from lymphadenectomy

PurposeIntraoperative identification of lymph node (LN) metastases (LNM) detected on preoperative PSMA PET/CT may be facilitated by PSMA radioguided surgery with the use of a gamma probe. We evaluated the uptake of 111In-labelled PSMA ligand DKFZ-617 (referred to as 111In-PSMA-617) in unaffected LN and LNM at the level of single LN.MethodsSix patients with prostate cancer (PCa) with suspicion of LNM on preoperative PSMA PET/CT underwent 111In-PSMA-617-guided lymphadenectomy (LA; four salvage LA and two primary LA). 111In-PSMA-617 (109u2009±u20095xa0MBq). was injected Intravenously 48xa0h prior to surgery Template LAs were performed in small subregions: common, external, obturator and internal iliac vessels, and presacral and retroperitoneal subregions (nxa0=u20094). Samples from each subregion were isolated aiming at the level of single LN. Uptake was measured ex situ using a germanium detector. Receiver operating characteristic (ROC) analysis was performed based on 111In-PSMA-617 uptake expressed as standardized uptake values normalized to lean body mass (SUL).ResultsOverall 310 LN (mean 52u2009±u200919.7) were removed from 74 subregions (mean 12u2009±u20093.7). Of the 310 LN, 35 turned out to be LNM on histopathology. Separation of the samples from all subregions resulted in 318 single specimens: 182 PCa-negative LN samples with 275 LN, 35 single LNM samples, 3 non-nodal PCa tissue samples and 98 fibrofatty tissue samples. The median SULs of nonaffected LN (0.16) and affected LN (13.2) were significantly different (pxa0<u20090.0001). Based on 38 tumour-containing and 182 tumour-free specimens, ROC analysis revealed an area under the curve of 0.976 (95% CI 0.95–1.00, pxa0<u20090.0001). Using a SUL cut-off value of 1.136, sensitivity, specificity, positive predictive value, negative predictive value and accuracy in discriminating affected from nonaffected LN were 92.1% (35/38), 98.9% (180/182), 94.6% (35/37), 98.4% (180/183) and 97.7% (215/220), respectively.ConclusionEx situ analysis at the level of single LN showed that 111In-PSMA-617 had excellent ability to discriminate between affected and nonaffected LN in our patients with PCa. This tracer characteristic is a prerequisite for in vivo real-time measurements during surgery.