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Abstract Background Estimation of beta cell mass is currently restricted to evaluating pancreatic tissue samples, which provides limited information. A non-invasive imaging technique that reliably quantifies beta cell mass enables monitoring of changes of beta cell mass during the progression of diabetes mellitus and may contribute to monitoring of therapy effectiveness. We assessed the specificity of radiolabelled exendin for beta cell mass quantification in humans. Fourteen adults with pancreas tumours were injected with 111In-labeled exendin-4 prior to pancreatic resection. In resected pancreas tissue, endocrine-exocrine ratios of tracer uptake were determined by digital autoradiography and accumulation of 111In-labeled exendin-4 was compared to insulin and GLP-1 receptor staining. Of four participants, abdominal single photon emission computed tomography/computed tomography (SPECT/CT) images were acquired to quantify pancreatic uptake in vivo Results Tracer uptake was predominantly present in the endocrine pancreas (endocrine-exocrine ratio: 3.6 [2.8–10.8]. Tracer accumulation showed overlap with insulin-positive regions, which overlapped with GLP-1 receptor positive areas. SPECT imaging showed pancreatic uptake of radiolabelled exendin in three participants. Conclusion Radiolabelled exendin specifically accumulates in the islets of Langerhans in human pancreas tissue. The clear overlap between regions positive for insulin and the GLP-1 receptor substantiate the beta cell specificity of the tracer. Radiolabelled exendin is therefore a valuable imaging agent for human beta cell mass quantification and has the potential to be used for a range of applications, including improvement of diabetes treatment by assessment of the effects of current and novel diabetes therapies on the beta cell mass. Trial registration ClinicalTrials.gov NCT03889496, registered 26,032,019, URL https://clinicaltrials.gov/study/NCT03889496?term=NCT03889496 . ClinicalTrials.gov NCT04733508, registered 02022021, URL https://clinicaltrials.gov/study/NCT04733508 .

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein’s dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

Abstract Pancreatic islet transplantation is a promising therapy for patients with type 1 diabetes. However, the duration of long-term graft survival is limited due to inflammatory as well as non-inflammatory processes and routine clinical tests are not suitable to monitor islet survival. 111In-exendin-SPECT (single photon emission computed tomography) is a promising method to non-invasively image islets after transplantation and has the potential to help improve the clinical outcome. Whether 111In-exendin-SPECT allows detecting small differences in beta-cell mass (BCM) and measuring the actual volume of islets that were successfully engrafted has yet to be demonstrated. Here, we evaluated the performance of 111In-exendin-SPECT using an intramuscular islet transplantation model in C3H mice. In vivo imaging of animals transplanted with 50, 100, 200, 400 and 800 islets revealed an excellent linear correlation between SPECT quantification of 111In-exendin uptake and insulin-positive area of islet transplants, demonstrating that 111In-exendin-SPECT specifically and accurately measures BCM. The high sensitivity of the method allowed measuring small differences in graft volumes, including grafts that contained less than 50 islets. The presented method is reliable, convenient and holds great potential for non-invasive monitoring of BCM after islet transplantation in humans.

Nanobodies are well-established targeting ligands for molecular imaging and therapy. Their short circulation time enables early imaging and reduces systemic radiation exposure. However, shorter circulation time leads to lower tracer accumulation in the target tissue. Cell-penetrating peptides (CPPs) improve cellular uptake of various cargoes, including nanobodies. CPPs could enhance tissue retention without compromising rapid clearance. However, systematic investigations on how the functionalities of nanobody and CPP combine with each other at the level of 2D and 3D cell cultures and in vivo are lacking. Here, we demonstrate that conjugates of the epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with different CPPs (nonaarginine, penetratin, Tat and hLF) differ with respect to cell binding and induction of endocytosis. For nonaarginine and penetratin we compared the competition of EGF binding and performance of L- and D-peptide stereoisomers, and tested the D-peptide conjugates in tumor cell spheroids and in vivo. The D-peptide conjugates showed better penetration into spheroids than the unconjugated 7D12. Both in vivo and in vitro, the behavior of the agent reflects the combination of both functionalities. Although CPPs cause promising increases in in vitro uptake and 3D penetration, the dominant effect of the CPP in the control of biodistribution warrants further investigation.

Renal ischemia/reperfusion injury (IRI) frequently complicates shock, renal transplantation and cardiac and aortic surgery, and has prognostic significance. The translocation of phosphatidylserines to cell surfaces is an important pro-inflammatory signal for cell-stress after IRI. We hypothesized that shielding of exposed phosphatidylserines by the annexin A5 (ANXA5) homodimer Diannexin protects against renal IRI. Protective effects of Diannexin on the kidney were studied in a mouse model of mild renal IRI. Diannexin treatment before renal IRI decreased proximal tubule damage and leukocyte influx, decreased transcription and expression of renal injury markers Neutrophil Gelatinase Associated Lipocalin and Kidney Injury Molecule-1 and improved renal function. A mouse model of ischemic hind limb exercise was used to assess Diannexin biodistribution and targeting. When comparing its biodistribution and elimination to ANXA5, Diannexin was found to have a distinct distribution pattern and longer blood half-life. Diannexin targeted specifically to the ischemic muscle and its affinity exceeded that of ANXA5. Targeting of both proteins was inhibited by pre-treatment with unlabeled ANXA5, suggesting that Diannexin targets specifically to ischemic tissues via phosphatidylserine-binding. This study emphasizes the importance of phosphatidylserine translocation in the pathophysiology of IRI. We show for the first time that Diannexin protects against renal IRI, making it a promising therapeutic tool to prevent IRI in a clinical setting. Our results indicate that Diannexin is a potential new imaging agent for the study of phosphatidylserine-exposing organs in vivo.

Arginine-glycine-aspartic acid (RGD) peptides preferentially bind to ?v?3 integrin, an integrin expressedon newly formed endothelial cells and on various tumor cells. When labeled with ?-emitting radionuclides,these peptides can be used for peptide-receptor radionuclide therapy of malignant tumors. Thesestudies aimed to investigate whether tumor targeting and tumor therapy could be optimized by dose fractionation.The RGD-peptide DOTA-E-[c(RGDfK)]2 was labeled with 111In for biodistribution experiments andwith 90Y for therapy experiments. In mice with NIH:OVCAR-3 ovarian carcinoma xenografts, optimal tumoruptake was obtained at peptide doses up to 1.0 mg (4.8 %ID/g). A peptide dose of 5 mg, required toadminister the maximum tolerable dose (MTD) 90Y-DOTA-E-[c(RGDfK)]2, was administered as 5 portionsof 1.0 mg. Tumor uptake of the fifth portion was significantly higher than that of the single 5.0 mgportion (3.3 %ID/g versus 2.1 %ID/g). The therapeutic efficacy of 37 MBq 90Y-DOTA-E-[c(RGDfK)]2(1 × 5.0 mg) was compared with that of 37 MBq administered in five equal portions (5 × 1.0 mg). Nodifference in tumor growth between the fractionated and the nonfractionated therapy was observed. Inconclusion, dose fractionation resulted in higher radiation doses. However, therapeutic efficacy of the radiolabeledpeptide was not significantly improved by dose fractionation. [ABSTRACT FROM AUTHOR]

Background: Interleukin-2 (IL-2) and interferon-α (IFN-α) can induce therapeutic responses in a minority (5-25%) of patients with metastatic renal cell carcinoma (RCC). G250-MN/CA IX, a tumor-associated antigen expressed on the majority of clear cell RCCs, is a potential (radio)immunotherapeutic target for G250-antibody based (radio)immunotherapy. We investigated the effect of the biological response modifiers (BRMs) IL-2, IFN-α, and IFN-γ on the expression of the G250 antigen on RCC cells. Methods: In vitro , the expression of the G250 antigen was measured by flow cytometry (FCM) after culturing RCC cells in the presence of various concentrations of the BRMs. Additionally, the number of G250 epitopes per cell was determined quantitatively by Scatchard analysis. Results: Upregulation of G250 expression was observed on RCC cells cultured in the presence of IFN-α or IFN-γ, whereas the addition of IL-2 had no effect. For both IFNs a clear dose-response relation between G250 antigen expression and IFN dose was observed, with IFN-γ being the more potent agent. G250 expression could be upregulated four-fold. Interestingly, the effect of combining IFN-α and IFN-γ revealed a more pronounced upregulation of G250 expression than either one of the IFNs alone. Conclusions: On the basis of in vitro experiments, G250 expression can be upregulated by IFN-α and IFN-γ. In vivo studies are warranted to investigate whether due to IFN treatment increased G250 expression occurs, and whether increased G250 expression can enhance the therapeutic efficacy of G250-antibody based (radio)immunotherapy. [ABSTRACT FROM AUTHOR]

Radioimmunotherapy (RIT) is a new and effective treatment modality in patients with non-Hodgkin's lymphoma. The monoclonal antibody (mAb) hLL2 (epratuzumab), a humanized mAb directed against the CD22 antigen, and which internalizes, can be labeled with various radionuclides. The biodistribution of hLL2 labeled with 131I, 186Re, 177Lu, and 88Y was studied in nude mice with subcutaneous human lymphoma xenografts in order to determine the most suitable of these four radionuclides for RIT with hLL2. Methods: Human Ramos lymphoma xenografts were transplanted in cyclophosphamide-pretreated athymic BALB/c mice. Four groups of mice were injected intravenously with 131I-, 186Re-, 88Y-, or 177Lu-labeled hLL2, respectively. To determine the nonspecific tumor uptake, two groups of mice received 88Y-labeled or 131I-labeled control antibody, cG250. The biodistribution of the radiolabel was determined 1, 3, and 7 days postinjection (p.i.). Results: Radiolabeled hLL2 had a higher tumor uptake than the nonspecific mAb at all time-points, irrespective of the radiolabel used. Tumor accretion of 88Y- and 177Lu-hLL2 was higher than tumor uptake of 131I- and 186Re-hLL2. Activity in the bone, represented by the femur without bone marrow, was higher for 177Lu- and 88Y-hLL2 than for 131I- and 186Re-hLL2 on day 7 p.i. Conclusion: The use of the residualizing radiolabels 88Y and 177Lu in combination with a mAb directed against an internalizing antigen resulted in higher uptake and better retention of the radiolabel in the tumor. [ABSTRACT FROM AUTHOR]