Humanization, Radiolabeling and Biodistribution Studies of an IgG1-Type Antibody Targeting Uncomplexed PSA for Theranostic Applications

Metastatic castration-resistant prostate cancer is today incurable. Conventional imaging methods have limited detection, affecting their ability to give an accurate outcome prognosis, and current therapies for metastatic prostate cancer are insufficient. This inevitably leads to patients relapsing with castration-resistant prostate cancer. Targeting prostate-specific antigens whose expression is closely linked to the activity in the androgen receptor pathway, and thus the pathogenesis of prostate cancer, is a possible way to increase specificity and reduce off-target effects. We have humanized and evaluated radioimmunoconjugates of a previously murine antibody, m5A10, targeting PSA intended for theranostics of hormone-refractory prostate cancer. The humanized antibody h5A10 was expressed in mammalian HEK293 cells transfected with the nucleotide sequences for the heavy and light chains of the antibody. Cell culture medium was filtered and purified by Protein G chromatography, and the buffer was changed to PBS pH 7.4 by dialysis. Murine and humanized 5A10 were conjugated with p-SCN-Bn-CHX-A”-DTPA. Surface plasmon resonance was used to characterize the binding to PSA of the immunoconjugates. Immunoconjugates were labeled with either indium-111 or lutetium-177. Biodistribution studies of murine and humanized 5A10 were performed in mice with LNCaP xenografts. 5A10 was successfully humanized, and in vivo targeting showed specific binding in xenografts. The results thus give an excellent platform for further theranostic development of humanized 5A10 for clinical applications.

Report of unexpected findings after cardiac stem cell injections in a preclinical model

Introduction Cardiac regenerative therapy is a proposed therapy for ischemic heart disease. So far efficacy has been low and this might partly be explained by low cardiac cell retention. In this study we aimed to investigate if cardiac cell retention improves using ureido-pyrimidinone units (UPy-gel) as a cell carrier. Methods We used an ischemia-reperfusion model. Pigs were randomized to intramyocardial injections with mesenchymal stromal cells (MSC) labelled with both Indium-111 and a fluorescent tracer in either PBS or in the UPy-gel. After 4 hours, a total body scintigraphy was performed to determine the cardiac cell retention and histology was obtained. Results In the first 4 pigs, we noticed focused areas of radio activity (hotspots) outside the heart in both the control and UPy-gel arm, and decided to interrupt the study. At histology we confirmed one hotspots to be located in a lymph node. No satisfactory explanation for these, potentially harmful, hotspots was found. Conclusion This study was interrupted due to unexpected extra-cardiac hotspots. Although we do not have a conclusive explanation for these findings, we find that sharing these results is important for future research. We recommend to use total body imaging in future retention studies to confirm of reject the occurrence of extra-cardiac cell accumulation after intramyocardial cell injection and discover the pathophysiology and its clinical implications.

Anti-Glycoprotein Antibodies and Sequestration Pattern of Indium Labeled Platelets in Immune Thrombocytopenia

Anti-glycoprotein antibodies play an important role in the pathophysiology of immune thrombocytopenia (ITP). The sequestration pattern of platelets in spleen and liver can be studied with Indium-111 labeled autologous platelet scans. No studies have investigated the role of anti-GP antibodies in sequestration pattern in ITP patients. In this study we examined the association between antibodies and 1) platelet sequestration site and 2) clearance rate of platelets. All ITP patients receiving an Indium-111 labeled autologous platelet study between 2014 and 2018 were included. Antibodies were measured using the direct MAIPA method to determine the presence and titer of anti-GPIIb/IIIa, anti-GPIb/IX and anti-GPV antibodies. Multivariate regression models were used to study the association between anti-GP antibodies, sequestration site and clearance rate. Seventy-four patients were included, with a mean age of 36 years. Forty-seven percent of the patients showed a predominantly splenic sequestration pattern, 29% mixed and 25% a hepatic pattern. In 53% of the patients anti-GP antibodies were detected. Regression models showed a significant association between splenic sequestration and GPV autoantibodies.. Furthermore, in patients where antibodies were present the clearance rate was higher in patients with a splenic sequestration. Anti-GPV antibodies are associated with a splenic sequestration pattern in ITP patients. These associations provide insight in the possible pathophysiological mechanisms of ITP, which may lead to better detection and treatment of this partly idiopathic and prevalent disease.

Discovery, optimization and biodistribution of an Affibody molecule for imaging of CD69

Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a Kd of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant ZCD69:4 had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule ZCD69:4 is suitable for further development for imaging of activated immune cells.

Radioguided Surgery for Gastroenteropancreatic Neuroendocrine Tumours: a Systematic Literature Review

Background Radioguided surgery (RGS) for gastroenteropancreatic neuroendocrine tumours (GEP-NETs) has been suggested as a way to improve intraoperative lesion detection. This systematic literature review of reports of the use of RGS for GEP-NETs was performed to determine if there is a benefit. Methods A literature search was conducted using Google Scholar and PubMed, and snowballing from any relevant literature. Full-text studies were included if they were published in the English language and reported outcomes of RGS on human subjects with GEP-NETs. Qualitative data synthesis was performed. Results Twenty-six papers including a total of 209 patients were included. The tracers used were predominantly indium-111 pentetreotide, gallium-68 DOTA-peptides, and technetium-99m EDDA/HYNIC-peptides. Heterogeneous protocols make comparisons difficult, but most papers reported a benefit from the use of RGS in tumours in the gastrointestinal tract; utility in localisation of pancreatic tumours was less clear. Time between tracer administration and operation varied: from 16 h to 8 days with indium-111, 0–24 h with technetium-99m, and 19–193 min with gallium-68. Eight teams reported the thresholding technique used for discrimination—four used a ratio, four statistical methods, and one looked at the sensitivity and specificity of different cut-offs. Six teams performed follow-up of 24 patients (three pancreas, eight gastrinoma, 13 gastrointestinal tract) for between 3 months and 3 years. Two patients relapsed (one pancreas, one gastrinoma) between 6 and 12 months post-surgery. Conclusions RGS appears to aid in localisation of gastrointestinal NETs, but the benefit is more equivocal in pancreatic NETs. Further work into outcomes is warranted.

Radioguided Surgery for Gastroenteropancreatic Neuroendocrine Tumours; a Systematic Literature Review

Background:Radioguided surgery (RGS) for gastroenteropancreatic neuroendocrine tumours (GEP-NETs) has been suggested as a way to improve intraoperative lesion detection. This systematic literature review of reports of the use of RGS for GEP-NETs was performed to determine if there is a benefit.Methods: A literature search was conducted using Google Scholar and PubMed, and snowballing from any relevant literature. Full-text studies were included if they were published in the English Language and reported outcomes of RGS on human subjects with GEP-NETs. Qualitative data synthesis was performed. Results: 26 papers including a total of 209 patients were included. The tracers used were predominantly indium-111 pentetreotide, gallium-68 DOTA-Peptides, and technetium-99m EDDA/HYNIC-Peptides. Heterogeneous protocols make comparisons difficult, but most papers reported a benefit from the use of RGS in tumours in the gastrointestinal tract; utility in localisation of pancreatic tumours was less clear. Time between tracer administration and operation varied; from 16 hours to 8 days with indium-111, 0-24 hours with technetium-99m and 19-193 minutes with gallium-68. Eight teams reported the thresholding technique used for discrimination – four used a ratio, four statistical methods, and one looked at the sensitivity and specificity of different cut-offs. Six teams performed follow-up of 24 patients (three pancreas, eight gastrinoma, 13 gastrointestinal tract) for between 3 months and 3 years. Two patients relapsed (one pancreas, one gastrinoma) between six and 12 months post-surgery. Conclusions:RGS appears to aid in localisation of gastrointestinal NETs, but the benefit is more equivocal in pancreatic NETs. Further work into outcomes is warranted.

Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE−/− mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.

Generation of Proton- and Alpha-Induced Nuclear Cross-Section Data via Random Forest Algorithm: Production of Radionuclide 111In

We investigated the generation of proton- and alpha-induced nuclear cross-section data in the production of Indium-111 (111In) for application in nuclear medicine. Here, we are interested in three reaction channels, which are 109Ag (α, 2n), 111Cd (p, n) and 112Cd (p, 2n), in the production of 111In. A random forest algorithm was used to generate nuclear cross-section data by using an experimental nuclear cross-section from the Experimental Nuclear Reaction Data (EXFOR) database as input. Hence, reasonably accurate regression curves of nuclear cross-section data could be produced with the evaluated nuclear data library ENDF/B-VII.0 set as the benchmark.