Purpose: Using our chelate-free, heat-induced radiolabeling (HIR) method, we show that a wide range of metals, including those with radioactive isotopologues used for diagnostic imaging and radionuclide therapy, bind to the Feraheme (FH) nanoparticle (NP), a drug approved for the treatment of iron anemia. Material and methods: FH NPs were heated (120°C) with nonradioactive metals, the resulting metal-FH NPs were characterized by inductively coupled plasma mass spectrometry (ICP-MS), dynamic light scattering (DLS), and r 1 and r 2 relaxivities obtained by nuclear magnetic relaxation spectrometry (NMRS). In addition, the HIR method was performed with [ 90 Y]Y 3+ , [ 177 Lu]Lu 3+ , and [ 64 Cu]Cu 2+ , the latter with an HIR technique optimized for this isotope. Optimization included modifying reaction time, temperature, and vortex technique. Radiochemical yield (RCY) and purity (RCP) were measured using size exclusion chromatography (SEC) and thin-layer chromatography (TLC). Results: With ICP-MS, metals incorporated into FH at high efficiency were bismuth, indium, yttrium, lutetium, samarium, terbium and europium (>75% @ 120 o C). Incorporation occurred with a small (less than 20%) but statistically significant increases in size and the r 2 relaxivity. An improved HIR technique (faster heating rate and improved vortexing) was developed specifically for copper and used with the HIR technique and [ 64 Cu] Cu 2+. Using SEC and TLC analyses with [ 90 Y]Y 3+ , [ 177 Lu]Lu 3+ and [ 64 Cu]Cu 2+ , RCYs were greater than 85% and RCPs were greater than 95% in all cases. Conclusion: The chelate-free HIR technique for binding metals to FH NPs has been extended to a range of metals with radioisotopes used in therapeutic and diagnostic applications. Cations with f-orbital electrons, more empty d-orbitals, larger radii, and higher positive charges achieved higher values of RCY and RCP in the HIR reaction. The ability to use a simple heating step to bind a wide range of metals to the FH NP, a widely available approved drug, may allow this NP to become a platform for obtaining radiolabeled nanoparticles in many settings.

Serotonin 5-HT3 receptors are involved in various brain functions including as an emesis target during cancer chemotherapy. We report here the development of (S)-2,3-dimethoxy-5-(3′-[18F]fluoropropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide ([18F]fesetron) as a potential PET imaging agent for serotonin 5-HT3 receptors. By radiolabeling((S)-2,3-dimethoxy-5-(3′-tosyloxypropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide) with fluorine-18, (S)-[18F]fesetron was obtained in 5 to 10% decay-corrected yields and with specific activities >74 GBq/μmol at the end of radiosynthesis. PET imaging in rats showed low uptake of [18F]fesetron in the brain with retention of binding in the striatal and cerebellar regions. Using colliculi as a reference region, ratios were 3.4 for striata and 2.5 for cerebellum. Ex vivo brain PET analysis displayed binding of [18F]fesetron in the hippocampus, striatum and cerebellar regions. Cerebellar regions corresponded to area postrema and nucleus tract solitaris known to contain 5-HT3 receptors. Dorsal hippocampus showed the highest uptake with ratio of >17 with respect to colliculi, while area postrema and striata had ratios of >10. Thus, [18F]fesetron exhibited a unique binding profile to rat brain regions known to contain significant amounts of serotonin 5-HT3 receptors. However, the very low brain uptake limits its usefulness as a PET radiotracer in this animal model.

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ACKGROUND: Antiphospholipid syndrome (APS) is defined by arterial and/or venous thrombosis and/or pregnancy morbidity along with persistent circulating antiphospholipid antibodies (aPL). Central to APS pathogenesis are B cells that generate autoreactive antibodies, including anticardiolipin antibodies (aCL), and anti-β2-glycoprotein-I antibodies (anti-B2GPI). Several studies have implicated specific germline and/or antigen-driven somatic hypermutations within the complementarity determining regions (CDRs) of pathogenic antibodies in APS, as well as other thrombotic disorders. Nevertheless, the B cell repertoire in thrombotic APS remains poorly characterized. Recently, autoreactive and platelet activating pathogenic antibodies harboring elongated tyrosine rich CDR3 motifs were reported in patients with heparin-induced thrombocytopenia (HIT) (Zhu et.al, Blood 2019, 2020). Whether such antibodies are present in APS and their potential role, if so, has not been determined. APPROACH : We leveraged single cell immunoprofiling to characterize the presence of antibodies harboring elongated tyrosine rich CDR3 motifs in the circulating B cell repertoire obtained from peripheral blood mononuclear cells (PBMC) from patients with thrombotic (n=4) and catastrophic APS (n=3) as well as healthy individuals (n=3). We also correlated the abundance of these antibodies with criteria aPL and inflammatory cytokines in plasma. The modified Ham (mHam) test was used to assess functional complement activation in plasma in plasma from all of the subjects studied (Chaturvedi et al. Blood 2020). RESULTS : B cell clones containing elongated polytyrosine rich CDR3 motifs were most abundant in patients with a diagnosis of catastrophic APS (n=3) , and were markedly elevated in patients with APS (n=4) compared to healthy counterparts (n=3) (Table 1). Elongated polytyrosine rich CDR3 motifs from CAPS patients contained penta-tyrosine (Y5), hexa-tyrosine (Y6), hepta-tyrosine (Y7), and octa-tyrosine (Y8) motifs (table 2) and contained more tyrosine residues than the penta-tyrosines previously reported in HIT. In addition, all of the polytyrosine containing motifs CDR3 motifs were present on the heavy chain and was followed by MDVW motif of IGHJ6. The presence of B cell clones containing elongated polytyrosine rich CDR3 motifs exhibited significant positive correlations with complement activation measured by mHAM test (r=0.728, p=0.0032), criteria aPLs including anticardiolipin antibodies (aCL) and anti-β2-glycoprotein-I antibodies (anti-B2GPI), as well as plasma inflammatory cytokines; TNFα (r=0.901, p=1.5x10 -5), IL-23 (r=0.780, p=0.0016), IL-17C cytokines (r=0.729, p=0.0033), sICAM1 (r=0.740, p=0.0025), sVCAM1 (r=0.764, p=0.0015), (Figure 1). The presence of elongated polytyrosine rich CDR3 motifs was also associated with the increase abundance of polyserine rich CDR3 motifs that were present predominantly in patients with CAPS. CONCLUSION : These preliminary studies provide the first characterization of the prevalence of B cell clones containing elongated polytyrosine rich CDR3 motifs, previously reported in HIT, in the B cell repertoire of APS patients. Notably, the abundance of these B cell clones is markedly elevated in patients with CAPS and is associated with elevated levels of inflammatory cytokines TNFa, IL-23, IL-17C, sICAM1, and sVCAM1. Validation studies in larger cohorts from APS patients and other thrombotic disorders are ongoing. Figure 1 Figure 1. Disclosures Chaturvedi: Alexion: Other: Advisory board member; Dova: Other: Advisory board member; Sanofi Genzyme: Other: Advisory board member; Argenx: Other: Advisory board member; UCB: Other: Advisory board participation. Khorana: Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Anthos: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. McCrae: Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria; Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy.