The Test and Protect Program: A Data-Driven, Community-Engaged Approach to COVID-19 Testing Site Localization.

As the COVID-19 pandemic progressed, reliable, accessible, and equitable community-based testing strategies were sought that did not flood already overburdened hospitals and emergency departments. In Hamilton County, Ohio, home to ~800 000 people across urban, suburban, and rural areas, we sought to develop and optimize an accessible, equitable county-wide COVID-19 testing program. Using Coronavirus Aid, Relief, and Economic Security Act funding, multidisciplinary, multiorganization partners created the test and protect program to deliver safe, reliable testing in neighborhoods and organizations needing it most. Our approach involved: (1) use of geospatial analytics to identify testing locations positioned to optimize access; (2) community engagement to ensure sites were in trusted places; and (3) tracking of data over time to facilitate ongoing improvement. Between August 2020 and December 2021, more than 65 000 tests were completed for nearly 46 000 individuals at community-based testing sites. These methods could have application beyond COVID-19 and our region.

Biotin reagents in antibody pretargeting. 6. Synthesis and in vivo evaluation of astatinated and radioiodinated aryl- and nido-carboranyl-biotin derivatives.

An investigation has been conducted to prepare and evaluate several radiohalogenated biotin derivatives as part of our studies to develop reagents for carrying (211)At in cancer pretargeting protocols. The primary goal of the investigation was to determine the in vivo stability and distribution properties of astatinated biotin derivatives. In addition to astatination, the biotin derivatives were radioiodinated for in vitro and in vivo comparison. Biodistributions were conducted in athymic mice, with sacrifice times of 1, 4, and 24 h to correspond to 9%, 32%, and 90% of (211)At decay (t(1/2) = 7.21 h). In the investigation, two biotin derivatives, 1a and 2a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, an ether linker moiety, and an aryl stannane moiety for radiohalogenation. Biotin derivatives 1a and 2a were radiolabeled with (125/131)I to give [(125)/(131)I]1b or [(125)I]2b and with (211)At to give [(211)At]1c or [(211)At]2c. In vivo studies demonstrated that co-injected [(125)I]2b and [(131)I]1b had very similar tissue distributions in athymic mice. Co-injection of [(211)At]2c and [(125)I]2b provided data that indicated that rapid deastatination occurred in vivo. A second set of biotin derivatives, 3a, 4a, and 5a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, and an anionic nido-carborane moiety for radiohalogenation. The biotin derivatives 4a and 5a contained an aryl moiety not present in 3a, and 5a had a trialkylamine functionality not present in 3a or 4a. Biotin derivative 3a was radioiodinated, but was not further investigated. Biotin derivatives 4a and 5a were radiolabeled with (211)At and (125)I to produce [(125)I]4b/[(211)At]4c and [(125)I]5b/[(211)At]5c. Comparison of [(125)I]4b and (separately) [(125)I]5b with [(131)I]1b showed that the nido-carborane containing biotin derivatives were retained in blood and tissue more than the aryl iodide derivative. In vivo evaluations of [(211)At]4c/[(125)I]4b and (separately) [(211)At]5c/[(125)I]5b indicated that some deastatination occurred in these compounds, but it was much less than observed for the aryl derivative [(211)At]2c. While the nido-carborane containing biotin derivatives provide a significant improvement in astatine stability over biotin derivatives previously studied, additional derivatives need to be prepared and studied to further improve the in vivo stability and blood/tissue clearance of these compounds.

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies.

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.

A streptavidin-biotin binding system that minimizes blocking by endogenous biotin.

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.

Reagents for astatination of biomolecules: comparison of the in vivo distribution and stability of some radioiodinated/astatinated benzamidyl and nido-carboranyl compounds.

An investigation has been conducted to assess the in vivo stability of a series of astatinated benzamides and astatinated nido-carborane compounds in mice. It was hypothesized that the higher bond strength of boron-astatine bonds in the nido-carboranes might provide increased stability toward in vivo deastatination. Four tri-n-butylstannylbenzamides were prepared for radiohalogenation and evaluation in vivo. Those compounds were N-propyl-4-(tri-n-butylstannyl)benzamide 1a, N-propyl-3-(tri-n-butylstannyl)benzamide 2a, ethyl 4-tri-n-butylstannylhippurate 3a, and 4-tri-n-butylstannyl-hippuric acid 4a. Seven mono-nido-carboranyl derivatives were prepared for radiohalogenation and in vivo evaluation. Four of the seven mono-carboranyl derivatives (5a, 6a, 7a, 13a) contained a 3-(nido-carboranyl)propionamide functionality, and the remaining compounds (8a, 8g, 10a) contained a 4-(nido-carboranyl)aniline functionality. Two additional derivatives (11a, 12a) were prepared that contained bis-(nido-carboranylmethyl)benzene moieties (also referred to as Venus flytrap complexes (VFCs). All benzamide and nido-carborane compounds underwent facile iodination and radiohalogenation, except a 4-(nido-carboranyl)aniline derivative, 8a. Iodination of 8a resulted in a mixture, of which the desired iodinated product was a minor component. Therefore, radiohalogenation was not attempted. It is believed that the mixture of products is due to the presence of a thiourea bond. Previous studies have shown that thiourea bonds can interfere with halogenation reactions. In vivo comparisons of the compounds were conducted by co-injection of dual labeled (125/131I and 211At) compounds. Tissue distribution data were obtained at 1 and 4 h postinjection of the radiolabeled compounds, as that was sufficient to determine if astatine was being released. Stability of the astatinated compound was assessed by the difference in concentration of radioiodine and astatine in lung and spleen. All of the benzamides were found to undergo rapid deastatination in vivo. The nido-carborane derivatives appeared to be slightly more stable to in vivo deastatination; however, they had long blood residence times. The surprising finding was that the VFC derivatives did not release 211At in vivo, even though they rapidly localized to liver. This finding provides encouragement that stable conjugates of 211At may be attained if appropriate modifications of the VFC can be made to redirect their excretion through the renal system.