Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment.

In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. Here we present INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity vary across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identifies a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing reveals a connection with cDC1s, and immunofluorescence confirms nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assist binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties.

Response of Canine Soft Tissue Sarcoma to Stereotactic Body Radiotherapy.

Canine soft tissue sarcoma (STS) has served as a preclinical model for radiation, hyperthermia, experimental therapeutics, and tumor microenvironmental research for decades. Stereotactic body radiotherapy (SBRT) demonstrates promising results for the control of various tumors in human and veterinary medicine; however, there is limited clinical data for the management of STS with SBRT. In this retrospective study, we aimed to define overall efficacy and toxicity of SBRT for the treatment of macroscopic canine STS to establish this preclinical model for comparative oncology research. Fifty-two canine patients met inclusion criteria. Total radiation dose prescribed ranged from 20-50 Gy delivered in 1-5 fractions. Median progression-free survival time (PFST) was 173 days and overall survival time (OST) 228 days. Best overall response was evaluable in 46 patients, with 30.4% responding to treatment (complete response n = 3; partial response n = 11). For responders, OST significantly increased to 475 days vs. 201 days (P = 0.009). Prognostic factors identified by multivariable Cox regressions included size of tumor and metastasis at presentation. Dogs were 3× more likely to progress (P = 0.009) or 3.5× more likely to experience death (P = 0.003) at all times of follow up if they presented with metastatic disease. Similarly, every 100-cc increase in tumor volume resulted in a 5% increase in the risk of progression (P = 0.002) and death (P = 0.001) at all times of follow up. Overall, 30.8% of patients developed acute toxicities, 7.7% grade 3; 28.8% of patients developed late toxicities, 11.5% grade 3. Increased dose administered to the skin significantly affected toxicity development. SBRT serves as a viable treatment option to provide local tumor control for canine macroscopic STS, particularly those with early-stage disease and smaller tumors. The results of this study will help to define patient inclusion criteria and to set dose limits for preclinical canine STS trials involving SBRT.

Isoniazid: Radical-induced oxidation and reduction chemistry.

Isoniazid is a potent and selective therapeutic prodrug agent used to treat infections by Mycobacterium tuberculosis. Although it has been used clinically for over five decades its full mechanism of action is still being elucidated. Essential to its mechanism of action is the activation of isoniazid to a reactive intermediate, the isonicotinyl acyl radical, by the catalase-peroxidase KatG. The isonicotinyl acyl radical then reacts with NAD producing an inhibitor of the NADH-dependent enoyl ACP reductase responsible for mycolic acid synthesis as its primary target. However, the initial oxidation of isoniazid by KatG has also revealed alternative reaction pathways leading to an array of carbon-, oxygen-, and nitrogen-centered radical intermediates. It has also been reported that isoniazid produces nitric oxide in the presence of KatG and hydrogen peroxide. In this study, the temperature-dependent rate constants for the hydroxyl radical oxidation and solvated electron reduction of isoniazid and two model compounds have been studied. Based on these data the initial oxidation of isoniazid by the hydroxyl radical has been shown to predominantly occur at the primary nitrogen of the hydrazyl moiety, consistent with the postulated mechanism for the formation of the isonicotinyl radical. The hydrated electron reduction occurred mostly at the pyridine ring. Concomitant EPR spin-trap measurements under a variety of oxidizing and reducing conditions did not show any evidence of nitric oxide production as had been previously reported. Finally, examination of the transient absorption spectra obtained for hydrated electron reaction with isoniazid demonstrated for the first time an initial reduced transient identified as the isonicotinyl acyl radical produced from isoniazid.

Radical-based destruction of nitramines in water: kinetics and efficiencies of hydroxyl radical and hydrated electron reactions.

In support of the potential use of advanced oxidation and reduction process technologies for the removal of carcinogenic nitro-containing compounds in water reaction rate constants for the hydroxyl radical and hydrated electron with a series of low molecular weight nitramines (R(1)R(2)-NNO(2)) have been determined using a combination of electron pulse radiolysis and transient absorption spectroscopy. The hydroxyl radical reaction rate constant was fast, ranging from 0.54-4.35 × 10(9) M(-1) s(-1), and seen to increase with increasing complexity of the nitramine alkyl substituents suggesting that oxidation primarily occurs by hydrogen atom abstraction from the alkyl chains. In contrast, the rate constant for hydrated electron reaction was effectively independent of compound structure, (k(av) = (1.87 ± 0.25) × 10(10) M(-1) s(-1)) indicating that the reduction predominately occurred at the common nitramine moiety. Concomitant steady-state irradiation and product measurements under aerated conditions also showed a radical reaction efficiency dependence on compound structure, with the overall radical-based degradation becoming constant for nitramines containing more than four methylene groups. The quantitative evaluation of these efficiency data suggest that some (~40%) hydrated electron reduction also results in quantitative nitramine destruction, in contrast to previously reported electron paramagnetic measurements on these compounds that proposed that this reduction only produced a transient anion adduct that would transfer its excess electron to regenerate the parent molecule.

Absolute kinetics and reaction efficiencies of hydroxyl-radical-induced degradation of methyl isothiocyanate (MITC) in different quality waters.

Methyl isothiocyanate (MITC), a toxic and corrosive skin and respiratory irritant, is a common soil fumigant byproduct which has become an atmospheric, aqueous, and soil contaminant. The work described here examines the degradation and potential removal of MITC from contaminated waters via free radical reactions. We have measured the oxidizing hydroxyl radical ((·)OH) reaction rate constant with MITC over a range of temperatures relevant to wastewater treatment conditions, determining a room temperature value of (5.69±0.56) x 10(8)M(-1)s(-1) and a corresponding Arrhenius activation energy of 12.90±0.82 kJ mol(-1). Hydroxyl radical reaction efficiencies with MITC in pure water, an associated matrix of model real-world waters, and a reverse osmosis permeate water have also been determined. While solutions containing these constituents had significantly decreased MITC removal efficiencies (5.5-14.7%) as compared to pure water (54.4±3.4%), relative rate calculation corrections showed that the (·)OH radical efficiencies for solutions containing DOM were the same as in pure water. However, the slightly higher efficiencies for carbonate-containing solutions indicated that some additional MITC degradation occurred from carbonate radical reactions.

Free radical-induced redox chemistry of Nedaplatin and Satraplatin under physiological conditions.

Temperature-dependent kinetics for the reactions of hydroxyl radicals and hydrated electrons with the anti-cancer drug nedaplatin have been determined using a combination of electron pulse radiolysis and absorption spectroscopy. Under physiological pH and chloride concentrations, the kinetics was well described by the equations [Formula: see text]and [Formula: see text]corresponding to Arrhenius activation energies of 15.88 +/- 1.16 and 14.14 +/- 1.41 kJ mol(-1) for hydroxyl radical oxidation and hydrated electron reduction, respectively. Through a comparison of spectral and kinetic literature it is believed that the oxidation reaction gives predominantly an intermediate Pt(III) species, whereas reduction gives a Pt(I) moiety. Analogous hydrated electron measurements for the Pt(IV) drug satraplatin showed multiple-component decays at higher temperatures (>20 degrees C), indicating that significant thermal degradation of this chemical occurs. From double-exponential curve fitting, the satraplatin reduction kinetics was found to be well described by the equation [Formula: see text]giving an activation energy of 22.78 +/- 1.78 kJ mol(-1) for this reaction. This measured temperature dependence was consistent with several model Pt(IV) compounds also investigated in this study, with all these data suggesting that the metal ion reduction to give Pt(III) was the dominant reaction occurring.

Free radical-induced redox chemistry of platinum-containing anti-cancer drugs.

Arrhenius parameters for the reactions of oxidizing hydroxyl radicals and reducing hydrated electrons with cisplatin, transplatin and carboplatin in aqueous solution have been determined using pulsed electron radiolysis and absorption spectroscopy techniques. Under physiological pH and chloride concentration conditions, hydroxyl radical reaction rate constants of (9.99 +/- 0.20) x 10(9), (8.38 +/- 0.55) x 10(9), and (6.03 +/- 0.08) x 10(9) M(-1) s(-1) at 24.0, 20.7 and 24.0 degrees C, respectively, with corresponding activation energies of 12.79 +/- 0.57, 13.88 +/- 1.14, and 14.35 +/- 0.56 kJ mol(-1) for these three reactions, were determined. These oxidations of cisplatin and transplatin to form a Pt(III) transient are significantly faster than reported previously at room temperature. The lower rate constant for carboplatin is consistent with hydroxyl radical reaction partitioning between reaction at the platinum center and the cyclobutanedicarboxylate ligand. The equivalent reductive hydrated electron reaction rate constants measured were (1.99 +/- 0.04) x 10(10) (24.0 degrees C), (1.77 +/- 0.08) x 10(10) (22.0 degrees C), and (8.92 +/- 0.06) x 10(9) M(-1) s(-1) (24.0 degrees C), with corresponding activation energies of 15.75 +/- 1.00, 19.74 +/- 1.82, and 19.99 +/- 0.34 kJ mol(-1). Again, the values determined for cisplatin and transplatin are faster than reported; however, all three values are consistent with direct reduction of the platinum center to form a Pt(I) moiety.

Free radical chemistry of advanced oxidation process removal of nitrosamines in water.

Absolute rate constants and degradation efficiencies for hydroxyl radical reactions with seven low-molecular-weight nitrosamines in water have been evaluated using a combination of electron-pulse radiolysis/absorption spectroscopy and steady-state radiolysis/GCMS measurements. The hydroxyl radical oxidation rate constants were found to depend upon nitrosamine size and to have a very good linear correlation with the number of methylene groups in these compounds. This correlation, given by In(k x OH) = (19.72 +/- 0.14) + (0.424 +/- 0.033) (#CH2), suggests that hydroxyl radical oxidation predominantly occurs by hydrogen atom abstraction from constituent methylene groups in each of these nitrosamines. In contrast, the hydrated electron reduction rate constants measured for these compounds were remarkably consistent, with an average value of (1.67 +/- 0.22) x 10(10) M(-1) s(-1). These reduction kinetic data are consistent with this predominantly diffusion-controlled reaction occurring at the N-NO moiety in these carcinogens. From steady-state radiolysis measurements under aerated conditions, specific hydroxyl radical degradation efficiencies for each nitrosamine were evaluated. For larger nitrosamines, the efficiency was constant at 100%; however, for the smaller alkyl substituted species, the efficiency was significantly lower, with a minimum value of only 80% determined for N-nitrosodimethylamine. The reduced efficiency is attributed to radical repair reactions competing with the slow peroxyl radical formation.