Saturable Binding Research Articles

Comparison of ultrafiltration and microdialysis for ceftriaxone protein-binding determination.

High protein binding (PB) of antibiotics has an impact on their antimicrobial activity. It has been questioned whether in vitro PB determination can capture the dynamic and concentration-dependent PB of highly bound antibiotics. This clinical study compared in vitro ultrafiltration (UF) and in vivo IV microdialysis (MD) methods to determine ceftriaxone PB. Six healthy male volunteers received a single IV 2 g dose of ceftriaxone. Unbound ceftriaxone plasma concentrations were measured with MD and venous plasma sampling with subsequent UF. Pharmacokinetic parameters were determined using non-compartmental pharmacokinetic analysis. Non-linear mixed-effects modelling was used to quantify the PB. The PTA was estimated. The Cmax of ceftriaxone total plasma concentration (297.42 ± 21.0 mg/L) was approximately 5.5-fold higher than for free concentrations obtained with UF (52.83 ± 5.07 mg/L), and only 3.5-fold higher than for free concentrations obtained with MD (81.37 ± 26.93 mg/L). Non-linear, saturable PB binding was confirmed for both UF and MD. Significantly different dissociation constants (Kd) for the albumin/ceftriaxone complex were quantified: in UF it was 23.7 mg/L (95% CI 21.3-26.2) versus 15.9 mg/L (95% CI 13.6-18.6) in MD. Moreover, the estimated number of binding sites (95% CI) per albumin molecule was 0.916 (0.86-0.97) in UF versus 0.548 in MD (0.51-0.59). The PTA obtained with MD was at most 27% higher than with UF. In vitro UF versus in vivo intravasal MD revealed significantly different PB, especially during the distribution phase. The method of PB determination could have an impact on the breakpoint determination and dose optimisation of antibiotics.

Prospective population pharmacokinetic study of tacrolimus in adult recipients early after liver transplantation: A comparison of Michaelis-Menten and theory-based pharmacokinetic models.

Background and Objective: Tacrolimus, a calcineurin inhibitor widely used as a potent immunosuppressant to prevent graft rejection, exhibits nonlinear kinetics in patients with kidney transplantation and nephrotic syndrome. However, whether nonlinear drug metabolism occurs in adult patients undergoing liver transplantation remains unclear, as do the main underlying mechanisms. Therefore, here we aimed to further confirm the characteristics of nonlinearity through a large sample size, and determine the potential influence of nonlinearity and its possible mechanisms. Methods: In total, 906 trough concentrations from 176 adult patients (150 men/26 women; average age: 50.68 ± 9.71 years, average weight: 64.54 ± 11.85kg after first liver transplantation) were included in this study. Population pharmacokinetic analysis was performed using NONMEM®. Two modeling strategies, theory-based linear compartmental and nonlinear Michaelis-Menten (MM) models, were evaluated and compared. Potential covariates were screened using a stepwise approach. Bootstrap, prediction-, and simulation-based diagnostics (prediction-corrected visual predictive checks) were performed to determine model stability and predictive performance. Finally, Monte Carlo simulations based on the superior model were conducted to design dosing regimens. Results: Postoperative days (POD), Aspartate aminotransferase (AST), daily tacrolimus dose, triazole antifungal agent (TAF) co-therapy, and recipient CYP3A5*3 genotype constituted the main factors in the theory-based compartmental final model, whereas POD, Total serum bilirubin (TBIL), Haematocrit (HCT), TAF co-therapy, and recipient CYP3A5*3 genotype were important in the nonlinear MM model. The theory-based final model exhibited 234Lh-1 apparent plasma clearance and 11,000L plasma distribution volume. The maximum dose rate (V max ) of the nonlinear MM model was 6.62 mgday-1; the average concentration at steady state at half-V max (K m ) was 6.46ngml-1. The nonlinear MM final model was superior to the theory-based final model and used to propose dosing regimens based on simulations. Conclusion: Our findings demonstrate that saturated tacrolimus concentration-dependent binding to erythrocytes and the influence of daily tacrolimus dose on metabolism may partly contribute to nonlinearity. Further investigation is needed is need to explore the causes of nonlinear pharmacokinetic of tacrolimus. The nonlinear MM model can provide reliable support for tacrolimus dosing optimization and adjustment in adult patients undergoing liver transplantation.

Synthesis, radiolabeling and evaluation of [99mTc][Tc-HYNIC/EDDA]-Met(O) as a early agent for amino acid metabolic imaging in C6 glioblastoma tumor

Amino acid metabolism is recognized as a target for medical imaging due to its increase in malignant cells. Several radiotracers with primary achievement and possible subsequent chances have been designed and tested to image amino acid metabolism. Here, we report a new amino acid conjugate, with the purpose of extending [99mTc][Tc-HYNIC/EDDA]-Met(O) for single photon emission tomography (SPECT) imaging.The S-oxo-l-methionine (Met(O)) amino acid hydrazinonicotinamide (HYNIC) chelator conjugate (HYNIC-Met(O)) was prepared, using Fmoc solid-phase synthesis, and was radiolabeled with [99mTc]technetium pertechnetate, using tricine and ethylenediamine-N,N-diacetic acid (EDDA) as co-ligands. In vitro cellular uptake profile and saturation binding of radiotracer were determined on C6 glioma cells. Biodistribution and imaging studies were carried out on rat bearing C6 tumor tissue grafts.[99mTc][Tc-HYNIC/EDDA]-Met(O) was prepared in high yield and radiochemical purity (>98 %). The partition coefficient result showed that radioconjugate was very hydrophilic. The radioconjugate indicated both high cell uptake and in vitro internalization. Low nanomolar dissociation constant (66.02 nM) in C6 glioma cells was obtained for it as well. [99mTc][Tc-HYNIC/EDDA]-Met(O) revealed magnificent tumor uptake at early time points, with 1.98 ± 0.33 % injected activity per gram tumor (% IA/g) at 30 min post injection. The tumor uptake continued for 1 and 2 h and was 0.45 ± 0.33 % IA/g at 4 h. The uptake in other organs decreased much more rapidly causing high tumor to normal organ ratios so that the highest ratio of 13.25 of tumor-to-muscle at 60 min after injection was obtained with high contrast in gamma imaging.These results point out a very favorable [99mTc]Tc-labeled amino acid for targeting amino acid metabolism through target system L amino acid transporter (LAT1) in malignant cells especially C6 glioma cells. [99mTc][Tc-HYNIC/EDDA]-Met(O) manifests extremely good distribution, excretion and imaging attributes. So it seems to be an appropriate nominate for clinical imaging.

Insertion of Nanoluc into the Extracellular Loops as a Complementary Method To Establish BRET-Based Binding Assays for GPCRs.

Luminescence-based techniques play an increasingly important role in all areas of biochemical research, including investigations on G protein-coupled receptors (GPCRs). One quite recent and popular addition has been made by introducing bioluminescence resonance energy transfer (BRET)-based binding assays for GPCRs, which are based on the fusion of nanoluciferase (Nluc) to the N-terminus of the receptor and the occurring energy transfer via BRET to a bound fluorescent ligand. However, being based on BRET, the technique is strongly dependent on the distance/orientation between the luciferase and the fluorescent ligand. Here we describe an alternative strategy to establish BRET-based binding assays for GPCRs, where the N-terminal fusion of Nluc did not result in functioning test systems with our fluorescent ligands (e.g., for the neuropeptide Y Y1 receptor (Y1R) and the neurotensin receptor type 1 (NTS1R)). Instead, we introduced Nluc into their second extracellular loop and we obtained binding data for the fluorescent ligands and reported standard ligands (in saturation and competition binding experiments, respectively) comparable to data from the literature. The strategy was transferred to the angiotensin II receptor type 1 (AT1R) and the M1 muscarinic acetylcholine receptor (M1R), which led to affinity estimates comparable to data from radioligand binding experiments. Additionally, an analysis of the binding kinetics of all fluorescent ligands at their respective target was performed using the newly described receptor/Nluc-constructs.

Effect of Iron Deficiency Anemia on Glycated Albumin Levels: A Comparative Study in Nondiabetic Subjects with Iron Deficiency Anemia.

Objective Glycated hemoglobin A1c (HbA1c), used for monitoring glycemia control, is altered in iron deficiency anemia (IDA). Glycated albumin (GA) is considered an alternate biomarker to HbA1c. However, effect of IDA on GA needs to be studied. Materials and Methods Thirty nondiabetic cases with IDA and 30 healthy controls were included. Fasting plasma glucose (FPG), creatinine, urea, albumin, total protein, ferritin, iron, unsaturated iron binding capacity, hemoglobin (Hb), HbA1c, complete hemogram, and GA were estimated. Transferrin saturation and total iron binding capacity (TIBC) were calculated. Statistical analysis was done using unpaired two-tailed t -test/Mann-Whitney U -test and Pearson's correlation/Spearman-rank correlation, as appropriate. Results Total protein, albumin, Hb, iron, ferritin, and transferrin saturation were significantly lower while FPG, GA, TIBC, and HbA1c were significantly higher in cases compared to controls. HbA1C and GA have a significant negative correlation with iron, transferrin saturation, and ferritin. Significant negative correlations of GA with albumin ( r = -0.754; p < 0.001) and Hb ( r = -0.435; p = 0.001) and that of HbA1c with albumin ( r = -0.271; p = 0.03) and Hb ( r = -0.629; p < 0.001) while significant positive correlation of Hb with albumin ( r = 0.395; p = 0.002) and HbA1c with FPG ( r = 0.415; p = 0.001) were observed. Conclusion Low albumin levels increase plasma protein glycation, including albumin. Hence, elevated GA levels indicate false elevation of GA in scenario of lowered albumin observed in IDA, similar to HbA1c. Thus, using GA in diabetes mellitus with IDA should be avoided or used with caution to prevent potentially inappropriate treatment intensification and risk of hypoglycemia.

Sporozoite motility as a quantitative readout for anti-CSP antibody inhibition

Antibodies can prevent malaria by neutralizing the infectious Plasmodium falciparum sporozoites (SPZ) before they establish an infection in the liver. Circumsporozoite protein (CSP), the most abundant surface protein of SPZ is the leading candidate for passive (and subunit) immunization approaches against malaria. Comprehensive assessment of the parasite-inhibitory capacity of anti-CSP monoclonal antibodies (mAbs) is an important step in advancing CSP-based immunization strategies. In this study, we employed a quantitative imaging-based motility assay to quantify the effect of anti-CSP mAbs on SPZ motility, both in vitro and in human skin.Our assay provided a quantitative measure of mAb parasite-inhibitory capacity through measurement of the half-maximal motility inhibitory concentration (IC50M) value for anti-CSP mAbs (IC50M 2A10: 24 nM, IC50M 3SP2: 71 nM). We found a sevenfold discrepancy between the IC50M and the binding saturation concentration measured by ELISA, possibly related to the observed shedding of CSP-mAb complexes during SPZ movement. In a subset of SPZ (5%), in vitro motility was unaffected by the presence of 2A10 while 3SP2 was able to completely block movement. In our ex vivo skin explant model, SPZ proved less susceptible to anti-CSP mAbs compared to SPZ in an in vitro environment. By quantitatively assessing motility, we created a valuable tool that can be used for comprehensive assessment of anti-CSP mAb potency. Insight that will help deepen our understanding of anti-CSP mAb potency and guide selection of the most promising anti-CSP mAbs for downstream clinical development.

Iron status and the risk of sepsis and severe COVID-19: a two-sample Mendelian randomization study

Observational studies have indicated an association between iron status and risk of sepsis and COVID-19. We estimated the effect of genetically-predicted iron biomarkers on risk of sepsis and risk of being hospitalized with COVID-19, performing a two-sample Mendelian randomization study. For risk of sepsis, one standard deviation increase in genetically-predicted serum iron was associated with odds ratio (OR) of 1.14 (95% confidence interval [CI] 1.01–1.29, P = 0.031). The findings were supported in the analyses for transferrin saturation and total iron binding capacity, while the estimate for ferritin was inconclusive. We found a tendency of higher risk of hospitalization with COVID-19 for serum iron; OR 1.29 (CI 0.97–1.72, P = 0.08), whereas sex-stratified analyses showed OR 1.63 (CI 0.94–2.86, P = 0.09) for women and OR 1.21 (CI 0.92–1.62, P = 0.17) for men. Sensitivity analyses supported the main findings and did not suggest bias due to pleiotropy. Our findings suggest a causal effect of genetically-predicted higher iron status and risk of hospitalization due to sepsis and indications of an increased risk of being hospitalized with COVID-19. These findings warrant further studies to assess iron status in relation to severe infections, including the potential of improved management.

Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods

BackgroundReplacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen.ResultsHere we generated highly soluble and stable recombinant protein constructs GST-CB1414–472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB1 receptor that corresponds to residues 442–472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay.ConclusionsCollectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

Adsorption and detoxification of glyphosate and aminomethylphosphonic acid by montmorillonite clays.

The co-occurrence of mixtures of glyphosate (GLP) and aminomethylphosphonic acid (AMPA) in contaminated water, soil, sediment, and plants is a cause for concern due to potential threats to the ecosystem and human health. Major routes of exposure include contact with contaminated water and soil and through consumption of crops containing GLP and AMPA residues. Calcium montmorillonite (CM) and acid-processed montmorillonite (APM) clays were investigated for their ability to tightly sorb and detoxify GLP and AMPA mixtures. In vitro adsorption and desorption isotherms and thermodynamic analysis indicated saturable Langmuir binding of both chemicals with high capacities, affinities, enthalpies, and free energies of sorption and low desorption rates. In silico computational modeling indicated that both GLP and AMPA can be readily absorbed onto clay surfaces through electrostatic interactions and hydrogen bonding. The safety and efficacy of the clays were confirmed using well-established living organisms, including an aquatic cnidarian (Hydra vulgaris), a soil nematode (Caenorhabditis elegans), and a floating plant (Lemna minor). Low levels of clay inclusion (0.05% and 0.2%) in the culture medium resulted in increased growth and protection against chemical mixtures based on multiple endpoints. Results indicated that montmorillonite clays may be used to bind mixtures of GLP and AMPA in water, soil, and plants.

Introducing micro physiological systems to evaluate new radiopharmaceuticals: A binding study with radiolabeled cetuximab

Abstract Radiopharmaceuticals can be used for targetspecific functional diagnostics, such as PET or SPECT imaging, or radionuclide therapy of diseased tissue, depending on the incorporated radionuclide. Following initial in vitro testing, radiopharmaceutical candidates are usually further characterized in small animals. Since reduction, replacement and refinement (3R) of animal testing is a central precept in preclinical research it would be beneficial to replace at least some of these tests by alternative methods. Using micro physiological system technology, various organ-on-chip models can be created with human cell systems/organoids, which are operated in a circulatory system under defined physiological conditions. Here we present first attempts to introduce micro physiological systems for evaluating radiopharmaceuticals using the radiolabeled anti-EGFR antibody cetuximab as reference compound. In a micro physiological system equipped with six 96-well plate-like microwells in a flow chamber, binding of 64Cu and 68Ga-labeled cetuximab to cells and spheroids grown from A431 (EGFR-positive) and MDA-MB435S (EGFR-negative) cells was measured and compared to conventional microplates. Specific saturation binding of radiolabeled cetuximab at increasing concentrations was analyzed using a phosphor imaging system. The affinity of radiolabeled cetuximab towards A431 spheroids measured in the micro physiological system was in the same range as that of the spheroids in conventional microplates. Within the assays in micro physiological systems, the results showed a trend towards increased affinity for A431 monolayers compared to the spheroids. The values of binding capacity for radiolabeled cetuximab on 2D and 3D A431 cell culture models were in the same order of magnitude when measured in micro physiological systems or in microplates. Building on these first promising results, the work will continue on MPS modules containing advanced human spheroid/ organoid models.

A Population Pharmacokinetic Modelling Approach to Unravel the Complex Pharmacokinetics of Vincristine in Children

BackgroundVincristine, a chemotherapeutic agent that extensively binds to β-tubulin, is commonly dosed at 1.4–2.0 mg/m2 capped at 2 mg. For infants, doses vary from 0.025–0.05 mg/kg or 50–80% of the mg/m2 dose. However, evidence for lower doses in infants compared to older children is lacking. This study was conducted to unravel the complex pharmacokinetics of vincristine, including the effects of age, to assist optimal dosing in this population.Methods206 patients (0.04–33.9 years; 25 patients < 1 years), receiving vincristine, with 1297 plasma concentrations were included. Semi-mechanistic population pharmacokinetic analyses were performed using non-linear mixed effects modelling.ResultsA three-compartment model, with one saturable compartment resembling saturable binding to β-tubulin and thus, saturable distribution, best described vincristine pharmacokinetics. Body weight and age were covariates significantly influencing the maximal binding capacity to β-tubulin, which increased with increasing body weight and decreased with increasing age. Vincristine clearance (CL) was estimated as 30.6 L/h (95% confidence interval (CI) 27.6–33.0), intercompartmental CL (Q) as 63.2 L/h (95%CI 57.2–70.1), volume of distribution of the central compartment as 5.39 L (95%CI 4.23–6.46) and of the peripheral compartment as 400 L (95%CI 357–463) (all parameters correspond to a patient of 70 kg). The maximal binding capacity was 0.525 mg (95%CI 0.479–0.602) (for an 18 year old patient of 70 kg), with a high association rate constant, fixed at 1300 /h and a dissociation constant of 11.5 /h.InterpretationA decrease of vincristine β-tubulin binding capacity with increasing age suggests that young children tolerate higher doses of vincristine.

Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry.

Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (Kd = 1.7 μM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular Ki = 22 nM), docetaxel (cellular Ki = 16 nM), cabazitaxel (cellular Ki = 6 nM), and ixabepilone (cellular Ki = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (Kb) of the microtubule destabilizers colchicine (Kb = 80 nM, α = 0.08), vinblastine (Kb = 7 nM, α = 0.18), and maytansine (Kb = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (Kb = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.

Streptococcal autolysin promotes dysfunction of swine tracheal epithelium by interacting with vimentin.

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium.

Structures, temperature effect, binding and clustering energies of Cu2+(MeOH)n=1-8 clusters and extrapolations

• The structure of solvated copper (II) ion in methanol is tetra and hexa-coordinated, depending on the temperature. • Compact and high coordinated structures dominate the population at all temperatures. • Absolute clustering free energies of solvated metal ions neither depend on the metal, nor on the ligand type. Copper plays an important role in many physiological biochemical and biological processes. This work sets out to investigate structures, relative stabilities, temperature-dependence of the relative population, saturated binding and clustering energies of the solvated Cu 2 + ion in methanol, using ab initio MP2 method associated with the 6-31++G** basis set. The isomers of the Cu 2 + ( MeOH ) n = 1 - 8 clusters are reported here for the first time in the whole literature, and relative stability obeys a well-defined stability rule. It is worth mentioning that the solvated Cu 2 + ion in methanol is tetra- or hexa-coordinated, depending on the temperature. The theoretical approach used in this work, combined with the structures pointed out and their corresponding relative populations leads to equatorial distances d Cu 2 + - O in methanol, in excellent agreement with experimental results. As far as temperature dependence isomers distribution are concerned, compact and high coordinated structures dominate the population of the studied clusters at all temperatures. We also predicted binding and clustering energies as a function of the cluster size, up to n = ∞ . The convergence, called the saturation, happens around n = 18 for clustering energies of Cu 2 + ion in methanol. For the case as ammonia and water as solvent, this saturation is reached at n = 20 and n = 25 , respectively. Thus, accurate solvation energies of Cu 2 + ion in those media using cluster continuum model could be derived clustering at least up to n = 18 , 20 and 25 respectively for methanol, ammonia and water as solvent. The corresponding binding electronic energy, binding enthalpy and binding free energy per methanol molecule are respectively, −158.9, −166.2 and −134.6 kJ mol −1 . As far as clustering energies per solvent molecule at the saturation are concerned, we reported −7.0, −6.5 and −2.9 kJ mol −1 for respectively the clustering electronic energy, clustering enthalpy and clustering free energy per methanol molecule of the solvated Cu 2 + ion.

Unravelling the Potential of Lutetium-177 Labeled Pertuzumab: Targeting HER 2 Receptors for Theranostic Applications

<h2>ABSTRACT</h2><h3>Purpose</h3> Pertuzumab, a humanized antibody against HER2 receptors has shown promising results in combination with trastuzumab and docetaxel for the neoadjuvant treatment of patients with HER2-positive breast cancer. The aim of the present study was to demonstrate the potential of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab for targeting HER2 positive tumors. Comparative studies with <b>[</b>¹⁷⁷Lu]-Lu-DTPA-trastuzumab and synergism in presence of cold trastuzumab was also envisaged. <h3>Methods & Materials</h3> Pertuzumab and trastuzumab were conjugated with p-NCS-CHX-A''-DTPA and radiolabeled with Lutetium-177. The stability of the formulations was assessed and <i>in-vitro</i> binding parameters (immunoreactivity, saturation and competitive binding) were examined in HER2 overexpressing and HER2 negative cell lines. SPECT imaging studies were performed in SCID mice bearing HER2 positive tumors. <h3>Results</h3> <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab and trastuzumab could be prepared with >95% radiochemical purity (% RCP). <i>In-vitro</i> binding studies revealed high affinity and specificity of the formulations towards HER2 receptors. Specific tumor uptake of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab in HER2 positive tumors was observed by SPECT imaging studies in tumor bearing mice, comparable to <b>[</b>¹⁷⁷Lu]-Lu-DTPA-trastuzumab. Significant retention of the formulations was observed in the tumor for up to 120 h. The studies also showed enhanced uptake of <b>[</b>¹⁷⁷Lu]-Lu-DTPA-pertuzumab in the presence of cold trastuzumab in HER2 positive tumors. <h3>Conclusions</h3> The present study revealed significant uptake of both ¹⁷⁷Lu labeled pertuzumab and trastuzumab in HER2 positive tumors. Further studies are warranted to determine efficacy of these formulations against tumor growth for confirming their potential as effective radioimmunotheranostic agents. Increase in uptake of radiolabeled pertuzumab in presence cold trastuzumab provides an avenue for more effective radionuclide imaging and therapy of HER2 positive tumors.

Synthesis and In Vivo Evaluation of a Site-specifically Labeled Radioimmunoconjugate for Dual-Modal (PET/NIRF) Imaging of MT1-MMP in Sarcomas.

Bone sarcomas are devastating primary bone cancers that mostly affect children, young adults, and the elderly. These aggressive tumors are associated with poor survival, and surgery remains the mainstay of treatment. Surgical planning is increasingly informed by positron emission tomography (PET), and tumor margin identification during surgery is aided by near-infrared fluorescence (NIRF) imaging, yet these investigations are confounded by probes that lack specificity for sarcoma biomarkers. We report the development of a dual-modal (PET/NIRF) immunoconjugate ([89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW) that targets MT1-MMP, a matrix metalloproteinase overexpressed in high-grade sarcomas. [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW was synthesized via site-specific chemoenzymatic glycan modification, characterized, and isolated in high specific activity and radiochemical purity. Saturation binding and immunoreactivity assays indicated only minor perturbation of binding properties. A novel mouse model of dedifferentiated chondrosarcoma based on intrafemoral inoculation of HT1080 WT or KO cells (high and low MT1-MMP expression, respectively) was used to evaluate target binding and biodistribution. Fluorescence and Cerenkov luminescence images of [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW showed preferential uptake in HT1080 WT tumors. Ex vivo gamma counting revealed that uptake in MT1-MMP-positive tumors was significantly higher than that in control groups. Taken together, [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW is a promising dual-modal sarcoma imaging agent for pre-operative surgical planning and intraoperative surgical guidance.

Structure-Activity Relationships and Pharmacokinetics of 111In-Labeled Glucagon-like Peptide-1 Receptor-Targeting Exendin-4 Derivatives Conjugated with Albumin Binder Moieties.

Insulinomas are neuroendocrine tumors that are derived from pancreatic β-cells, and they often overexpress the glucagon-like peptide-1 receptor (GLP-1R). Radiolabeled exendin-4 derivatives have been used to noninvasively detect the GLP-1R during the diagnosis and preoperative localization of insulinomas; however, their marked renal accumulation can hinder the imaging of pancreatic tail lesions. In this study, we designed and synthesized 111In-labeled exendin-4 derivatives that possessed 4-(4-substituted phenyl)-moieties as albumin binder (ALB) moieties ([111In]In-E4DA2-4), and studied their structure-activity relationships and pharmacokinetics (as well as those of [111In]In-E4DA1, which we previously reported) to determine their usefulness as radioligands for GLP-1R imaging. 111In-labeling was performed by reacting maleimide precursors with [111In]InCl3 in 2-(N-morpholino)ethanesulfonic acid buffer, and then, the products were conjugated with exendin-4-Cys40. A saturation binding assay using GLP-1R-expressing INS-1 cells was carried out to evaluate the in vitro affinity of the radioligands for the cells. In addition, the affinity of the 111In-labeled derivatives for human serum albumin (HSA) was evaluated in an HSA-binding assay. Furthermore, an in vivo biodistribution study and single-photon emission computed tomography (SPECT) imaging were performed using INS-1 tumor-bearing mice. [111In]In-E4DA1-4 were prepared at radiochemical yields of 6-17%. In the saturation binding assay, [111In]In-E4DA1-4 showed a similar affinity for the INS-1 cells, indicating that the kind of ALB moiety used had no effect on the affinity of the exendin-4 derivatives for the cells. In the HSA-binding assay, [111In]In-E4DA1-4 all bound to HSA. In the biodistribution assay, [111In]In-E4DA1-4 exhibited marked tumor accumulation and retention. In addition, they showed lower renal accumulation than previously reported exendin-4-based radioligands without ALB moieties. The pharmacokinetics of the 111In-labeled exendin-4 derivatives varied markedly according to the kind of ALB moiety used. In particular, [111In]In-E4DA2, which contained a 4-(4-bromophenyl)butyric acid derivative as an ALB moiety, showed the highest tumor accumulation. SPECT imaging with [111In]In-E4DA2 clearly visualized INS-1 tumors with no marked accumulation in normal organs. These results provide important information that will aid the design of novel exendin-4-based radioligands targeting the GLP-1R.

Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens.

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, KD, and can be calculated as the concentration of antibody at which half the antibody binding sites are occupied at equilibrium. This method can be generalized to any radiolabeled antibody or other protein and peptide scaffolds. In contrast to cell-based methods, the choice of immobilized antigens is particularly useful for validating binding affinities after long-term storage of antibodies, distinguishing binding affinities of fragment antigen-binding region (Fab) arms in bispecific antibody constructs, and determining if there is variability in antigen expression between different cell lines. This method involves immobilizing a fixed amount of antigen to specified wells on a breakable 96-well plate. Then, nonspecific binding was blocked in all wells with bovine serum albumin (BSA). Subsequently, the rAb was added in a concentration gradient to all wells. A range of concentrations was chosen to allow the rAb to reach saturation, i.e., a concentration of antibody at which all antigens are continuously bound by the rAb. In designated wells without immobilized antigen, nonspecific binding of the rAb can be determined. By subtracting nonspecific binding from total binding in the wells with immobilized antigen, specific binding of the rAb to the antigen can be determined. The KD of the rAb was calculated from the resulting saturation binding curve. As an example, binding affinity was determined using radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins.

Abstract 5577: PSMA targeting CAR T cell immunotherapeutic strategy for prostate cancer

Abstract Introduction: Prostate cancer is the third most frequent cause of cancer-related death in men worldwide. Since prostate-specific membrane antigen (PSMA) shows a significant overexpression on prostatic cancer cells, PSMA can be considered a promising target for prostate cancer imaging and treatment. Although chimeric antigen receptor (CAR) T cells have shown great clinical outcomes in hematological malignancies, there is still limited efficacy in solid tumors due to the lack of tumor-specific antigens, low T cell infiltration in the tumors, immune suppressive tumor microenvironment, and on-target off-tumor toxicity in healthy tissues. The antigen recognition module of CAR T cells is usually a single-chain variable fragment (scFv). In contrast, the variable domains of heavy-chain antibodies (VHH) are small, stable, single-domain antibody fragments with high affinity and do not require the supramolecular assembly. Severe fatalities recently halted clinical trials of PSMA CAR T cells, so affinity tuning may be required to avoid CAR T targeting normal tissues. Here, we developed VHH-based CAR T cells targeting PSMA, able to co-express human somatostatin receptor 2 (SSTR2) actioning as a PET reporter for tracking CAR T cell distribution, as an immunotherapeutic strategy for prostate cancer. Methods: Primary human T cells were isolated and transduced with PSMA CAR lentivirus at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. Mouse models of 8505C cell line transduced to overexpress PSMA were utilized to test the efficacy of CAR T cells. Tumor growth was monitored regularly by bioluminescence imaging. PET/CT imaging was performed to monitor T cell localization and biodistribution using ¹⁸F-NOTA-octreotide radiotracer targeting SSTR2 introduced in CAR T cells. Results: By alanine substitution of CDR3 in the VHH, we identified several low affinity candidates. The affinity of the VHH-based PSMA CAR was determined to be 22 nM by a saturation binding assay using monomeric human PSMA protein. PSMA CAR expression in primary human T cells was higher than 90%. Compared to non-treated mice, PSMA targeting CAR T cells led to tumor reduction and improved survival in animal models. Moreover, high levels of T cell infiltration and localization in tumor tissues were found, based on PET/CT imaging. Considering possible treatment-associated toxicities, which led to 2 patient deaths and the halt of one phase 1 trial of PSMA CAR T cells, we will affinity tune VHH-based CAR to improve the safety profile. Thus, additional studies will be performed to test both activity and safety of affinity variants of PSMA CAR T cells. Conclusions: We developed PSMA targeting CAR T cells able to induce potent and specific killing of prostate cancer cells. This strategy demonstrated significant therapeutic efficacy in vivo against animal models. Therefore, CAR T cells targeting PSMA may be a promising treatment strategy for patients with prostate cancer. Citation Format: Juan Gonzalez-Valdivieso, Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Moonsoo M. Jin. PSMA targeting CAR T cell immunotherapeutic strategy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5577.

Abstract 5568: Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12

Abstract Introduction: The application of chimeric antigen receptor (CAR) T cells in solid tumors has met many challenges, arising from the paucity of effective yet safe targets and T cell dysfunction within the immunosuppressive milieu. Clinical trials over the years have reported severe on-target off-tumor toxicities associated with CAR T cell therapies targeting tumor-associated antigens (TAAs). Here, we propose a strategy to target TAAs effectively and safely: 1) improve tumor-targeting specificity by affinity-tuning; 2) armor T cells with antigen-dependent expression of interleukin-12 (IL-12) to compensate loss of CAR T cell activity caused by affinity-tuning. In this system, IL-12 expression is tightly regulated by the nuclear factor of the activated T cells promoter (NFAT) to limit IL-12 to the environ of activated affinity-tuned CAR T cells within the tumor. We demonstrate here the feasibility of this strategy using epithelial cell adhesion molecule (EpCAM) as a model TAA. Methods: Affinity variants were generated by alanine scanning mutagenesis in the complementary-determining region 3 (CDR3) of an EpCAM antibody (UBS54). The affinity of CAR variants was determined by flow cytometry-based saturation binding assay. On-target off-tumor cytotoxicity of CAR T cells was examined against human primary normal epithelial cells. In vivo anti-tumor efficacy was evaluated in mouse models of gastric cancer with cell line-derived (SNU-638 and MKN-45) and patient-derived xenografts. Results: Substitution of alanine into the UBS54 CDR3 led to identification of a tyrosine (6th residue in heavy chain CDR3) as a hot spot for affinity tuning. Among several more subtle amino acid substitutions for Tyr-6, we found that CAR molecule with valine substituent (Y6V) possesses 10 µM affinity toward EpCAM, rendering CAR T cells to be selective to EpCAM-high tumors while being not reactive to primary normal epithelial cells in vitro. In gastric cancer mouse models, compared to UBS54 CAR T cells that mediated rapid tumor remission, Y6V CAR T cells produced mainly partial responses. To revitalize affinity-tuned CAR T cells, we further engineered CAR T cells to release IL-12 under NFAT. We found that both the activity of the NFAT promoter and the level of IL-12 release were tightly regulated by the antigen density of targets when inducible IL-12 is combined with 10 µM affinity Y6V CAR; however, such dependence is lost when 1 µM affinity UBS54 was examined. In mouse models of gastric cancer, inducible lL-12 armored Y6V CAR T cells produced enhanced anti-tumor responses without elevating systemic exposure to IL-12, evidenced by low levels of IL-12 in mouse sera (below 100 pg/ml). Conclusions: Combination of affinity-tuned CAR with inducible expression of IL-12 is a promising strategy to develop CAR T cell therapy against TAAs with the potential for reduced on-target off-tumor toxicity and tumor resistance. Citation Format: Yanping Yang, Huan Yang, Yago Alcaina, Jaclyn E. McCloskey, Janusz Puc, Alyssa Birt, Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5568.