Quantifying dynamic range in red blood cell energetics: Evidence of progressive energy failure during storage.

BACKGROUND: During storage, red blood cells (RBCs) undergo significant biochemical and morphologic changes, referred to collectively as the "storage lesion". It was hypothesized that these defects may arise from disrupted oxygen-based regulation of RBC energy metabolism, with resultant depowering of intrinsic antioxidant systems. STUDY DESIGN AND METHODS: As a function of storage duration, the dynamic range in RBC metabolic response to three models of biochemical oxidant stress (methylene blue, hypoxanthine/xanthine oxidase, and diamide) was assessed, comparing glycolytic flux by NMR and UHPLC-MS methodologies. Blood was processed/stored under standard conditions (AS-1 additive solution) with leukoreduction. Over a 6-week period, RBC metabolic and antioxidant status were assessed at baseline and following exposure to the three biochemical oxidant models. Comparison was made of glycolytic flux (1 H-NMR tracking of [2-13 C]-glucose and metabolomic phenotyping with [1,2,3-13 C3 ] glucose), reducing equivalent (NADPH/NADP+ ) recycling, and thiol-based (GSH/GSSG) antioxidant status. RESULTS: As a function of storage duration, we observed the following: (1) a reduction in baseline hexose monophosphate pathway (HMP) flux, the sole pathway responsible for the regeneration of the essential reducing equivalent NADPH; with (2) diminished stress-based dynamic range in both overall glycolytic as well as proportional HMP flux. In addition, progressive with storage duration, RBCs showed (3) constraint in reducing equivalent (NADPH) recycling capacity, (4) loss of thiol based (GSH) recycling capacity, and (5) dysregulation of metabolon assembly at the cytoplasmic domain of Band 3 membrane protein (cdB3). CONCLUSION: Blood storage disturbs normal RBC metabolic control, depowering antioxidant capacity and enhancing vulnerability to oxidative injury.

177Lu-labeled low-molecular-weight agents for PSMA-targeted radiopharmaceutical therapy.

PURPOSE: To develop a prostate-specific membrane antigen (PSMA)-targeted radiotherapeutic for metastatic castration-resistant prostate cancer (mCRPC) with optimized efficacy and minimized toxicity employing the ß-particle radiation of 177Lu. METHODS: We synthesized 14 new PSMA-targeted, 177Lu-labeled radioligands (177Lu-L1-177Lu-L14) using different chelating agents and linkers. We evaluated them in vitro using human prostate cancer PSMA(+) PC3 PIP and PSMA(-) PC3 flu cells and in corresponding flank tumor models. Efficacy and toxicity after 8 weeks were evaluated at a single administration of 111 MBq for 177Lu-L1, 177Lu-L3, 177Lu-L5 and 177Lu-PSMA-617. Efficacy of 177Lu-L1 was further investigated using different doses, and long-term toxicity was determined in healthy immunocompetent mice. RESULTS: Radioligands were produced in high radiochemical yield and purity. Cell uptake and internalization indicated specific uptake only in PSMA(+) PC3 cells. 177Lu-L1, 177Lu-L3 and 177Lu-L5 demonstrated comparable uptake to 177Lu-PSMA-617 and 177Lu-PSMA-I&T in PSMA-expressing tumors up to 72 h post-injection. 177Lu-L1, 177Lu-L3 and 177Lu-L5 also demonstrated efficient tumor regression at 8 weeks. 177Lu-L1 enabled the highest survival rate. Necropsy studies of the treated group at 8 weeks revealed subacute damage to lacrimal glands and testes. No radiation nephropathy was observed 1 year post-treatment in healthy mice receiving 111 MBq of 177Lu-L1, most likely related to the fast renal clearance of this agent. CONCLUSIONS: 177Lu-L1 is a viable clinical candidate for radionuclide therapy of PSMA-expressing malignancies because of its high tumor-targeting ability and low off-target radiotoxic effects.

MRI Assessment of Prostate-Specific Membrane Antigen (PSMA) Targeting by a PSMA-Targeted Magnetic Nanoparticle: Potential for Image-Guided Therapy.

Magnetic nanoparticle (MNP)-induced hyperthermia is currently being evaluated for localized prostate cancer. We evaluated the feasibility of tumor-selective delivery of prostate-specific membrane antigen (PSMA)-targeted MNPs in a murine model with high-resolution magnetic resonance imaging (MRI) after intravenous administration of MNPs at a concentration necessary for hyperthermia. A PSMA-targeted MNP was synthesized and evaluated using T2-weighted MRI, after intravenous administration of 50 mg/kg of the MNP. Significant contrast enhancement ( P < 0.0002, n = 5) was observed in PSMA(+) tumors compared to PSMA(-) tumors 24 h and 48 h after contrast agent administration. Mice were also imaged with near-infrared fluorescence imaging, to validate the MRI results. Two-photon microscopy revealed higher vascular density at the tumor periphery, which resulted in higher  peripheral accumulation of PSMA-targeted MNPs. These results suggest that the delivery of PSMA-targeted MNPs to PSMA(+) tumors is both actively targeted and passively mediated.

Airway glycomic and allergic inflammatory consequences resulting from keratan sulfate galactose 6-O-sulfotransferase (CHST1) deficiency.

Siglec-F is a pro-apoptotic receptor on mouse eosinophils that recognizes 6'-sulfated sialyl Lewis X and 6'-sulfated sialyl N-acetyl-lactosamine as well as multivalent sialyl N-acetyl-lactosamine structures on glycan arrays. We hypothesized that attenuation of the carbohydrate sulfotransferase 1 (CHST1) gene encoding keratan sulfate galactose 6-O-sulfotransferase, an enzyme likely required for 6'-sulfation of some of these putative Siglec-F glycan ligands, would result in decreased Siglec-F lung ligand levels and enhanced allergic eosinophilic airway inflammation. Tissue analysis detected CHST1 expression predominantly not only in parenchymal cells but not in airway epithelium, the latter being a location where Siglec-F ligands are located. Western blotting of lung extracts with Siglec-F-Fc fusion proteins detected ≈500 kDa and ≈200 kDa candidate Siglec-F ligands that were not appreciably altered in CHST1-/- lungs compared with normal mouse lungs. Characterization of the O-linked glycans of lung tissue and bronchoalveolar lavage fluid detected altered sialylation but minimal change in sulfation. Eosinophilic airway inflammation was induced in wild-type (WT) and CHST1-/- mice via sensitization to ovalbumin (OVA) and repeated airway challenge. After OVA sensitization and challenge, Siglec-F ligands on airway cells, and numbers of eosinophils and neutrophils accumulating in the airways, both increased to a similar degree in WT and CHST1-/- mouse lungs, while macrophages and lymphocytes increased significantly more in CHST1-/- mouse airway compared with normal mouse lungs. Therefore, keratan sulfate galactose 6-O-sulfotransferase does not contribute to the synthesis of glycan ligands for Siglec-F in the airways, although its absence results in exaggerated accumulation of airway macrophages and lymphocytes.

Endogenous airway mucins carry glycans that bind Siglec-F and induce eosinophil apoptosis.

BACKGROUND: Sialic acid-binding, immunoglobulin-like lectin (Siglec) F is a glycan-binding protein selectively expressed on mouse eosinophils. Its engagement induces apoptosis, suggesting a pathway for ameliorating eosinophilia in the setting of asthma and other eosinophil-associated diseases. Siglec-F recognizes sialylated sulfated glycans in glycan-binding assays, but the identities of endogenous sialoside ligands and their glycoprotein carriers in vivo are unknown. OBJECTIVES: To use mouse lung-derived materials to isolate, biochemically identify, and biologically characterize naturally occurring endogenous glycan ligands for Siglec-F. METHODS: Lungs from normal and mucin-deficient mice, as well as mouse tracheal epithelial cells, were investigated in vitro and in vivo for the expression of Siglec-F ligands. Western blotting and cytochemistry used Siglec-F-Fc as a probe for directed purification, followed by liquid chromatography-tandem mass spectrometry of recognized glycoproteins. Purified components were tested in mouse eosinophil-binding assays and flow cytometry-based cell death assays. RESULTS: We detected mouse lung glycoproteins that bound to Siglec-F; binding was sialic acid dependent. Proteomic analysis of Siglec-F binding material identified Muc5b and Muc4. Cross-affinity enrichment and histochemical analysis of lungs from mucin-deficient mice assigned and validated the identity of Muc5b as one glycoprotein ligand for Siglec-F. Purified mucin preparations carried sialylated and sulfated glycans, bound to eosinophils and induced their death in vitro. Mice conditionally deficient in Muc5b displayed exaggerated eosinophilic inflammation in response to intratracheal installation of IL-13. CONCLUSIONS: These data identify a previously unrecognized endogenous anti-inflammatory property of airway mucins by which their glycans can control lung eosinophilia through engagement of Siglec-F.

Mice deficient in the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) exhibit enhanced allergic eosinophilic airway inflammation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-F is a proapoptotic receptor on mouse eosinophils, but little is known about its natural tissue ligand. OBJECTIVE: We previously reported that the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) is required for constitutive Siglec-F lung ligand synthesis. We therefore hypothesized that attenuation of ST3Gal-III will decrease Siglec-F ligand levels and enhance allergic eosinophilic airway inflammation. METHODS: C57BL/6 wild-type mice and St3gal3 heterozygous or homozygous deficient (St3gal3(+/-) and St3gal3(-/-)) mice were used. Eosinophilic airway inflammation was induced through sensitization to ovalbumin (OVA) and repeated airway OVA challenge. Siglec-F human IgG1 fusion protein (Siglec-F-Fc) was used to detect Siglec-F ligands. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for inflammation, as well as various cytokines and chemokines. Serum was analyzed for allergen-specific immunoglobulin levels. RESULTS: Western blotting with Siglec-F-Fc detected approximately 500-kDa and approximately 200-kDa candidate Siglec-F ligands that were less abundant in St3gal3(+/-) lung extracts and nearly absent in St3gal3(-/-) lung extracts. After OVA sensitization and challenge, Siglec-F ligands were increased in wild-type mouse lungs but less so in St3gal3 mutants, whereas peribronchial and BALF eosinophil numbers were greater in the mutants, with the following rank order: St3gal3(-/-) ≥ St3gal3(+/-) > wild-type mice. Levels of various cytokines and chemokines in BALF were not significantly different among these 3 types of mice, although OVA-specific serum IgG1 levels were increased in St3gal3(-/-) mice. CONCLUSIONS: After OVA sensitization and challenge, St3gal3(+/-) and St3gal3(-/-) mice have more intense allergic eosinophilic airway inflammation and less sialylated Siglec-F ligands in their airways. One possible explanation for these findings is that levels of sialylated airway ligands for Siglec-F might be diminished in mice with attenuated levels of ST3Gal-III, resulting in a reduction in a natural proapoptotic pathway for controlling airway eosinophilia.

COVID-19 impairs oxygen delivery by altering red blood cell hematological, hemorheological, and oxygen transport properties.

Introduction: Coronavirus disease 2019 (COVID-19) is characterized by impaired oxygen (O2) homeostasis, including O2 sensing, uptake, transport/delivery, and consumption. Red blood cells (RBCs) are central to maintaining O2 homeostasis and undergo direct exposure to coronavirus in vivo. We thus hypothesized that COVID-19 alters RBC properties relevant to O2 homeostasis, including the hematological profile, Hb O2 transport characteristics, rheology, and the hypoxic vasodilatory (HVD) reflex. Methods: RBCs from 18 hospitalized COVID-19 subjects and 20 healthy controls were analyzed as follows: (i) clinical hematological parameters (complete blood count; hematology analyzer); (ii) O2 dissociation curves (p50, Hill number, and Bohr plot; Hemox-Analyzer); (iii) rheological properties (osmotic fragility, deformability, and aggregation; laser-assisted optical rotational cell analyzer (LORRCA) ektacytometry); and (iv) vasoactivity (the RBC HVD; vascular ring bioassay). Results: Compared to age- and gender-matched healthy controls, COVID-19 subjects demonstrated 1) significant hematological differences (increased WBC count-with a higher percentage of neutrophils); RBC distribution width (RDW); and reduced hematocrit (HCT), Hb concentration, mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC); 2) impaired O2-carrying capacity and O2 capacitance (resulting from anemia) without difference in p50 or Hb-O2 cooperativity; 3) compromised regulation of RBC volume (altered osmotic fragility); 4) reduced RBC deformability; 5) accelerated RBC aggregation kinetics; and (6) no change in the RBC HVD reflex. Discussion: When considered collectively, homeostatic compensation for these RBC impairments requires that the cardiac output in the COVID cohort would need to increase by ∼135% to maintain O2 delivery similar to that in the control cohort. Additionally, the COVID-19 disease RBC properties were found to be exaggerated in blood-type O hospitalized COVID-19 subjects compared to blood-type A. These data indicate that altered RBC features in hospitalized COVID-19 subjects burden the cardiovascular system to maintain O2 delivery homeostasis, which appears exaggerated by blood type (more pronounced with blood-type O) and likely plays a role in disease pathogenesis.

A Series of PSMA-Targeted Near-Infrared Fluorescent Imaging Agents.

We have synthesized a series of 10 new, PSMA-targeted, near-infrared imaging agents intended for use in vivo for fluorescence-guided surgery (FGS). Compounds were synthesized from the commercially available amine-reactive active NHS ester of DyLight800. We altered the linker between the PSMA-targeting urea moiety and the fluorophore with a view to improve the pharmacokinetics. Chemical yields for the conjugates ranged from 51% to 86%. The Ki values ranged from 0.10 to 2.19 nM. Inclusion of an N-bromobenzyl substituent at the ε-amino group of lysine enhanced PSMA+ PIP tumor uptake, as did hydrophilic substituents within the linker. The presence of a polyethylene glycol chain within the linker markedly decreased renal uptake. In particular, DyLight800-10 demonstrated high specific uptake relative to background signal within kidney, confirmed by immunohistochemistry. These compounds may be useful for FGS in prostate, renal or other PSMA-expressing cancers.

An Improved 211At-Labeled Agent for PSMA-Targeted α-Therapy.

α-Particle emitters targeting the prostate-specific membrane antigen (PSMA) proved effective in treating patients with prostate cancer who were unresponsive to the corresponding ß-particle therapy. 211At is an α-emitter that may engender less toxicity than other α-emitting agents. We synthesized a new 211At-labeled radiotracer targeting PSMA that resulted from the search for a pharmacokinetically optimized agent. Methods: A small series of 125I-labeled compounds was synthesized from tin precursors to evaluate the effect of the location of the radiohalogen within the molecule and the presence of lutetium in the chelate on biodistribution. On that basis, 211At-3-Lu was selected and evaluated in cell uptake and internalization studies, and biodistribution and PSMA-expressing (PSMA+) PC3 PIP tumor growth control were evaluated in experimental flank and metastatic (PC3-ML-Luc) models. A long-term (13-mo) toxicity study was performed for 211At-3-Lu, including tissue chemistries and histopathology. Results: The radiochemical yield of 211At-3-Lu was 17.8% ± 8.2%. Lead compound 211At-3-Lu demonstrated total uptake within PSMA+ PC3 PIP cells of 13.4 ± 0.5% of the input dose after 4 h of incubation, with little uptake in control cells. In SCID mice, 211At-3-Lu provided uptake that was 30.6 ± 4.8 percentage injected dose per gram (%ID/g) in PSMA+ PC3 PIP tumor at 1 h after injection, and this uptake decreased to 9.46 ± 0.96 %ID/g by 24 h. Tumor-to-salivary gland and tumor-to-kidney ratios were 129 ± 99 at 4 h and 130 ± 113 at 24 h, respectively. Deastatination was not significant (stomach, 0.34 ± 0.20 %ID/g at 4 h). Dose-dependent survival was demonstrated at higher doses (>1.48 MBq) in both flank and metastatic models. There was little off-target toxicity, as demonstrated by hematopoietic stability, unchanged tissue chemistries, weight gain rather than loss throughout treatment, and favorable histopathologic findings. Conclusion: Compound 211At-3-Lu or close analogs may provide limited and acceptable toxicity while retaining efficacy in management of prostate cancer.

Characterization of expression of glycan ligands for Siglec-F in normal mouse lungs.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-F, an inhibitory receptor on mouse eosinophils, preferentially recognizes the glycan ligand 6'-sulfated sialyl Lewis X, but little is known about the requirements for its lung expression. RT-PCR and immunohistochemistry were used to detect and localize the sulfotransferase keratin sulfate galactose 6-O sulfotransferase (KSGal6ST, also known as carbohydrate sulfotransferase 1; gene name, Chst1) that is putatively required for 6'-sulfated Sialyl Lewis X synthesis. RT-PCR detected the greatest constitutive expression of Chst1 in lung, liver, and spleen tissue. Immunohistochemistry localized the expression of KSGal6ST in lung tissue primarily to airway epithelium. Siglec-F-Ig fusion protein selectively bound in a similar pattern, and was unaffected in lung tissue treated with methanol or deficient in Type 2 α2,3 sialyltransferase (St3gal2), but was eliminated by proteinase K or sialidase, and was absent in tissue deficient in the Type 3 α2,3 sialyltransferase (St3gal3). Binding of the Siglec-F-Ig fusion protein was similar in pattern to, and completely blocked by, a plant lectin recognizing α2,3-linked sialic acid. Thus, α2,3-linked sialic acid-containing glycoprotein Siglec-F ligands and the enzymes required for their synthesis are constitutively expressed in murine lungs, especially by airway epithelium. St3gal3, but not St3gal2, is required for constitutive Siglec-F ligand synthesis. The survival of eosinophils entering the lung may be shortened by encountering these Siglec-F sialoside ligands.

A prostate-specific membrane antigen (PSMA)-targeted prodrug with a favorable in vivo toxicity profile.

Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed a non-radioactive prodrug, SBPD-1, composed of a small-molecule PSMA-targeting moiety, a cancer-selective cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SBPD-1 demonstrated high binding affinity to PSMA (Ki = 8.84 nM) and selective cytotoxicity to PSMA-expressing PC cell lines (IC50 = 3.90 nM). SBPD-1 demonstrated a significant survival benefit in two murine models of human PC relative to controls. The highest dose tested did not induce toxicity in immunocompetent mice. The high specific targeting ability of SBPD-1 to PSMA-expressing tumors and its favorable toxicity profile warrant its further development.

Scalable Purification of Plasmid DNA Nanoparticles by Tangential Flow Filtration for Systemic Delivery.

Plasmid DNA (pDNA) nanoparticles synthesized by complexation with linear polyethylenimine (lPEI) are one of the most effective non-viral gene delivery vehicles. However, the lack of scalable and reproducible production methods and the high toxicity have hindered their clinical translation. Previously, we have developed a scalable flash nanocomplexation (FNC) technique to formulate pDNA/lPEI nanoparticles using a continuous flow process. Here, we report a tangential flow filtration (TFF)-based scalable purification method to reduce the uncomplexed lPEI concentration in the nanoparticle formulation and improve its biocompatibility. The optimized procedures achieved a 60% reduction of the uncomplexed lPEI with preservation of the nanoparticle size and morphology. Both in vitro and in vivo studies showed that the purified nanoparticles significantly reduced toxicity while maintaining transfection efficiency. TFF also allows for gradual exchange of solvents to isotonic solutions and further concentrating the nanoparticles for injection. Combining FNC production and TFF purification, we validated the purified pDNA/lPEI nanoparticles for future clinical translation of this gene nanomedicine.

Preclinical Evaluation of 213Bi- and 225Ac-Labeled Low-Molecular-Weight Compounds for Radiopharmaceutical Therapy of Prostate Cancer.

Prostate-specific membrane antigen (PSMA)-targeted radiopharmaceutical therapy is a new option for patients with advanced prostate cancer refractory to other treatments. Previously, we synthesized a ß-particle-emitting low-molecular-weight compound, 177Lu-L1 which demonstrated reduced off-target effects in a xenograft model of prostate cancer. Here, we leveraged that scaffold to synthesize α-particle-emitting analogs of L1, 213Bi-L1 and 225Ac-L1, to evaluate their safety and cell kill effect in PSMA-positive (+) xenograft models. Methods: The radiochemical synthesis, cell uptake, cell kill, and biodistribution of 213Bi-L1 and 225Ac-L1 were evaluated. The efficacy of 225Ac-L1 was determined in human PSMA+ subcutaneous and micrometastatic models. Subacute toxicity at 8 wk and chronic toxicity at 1 y after administration were evaluated for 225Ac-L1. The absorbed radiation dose of 225Ac-L1 was determined using the biodistribution data and α-camera imaging. Results:213Bi- and 225Ac-L1 demonstrated specific cell uptake and cell kill in PSMA+ cells. The biodistribution of 213Bi-L1 and 225Ac-L1 revealed specific uptake of radioactivity within PSMA+ lesions. Treatment studies of 225Ac-L1 demonstrated activity-dependent, specific inhibition of tumor growth in the PSMA+ flank tumor model. 225Ac-L1 also showed an increased survival benefit in the micrometastatic model compared with 177Lu-L1. Activity-escalated acute and chronic toxicity studies of 225Ac-L1 revealed off-target radiotoxicity, mainly in kidneys and liver. The estimated maximum tolerated activity was about 1 MBq/kg. α-Camera imaging of 225Ac-L1 revealed high renal cortical accumulation at 2 h followed by fast clearance at 24 h. Conclusion:225Ac-L1 demonstrated activity-dependent efficacy with minimal treatment-related organ radiotoxicity. 225Ac-L1 is a promising therapeutic for further clinical evaluation.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

Auger radiopharmaceutical therapy targeting prostate-specific membrane antigen in a micrometastatic model of prostate cancer.

Auger radiopharmaceutical therapy is a promising strategy for micrometastatic disease given high linear energy transfer and short range in tissues, potentially limiting normal tissue toxicities. We previously demonstrated anti-tumor efficacy of a small-molecule Auger electron emitter targeting the prostate-specific membrane antigen (PSMA), 2-[3-[1-carboxy-5-(4-[125I]iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid), or 125I-DCIBzL, in a mouse xenograft model. Here, we investigated the therapeutic efficacy, long-term toxicity, and biodistribution of 125I-DCIBzL in a micrometastatic model of prostate cancer (PC). Methods: To test the therapeutic efficacy of 125I-DCIBzL in micrometastatic PC, we used a murine model of human metastatic PC in which PSMA+ PC3-ML cells expressing firefly luciferase were injected intravenously in NSG mice to form micrometastatic deposits. One week later, 0, 0.37, 1.85, 3.7, 18.5, 37, or 111 MBq of 125I-DCIBzL was administered (intravenously). Metastatic tumor burden was assessed using bioluminescence imaging (BLI). Long-term toxicity was evaluated via serial weights and urinalysis of non-tumor-bearing mice over a 12-month period, as well as final necropsy. Results: In the micrometastatic PC model, activities of 18.5 MBq 125I-DCIBzL and above significantly delayed development of detectable metastatic disease by BLI and prolonged survival in mice. Gross metastases were detectable in control mice and those treated with 0.37-3.7 MBq 125I-DCIBzL at a median of 2 weeks post-treatment, versus 4 weeks for those treated with 18.5-111 MBq 125I-DCIBzL (P<0.0001 by log-rank test). Similarly, treatment with ≥18.5 MBq 125I-DCIBzL yielded a median survival of 11 weeks, compared with 6 weeks for control mice (P<0.0001). At 12 months, there was no appreciable toxicity via weight, urinalysis, or necropsy evaluation in mice treated with any activity of 125I-DCIBzL, which represents markedly less toxicity than the analogous PSMA-targeted α-particle emitter. Macro-to-microscale dosimetry modeling demonstrated lower absorbed dose in renal cell nuclei versus tumor cell nuclei due to lower levels of drug uptake and cellular internalization in combination with the short range of Auger emissions. Conclusion: PSMA-targeted radiopharmaceutical therapy with the Auger emitter 125I-DCIBzL significantly delayed development of detectable metastatic disease and improved survival in a micrometastatic model of PC, with no long-term toxicities noted at 12 months, suggesting a favorable therapeutic ratio for treatment of micrometastatic PC.

Preclinical Evaluation of 203/212Pb-Labeled Low-Molecular-Weight Compounds for Targeted Radiopharmaceutical Therapy of Prostate Cancer.

Targeted radiopharmaceutical therapy (TRT) using α-particle radiation is a promising approach for treating both large and micrometastatic lesions. We developed prostate-specific membrane antigen (PSMA)-targeted low-molecular-weight agents for 212Pb-based TRT of patients with prostate cancer (PC) by evaluating the matching γ-emitting surrogate, 203Pb. Methods: Five rationally designed low-molecular-weight ligands (L1-L5) were synthesized using the lysine-urea-glutamate scaffold, and PSMA inhibition constants were determined. Tissue biodistribution and SPECT/CT imaging of 203Pb-L1-203Pb-L5 were performed on mice bearing PSMA(+) PC3 PIP and PSMA(-) PC3 flu flank xenografts. The absorbed radiation dose of the corresponding 212Pb-labeled analogs was determined using the biodistribution data. Antitumor efficacy of 212Pb-L2 was evaluated in PSMA(+) PC3 PIP and PSMA(-) PC3 flu tumor models and in the PSMA(+) luciferase-expressing micrometastatic model. 212Pb-L2 was also evaluated for dose-escalated, long-term toxicity. Results: All new ligands were obtained in high yield and purity. PSMA inhibitory activities ranged from 0.10 to 17 nM. 203Pb-L1-203Pb-L5 were synthesized in high radiochemical yield and specific activity. Whole-body clearance of 203Pb-L1-203Pb-L5 was fast. The absorbed dose coefficients (mGy/kBq) of the tumor and kidneys were highest for 203Pb-L5 (31.0, 15.2) and lowest for 203Pb-L2 (8.0, 4.2). The tumor-to-kidney absorbed dose ratio was higher for 203Pb-L3 (3.2) and 203Pb-L4 (3.6) than for the other agents, but with lower tumor-to-blood ratios. PSMA(+) tumor lesions were visualized through SPECT/CT as early as 0.5 h after injection. A proof-of-concept therapy study with a single administration of 212Pb-L2 demonstrated dose-dependent inhibition of tumor growth in the PSMA(+) flank tumor model. 212Pb-L2 also demonstrated an increased survival benefit in the micrometastatic model compared with 177Lu-PSMA-617. Long-term toxicity studies in healthy, immunocompetent CD-1 mice revealed kidney as the dose-limiting organ. Conclusion:203Pb-L1-203Pb-L5 demonstrated favorable pharmacokinetics for 212Pb-based TRT. The antitumor efficacy of 212Pb-L2 supports the corresponding 203Pb/212Pb theranostic pair for PSMA-based α-particle TRT in advanced PC.

Imaging CAR T cell therapy with PSMA-targeted positron emission tomography.

Chimeric antigen receptor (CAR) T cell therapy for hematologic malignancies is fraught with several unknowns, including number of functional T cells that engage target tumor, durability and subsequent expansion and contraction of that engagement, and whether toxicity can be managed. Non-invasive, serial imaging of CAR T cell therapy using a reporter transgene can address those issues quantitatively. We have transduced anti-CD19 CAR T cells with the prostate-specific membrane antigen (PSMA) because it is a human protein with restricted normal tissue expression and has an expanding array of positron emission tomography (PET) and therapeutic radioligands. We demonstrate that CD19-tPSMA(N9del) CAR T cells can be tracked with [18F]DCFPyL PET in a Nalm6 model of acute lymphoblastic leukemia. Divergence between the number of CD19-tPSMA(N9del) CAR T cells in peripheral blood and bone marrow and those in tumor was evident. These findings underscore the need for non-invasive repeatable monitoring of CAR T cell disposition clinically.

A side-by-side evaluation of [18F]FDOPA enantiomers for non-invasive detection of neuroendocrine tumors by positron emission tomography.

Neuroendocrine tumors (NETs) are an extremely heterogenous group of malignancies with variable clinical behavior. Molecular imaging of patients with NETs allows for effective patient stratification and treatment guidance and is crucial in selection of targeted therapies. Positron emission tomography (PET) with the radiotracer L-[18F]FDOPA is progressively being utilized for non-invasive in vivo visualization of NETs and pancreatic ß-cell hyperplasia. While L-[18F]FDOPA-PET is a valuable tool for disease detection and management, it also exhibits significant diagnostic limitations owing to its inherent physiological uptake in off-target tissues. We hypothesized that the D-amino acid structural isomer of that clinical tracer, D-[18F]FDOPA, may exhibit superior clearance capabilities owing to a reduced in vivo enzymatic recognition and enzyme-mediated metabolism. Here, we report a side-by-side evaluation of D-[18F]FDOPA with its counterpart clinical tracer, L-[18F]FDOPA, for the non-invasive in vivo detection of NETs. In vitro evaluation in five NET cell lines, including invasive small intestinal neuroendocrine carcinomas (STC-1), insulinomas (TGP52 and TGP61), colorectal adenocarcinomas (COLO-320) and pheochromocytomas (PC12), generally indicated higher overall uptake levels of L-[18F]FDOPA, compared to D-[18F]FDOPA. While in vivo PET imaging and ex vivo biodistribution studies in PC12, STC-1 and COLO-320 mouse xenografts further supported our in vitro data, they also illustrated lower off-target retention and enhanced clearance of D-[18F]FDOPA from healthy tissues. Cumulatively our results indicate the potential diagnostic applications of D-[18F]FDOPA for malignancies where the utility of L-[18F]FDOPA-PET is limited by the physiological uptake of L-[18F]FDOPA, and suggest D-[18F]FDOPA as a viable PET imaging tracer for NETs.

Evaluation of 111In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen.

5D3 is a new high-affinity murine monoclonal antibody specific for prostate-specific membrane antigen (PSMA). PSMA is a target for the imaging and therapy of prostate cancer. 111In-labeled antibodies have been used as surrogates for 177Lu/90Y-labeled therapeutics. We characterized 111In-DOTA-5D3 by SPECT/CT imaging, tissue biodistribution studies, and dosimetry. Methods: Radiolabeling, stability, cell uptake, and internalization of 111In-DOTA-5D3 were performed by established techniques. Biodistribution and SPECT imaging were done on male nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice bearing human PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate cancer xenografts on the upper right and left flanks, respectively, at 2, 24, 48, 72, and 192 h after injection. Biodistribution was also evaluated in tumor-free, healthy male CD-1 mice. Blocking studies were performed by coinjection of a 10-fold and 50-fold excess of 5D3 followed by biodistribution at 24 h to determine PSMA binding specificity. The absorbed radiation doses were calculated on the basis of murine biodistribution data, which were translated to a human adult man using organ weights as implemented in OLINDA/EXM. Results:111In-DOTA-5D3 was synthesized with specific activity of approximately 2.24 ± 0.74 MBq/µg (60.54 ± 20 µCi/µg). Distribution of 111In-DOTA-5D3 in PSMA(+) PC3 PIP tumor peaked at 24 h after injection and remained high until 72 h. Uptake in normal tissues, including the blood, spleen, liver, heart, and lungs, was highest at 2 h after injection. Coinjection of 111In-DOTA-5D3 with a 10- and 50-fold excess of nonradiolabeled antibody significantly reduced PSMA(+) PC3 PIP tumor and salivary gland uptake at 24 h but did not reduce uptake in kidneys and lacrimal glands. Significant clearance of 111In-DOTA-5D3 from all organs occurred at 192 h. The highest radiation dose was received by the liver (0.5 mGy/MBq), followed by the spleen and kidneys. Absorbed radiation doses to the salivary and lacrimal glands and bone marrow were low. Conclusion:111In-DOTA-5D3 is a new radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA.

Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles.

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at -20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.