Cell Binding Assays Research Articles

Exploration of commercial cyclen-based chelators for mercury-197m/g incorporation into theranostic radiopharmaceuticals.

A comprehensive investigation of the Hg2+ coordination chemistry and 197m/gHg radiolabeling capabilities of cyclen-based commercial chelators, namely, DOTA and DOTAM (aka TCMC), along with their bifunctional counterparts, p-SCN-Bn-DOTA and p-SCN-Bn-TCMC, was conducted to assess the suitability of these frameworks as bifunctional chelators for the 197m/gHg2+ theranostic pair. Radiolabeling studies revealed that TCMC and DOTA exhibited low radiochemical yields (0%-6%), even when subjected to harsh conditions (80°C) and high ligand concentrations (10-4M). In contrast, p-SCN-Bn-TCMC and p-SCN-Bn-DOTA demonstrated significantly higher 197m/gHg radiochemical yields (100% ± 0.0% and 70.9% ± 1.1%, respectively) under the same conditions. The [197m/gHg]Hg-p-SCN-Bn-TCMC complex was kinetically inert when challenged against human serum and glutathione. To understand the differences in labeling between the commercial chelators and their bifunctional counterparts, non-radioactive natHg2+ complexes were assessed using NMR spectroscopy and DFT calculations. The NMR spectra of Hg-TCMC and Hg-p-SCN-Bn-TCMC suggested binding of the Hg2+ ion through the cyclen backbone framework. DFT studies indicated that binding of the Hg2+ ion within the backbone forms a thermodynamically stable product. However, competition can form between isothiocyanate binding and binding through the macrocycle, which was experimentally observed. The isothiocyanate bound coordination product was dominant at the radiochemical scale as, in comparison, the macrocycle bound product was seen at the NMR scale, agreeing with the DFT result. Furthermore, a bioconjugate of TCMC (TCMC-PSMA) targeting prostate-specific membrane antigen was synthesized and radiolabeled, resulting in an apparent molar activity of 0.089 MBq/nmol. However, the complex demonstrated significant degradation over 24h when exposed to human serum and glutathione. Subsequently, cell binding assays were conducted, revealing a Ki value ranging from 19.0 to 19.6nM. This research provides crucial insight into the effectiveness of current commercial chelators in the context of 197m/gHg2+ radiolabeling. It underscores the necessity for the development of specific and customized chelators to these unique "soft" radiometals to advance 197m/gHg2+ radiopharmaceuticals.

Binding affinity between coronavirus spike protein and human ACE2 receptor

Coronaviruses (CoVs) pose a major risk to global public health due to their ability to infect diverse animal species and potential for emergence in humans. The CoV spike protein mediates viral entry into the cell and plays a crucial role in determining the binding affinity to host cell receptors. With particular emphasis on α- and β-coronaviruses that infect humans and domestic animals, current research on CoV receptor use suggests that the exploitation of the angiotensin-converting enzyme 2 (ACE2) receptor poses a significant threat for viral emergence with pandemic potential. This review summarizes the approaches used to study binding interactions between CoV spike proteins and the human ACE2 (hACE2) receptor. Solid-phase enzyme immunoassays and cell binding assays allow qualitative assessment of binding but lack quantitative evaluation of affinity. Surface plasmon resonance, Bio-layer interferometry, and Microscale Thermophoresis on the other hand, provide accurate affinity measurement through equilibrium dissociation constants (KD). In silico modeling predicts affinity through binding structure modeling, protein-protein docking simulations, and binding energy calculations but reveals inconsistent results due to the lack of a standardized approach. Machine learning and deep learning models utilize simulated and experimental protein-protein interaction data to elucidate the critical residues associated with CoV binding affinity to hACE2. Further optimization and standardization of existing approaches for studying binding affinity could aid pandemic preparedness. Specifically, prioritizing surveillance of CoVs that can bind to human receptors stands to mitigate the risk of zoonotic spillover.

Synthesis and Preliminary Study of 99mTc-Labeled HYNIC-FAPi for Imaging of Fibroblast Activation Proteins in Tumors.

Fibroblast activation protein (FAP) is an emerging target for cancer diagnosis. Different types of FAP inhibitor (FAPI)-based radiotracers have been developed and applied for tumor imaging. However, few FAPI tracers for single photon emission computed tomography (SPECT) imaging have been reported. SPECT imaging is less expensive and more widely distributed than positron emission tomography (PET), and thus, 99mTc-labeled FAPIs would be more available to patients in developing regions. Herein, we developed a FAPI-04-derived radiotracer, HYNIC-FAPi-04 (HFAPi), for SPECT imaging. 99mTc-HFAPi, with a radiochemical purity of >98%, was prepared using a kit formula within 30 min. The specificity of 99mTc-HFAPi for FAP was validated by a cell binding assay in vitro and SPECT/CT imaging in vivo. The binding affinity (Kd value) of 99mTc-HFAPi for human FAP and murine FAP was 4.49 and 2.07 nmol/L, respectively. SPECT/CT imaging in HT1080-hFAP tumor-bearing mice showed the specific FAP targeting ability of 99mTc-HFAPi in vivo. In U87MG tumor-bearing mice, 99mTc-HFAPi had a higher tumor uptake compared with that of HT1080-hFAP and 4T1-mFAP tumor models. Interestingly, 99mTc-HFAPi showed a relatively high uptake in some murine joints. 99mTc-HFAPi accumulated in tumor lesions with a high tumor-to-background ratio. A preliminary clinical study was also performed in breast cancer patients. Additionally, 99mTc-HFAPi exhibited an advantage over 18F-FDG in the detection of lymph node metastatic lesions in breast cancer patients, which is helpful in improving treatment strategies. In short, 99mTc-HFAPi showed excellent affinity and specificity for FAP and is a promising SPECT radiotracer for (re)staging and treatment planning of breast cancers.

Ad5-nCoV Vaccination Could Induce HLA-E Restricted CD8+ T Cell Responses Specific for Epitopes on Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein.

We evaluated cellular immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in an immunized population based on HLA-E-restricted CD8+ T cell epitope identification. HLA-E-restricted SARS-CoV-2 CD8+ T cell nonamer peptides were predicted with software. An HLA-E-transfected K562 cell binding assay was used to screen for high-affinity peptides. IFN-γ enzyme-linked immunospot assays were used to identify HLA-E-restricted epitopes. An HLA-E/epitope tetramer was employed to detect the frequencies of epitope-specific CD8+ T cells. Four CD8+ T cell epitopes on the spike protein of SARS-CoV-2 restricted by both HLA-E*0101 and E*0103 were identified. HLA-E-restricted epitope-specific IFN-γ-secreting CD8+ T cell responses could be detected in individuals vaccinated with SARS-CoV-2 vaccines. Importantly, the frequencies of epitope-specific CD8+ T cells in Ad5-nCoV vaccinated individuals were higher than in individuals vaccinated with recombinant protein or inactivated vaccines. Moreover, the frequencies of epitope-specific CD8+ T cells could be maintained for at least 120 days after only one dose of Ad5-nCoV vaccine, while the frequencies of epitope-specific CD8+ T cells decreased in individuals after two doses of Ad5-nCoV vaccine. These findings may contribute to a more comprehensive evaluation of the protective effects of vaccines for SARS-CoV-2; meanwhile, they may provide information to characterize HLA-E-restricted CD8+ T cell immunity against SARS-CoV-2 infection.

Prediction and identification of HLA-A*0201-restricted epitopes from cancer testis antigen CT23

ABSTRACT Cancer-testis antigen CT23 is a class of tumor-associated antigens (TAA) characterized by restricted expression in male germ cells and a variety of tumor tissues. Numerous studies have shown that CT23 is closely related to tumor cell viability, proliferation, metastasis and invasion. CT23 is immunogenic and can cause specific immune response in tumor patients. Therefore, it is considered to be one of the best target antigens for designing therapeutic tumor vaccines and T-cell-mediated tumor immunotherapy. In this study, we initially obtained seven HLA-A*0201-restricted CT23 epitope candidate peptides through the T cell epitope prediction program. Subsequently, a T2 cell binding assay revealed the potential binding of all candidate peptides with HLA-A2 molecules. Notably, peptide P7 (ALLVLCYSI) exhibited the highest affinity, as evidenced by a fluorescence index (FI) of 2.19. Dendritic cells (DCs) loaded with CT23 candidate peptide can stimulate CD8+T cell activation and proliferation, and compared with other candidate peptides, candidate peptide P7 is superior. The cytotoxic T lymphocytes (CTLs) stimulated by the peptide P7 had killing effect on tumor cells (HLA-A*0201+, CT23+), but no killing effect on tumor cells (HLA-A*0201−, CT23+). The CTLs induced by the peptide P7 also had a specific killing effect on T2 cells bearing the peptide P7. In summary, our findings suggest that the CT23 peptide P7 (ALLVLCYSI) can induce immune responses and holds potential for tumor-specific CTL therapy.

Design, Synthesis, and Evaluation of 18F-Labeling CYP1B1 PET Tracer Based on 2-Phenylquinazolin

Cytochrome P450 (CYP)1B1 has been identified to be specifically overexpressed in several solid tumors, thus it’s a potential target for the detection of tumors. Based on the 2-Phenylquinazolin CYP1B1 inhibitors, we designed and synthesized several positron emission computed tomography (PET) imaging probes targeting CYP1B1. Through IC50 determinations, most of these probes exhibited good affinity and selectivity to CYP1B1. Considering their affinity, solubility, and their 18F labeling methods, we chose compound 5c as the best candidate. The 18F radiolabeling of [18F] 5c was easy to handle with good radiolabeling yield and radiochemical purity. In vitro and in vivo stability study indicated that probe [18F]5c has good stability. In cell binding assay, [18F]5c could be specifically taken up by tumor cells, especially HCT-116 cells. Although the tumor-blood (T/B) and tumor-muscle (T/M) values and PET imaging results were unsatisfied, it is still possible to develop PET probes targeting CYP1B1 by structural modification on the basis of 5c in the future.

Low-Loaded Polyethylene Glycol (PEG) Resin for High-Purity Peptide Synthesis and Cell Binding Assays

Low-Loaded Polyethylene Glycol (PEG) Resin for High-Purity Peptide Synthesis and Cell Binding Assays

Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy.

Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions.

RUNX3 regulates the susceptibility against EGFR-targeted non-small cell lung cancer therapy using 47Sc-conjugated cetuximab

BackgroundRadioimmunotherapy with cetuximab and conjugates with various radioisotopes is a feasible treatment option for different tumor models. Scandium-47 (47Sc), one of several β−-particle-emitting radioisotopes, displays favorable physical and chemical properties for conjugation to monoclonal antibodies. However, the therapeutic efficacy of 47Sc in preclinical and clinical studies is largely unknown. Given that intrinsic alterations in tumors greatly contribute to resistance to anti-epidermal growth factor receptor (EGFR)-targeted therapy, research on overcoming resistance to radioimmunotherapy using cetuximab is required.Methods47Sc was produced by irradiation of a CaCO3 target at the HANARO research reactor in KAERI (Korea Atomic Energy Research Institute) and prepared by chromatographic separation of the irradiated target. Cetuximab was conjugated with 47Sc using the bifunctional chelating agent DTPA. Radiochemical purity was determined using instant thin-layer chromatography. The immunoreactivity of 47Sc-DTPA-cetuximab was evaluated using the Lindmo method and an in vitro cell-binding assay. The inhibitory effects of cetuximab and 47Sc-DTPA-cetuximab were confirmed using cell growth inhibition and BrdU cell proliferation assays. Differences in protein expression levels between cetuximab- and 47Sc-DTPA-cetuximab-treated cells were confirmed using western blotting. Complex formation between RUNX3 and DNA repair components was confirmed using immunoprecipitation and western blotting.ResultsCetuximab induces cell cycle arrest and cell death in EGFR-overexpressing NSCLC cells. Radiolabeling of cetuximab with 47Sc led to increased therapeutic efficacy relative to cetuximab alone. Application of 47Sc-DTPA-cetuximab induced DNA damage responses, and activation of RUNX3 significantly enhanced the therapeutic efficacy of 47Sc-DTPA-cetuximab. RUNX3 mediated susceptibility to EGFR-targeted NSCLC therapy using 47Sc-DTPA-cetuximab via interaction with components of the DNA damage and repair machinery.Conclusions47Sc-DTPA-cetuximab promoted cell death in EGFR-overexpressing NSCLC cells by targeting EGFR and inducing DNA damage as a result of β irradiation emitted from the conjugated 47Sc. Activation of RUNX3 played a key role in DNA damage and repair processes in response to the ionizing radiation and inhibited cell growth, thus leading to more effective tumor suppression. RUNX3 can potentially moderate susceptibility to 47Sc-conjugated cetuximab by modulating DNA damage and repair process mechanisms.

Complement Activation-Independent Attenuation of SARS-CoV-2 Infection by C1q and C4b-Binding Protein.

The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1β, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response.

Experimental study of 131I-caerin 1.1 and 131I-c(RGD)2 for internal radiation therapy of esophageal cancer xenografts

ObjectiveThe aim of this study was to analyze and compare the therapeutic effects of 131I-caerin 1.1 and 131I-c(RGD)2 on TE-1 esophageal cancer cell xenografts. Methods(1) The in vitro antitumor effects of the polypeptides caerin 1.1 and c(RGD)2 were verified by MTT and clonogenic assays. 131I-caerin 1.1 and 131I-c(RGD)2 were prepared by chloramine-T (Ch-T) direct labeling, and their basic properties were measured. The binding and elution of 131I-caerin 1.1, 131I-c(RGD)2, and Na131I (control group) in esophageal cancer TE-1 cells were studied through cell binding and elution assays. (2) The antiproliferative effect and cytotoxicity of 131I-caerin 1.1, 131I-c(RGD)2, Na131I, caerin 1.1 and c(RGD)2 on TE-1 cells were detected by Cell Counting Kit-8 (CCK-8) assay. (3) A nude mouse esophageal cancer (TE-1) xenograft model was established to study and compare the efficacy of 131I-caerin 1.1 and 131I-c(RGD)2 in internal radiation therapy for esophageal cancer. Results(1) Caerin 1.1 inhibited the in vitro proliferation of TE-1 cells in a concentration-dependent manner, with an IC50 of 13.00 µg/mL. The polypeptide c(RGD)2 had no evident inhibitory effect on the in vitro proliferation of TE-1 cells. Therefore, the antiproliferative effects of caerin 1.1 and c(RGD)2 on esophageal cancer cells were significantly different (P < 0.05). The clonogenic assay showed that the clonal proliferation of TE-1 cells decreased as the concentration of caerin 1.1 increased. Compared with the control group (drug concentration of 0 µg/mL), the caerin 1.1 group showed significantly lower clonal proliferation of TE-1 cells (P < 0.05). (2) The CCK-8 assay showed that 131I-caerin 1.1 inhibited the in vitro proliferation of TE-1 cells, while 131I-c(RGD)2 had no evident inhibitory effect on proliferation. The two polypeptides showed significantly different antiproliferative effects on esophageal cancer cells at higher concentrations (P < 0.05). Cell binding and elution assays showed that 131I-caerin 1.1 stably bound to TE-1 cells. The cell binding rate of 131I-caerin 1.1 was 15.8 % ± 1.09 % at 24 h and 6.95 % ± 0.22 % after 24 h of incubation and elution. The cell binding rate of 131I-c(RGD)2 was 0.06 % ± 0.02 % at 24 h and 0.23 % ± 0.11 % after 24 h of incubation and elution. (3) In the in vivo experiment, 3 days after the last treatment, the tumor sizes of the phosphate-buffered saline (PBS) group, caerin 1.1 group, c(RGD)2 group, 131I group, 131I-caerin 1.1 group, and 131I-c(RGD)2 group were 68.29 ± 2.67 mm3, 61.78 ± 3.58 mm3, 56.67 ± 5.65 mm3, 58.88 ± 1.71 mm3, 14.40 ± 1.38 mm3, and 60.14 ± 0.47 mm3, respectively. Compared with the other treatment groups, the 131I-caerin 1.1 group had significantly smaller tumor sizes (P < 0.001). After treatment, the tumors were isolated and weighed. The tumor weights in the PBS group, caerin 1.1 group, c(RGD)2 group, 131I group, 131I-caerin 1.1 group, and 131I-c(RGD)2 group were 39.50 ± 9.54 mg, 38.25 ± 5.38 mg, 38.35 ± 9.53 mg, 28.25 ± 8.50 mg, 9.50 ± 4.43 mg, and 34.75 ± 8.06 mg, respectively. The tumor weights in the 131I-caerin 1.1 group were significantly lighter than those in the other groups (P < 0.01). Conclusion131I-caerin 1.1 has tumor-targeting properties, is capable of targeted binding to TE-1 esophageal cancer cells, can be stably retained in tumor cells, and has an evident cytotoxic killing effect, while 131I-c(RGD)2 has no evident cytotoxic effect. 131I-caerin 1.1 better suppressed tumor cell proliferation and tumor growth than pure caerin 1.1, 131I-c(RGD)2, and pure c(RGD)2.

Bioactive Natural Product Discovery via Deuterium Adduct Bioactivity Screening.

The discovery of bioactive natural products lies at the forefront of human medicine. The continued discovery of these molecules is imperative in the fight against infection and disease. While natural products have historically dominated the drug market, discovery in recent years has slowed significantly, partly due to limitations in current discovery methodologies. This work demonstrates a new workflow, deuterium adduct bioactivity screening (DABS), which pairs untargeted isotope labeling with whole cell binding assays for bioactive natural product discovery. DABS was validated and led to the discovery of a new isoprenyl guanidine alkaloid, zillamycin, which showed anti-cancer and anti-microbial activities. DABS thus represents a new workflow to accelerate discovery of natural products with a wide range of bioactive potentials.

Dried Blood Spot Collection for AQP4-IgG testing provides opportunity for improving global health NMOSD diagnostics (S50.001)

Dried Blood Spot Collection for AQP4-IgG testing provides opportunity for improving global health NMOSD diagnostics (S50.001)

Mobilization of monocytic myeloid-derived suppressor cells is regulated by PTH1R activation in bone marrow stromal cells

Myeloid-derived suppressor cells (MDSCs) are bone marrow (BM)-derived immunosuppressive cells in the tumor microenvironment, but the mechanism of MDSC mobilization from the BM remains unclear. We investigated how BM stromal cell activation by PTH1R contributes to MDSC mobilization. PTH1R activation by parathyroid hormone (PTH) or PTH-related peptide (PTHrP), a tumor-derived counterpart, mobilized monocytic (M-) MDSCs from murine BM without increasing immunosuppressive activity. In vitro cell-binding assays demonstrated that α4β1 integrin and vascular cell adhesion molecule (VCAM)-1, expressed on M-MDSCs and osteoblasts, respectively, are key to M-MDSC binding to osteoblasts. Upon PTH1R activation, osteoblasts express VEGF-A and IL6, leading to Src family kinase phosphorylation in M-MDSCs. Src inhibitors suppressed PTHrP-induced MDSC mobilization, and Src activation in M-MDSCs upregulated two proteases, ADAM-17 and MMP7, leading to VCAM1 shedding and subsequent disruption of M-MDSC tethering to osteoblasts. Collectively, our data provide the molecular mechanism of M-MDSC mobilization in the bones of tumor hosts.

SARS-CoV-2 Enters Human Leydig Cells and Affects Testosterone Production In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a SARS-like coronavirus, continues to produce mounting infections and fatalities all over the world. Recent data point to SARS-CoV-2 viral infections in the human testis. As low testosterone levels are associated with SARS-CoV-2 viral infections in males and human Leydig cells are the main source of testosterone, we hypothesized that SARS-CoV-2 could infect human Leydig cells and impair their function. We successfully detected SARS-CoV-2 nucleocapsid in testicular Leydig cells of SARS-CoV-2-infected hamsters, providing evidence that Leydig cells can be infected with SARS-CoV-2. We then employed human Leydig-like cells (hLLCs) to show that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 is highly expressed in hLLCs. Using a cell binding assay and a SARS-CoV-2 spike-pseudotyped viral vector (SARS-CoV-2 spike pseudovector), we showed that SARS-CoV-2 could enter hLLCs and increase testosterone production by hLLCs. We further combined the SARS-CoV-2 spike pseudovector system with pseudovector-based inhibition assays to show that SARS-CoV-2 enters hLLCs through pathways distinct from those of monkey kidney Vero E6 cells, a typical model used to study SARS-CoV-2 entry mechanisms. We finally revealed that neuropilin-1 and cathepsin B/L are expressed in hLLCs and human testes, raising the possibility that SARS-CoV-2 may enter hLLCs through these receptors or proteases. In conclusion, our study shows that SARS-CoV-2 can enter hLLCs through a distinct pathway and alter testosterone production.

Assessing the allogenic realness of the Cw1/12/15 pattern occurring in the LABScreen single antigen assay.

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer…). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay.

Abstract 5049: Pb-203 image guided Pb-212 receptor targeted alpha particle therapy for cancer - a powerful emerging paradigm

Abstract Objective: Pb-203 and Pb-212 have emerged as a promising elementally-matched theranostic pair for imaging-guided alpha-particle radiotherapy for cancer. A somatostatin receptor 2 (SSTR2)-targeted peptide coupled with novel Pb specific chelator (PSC) has been developed (PSC-PEG2-TOC). Here, preclinical in vitro and in vivo evaluation of 203Pb/212Pb-labeled PSC-PEG2-TOC was conducted including head to head therapy of SSTR2+ tumors in mice vs standard of care beta particle therapy and first in humans clinical imaging of SSTR2+ tumors. Method: [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC was prepared by published methods. Radiochemical stability of [203Pb]PSC-PEG2-TOC, [212Pb]PSC-PEG2-TOC and [212Bi]PSC-PEG2-TOC in human serum was determined. Binding affinity of [203Pb]PSC-PEG2-TOC was determined by binding assays in AR42J cells. In vivo SSTR2-mediated tumor targeting of [203Pb]PSC-PEG2-TOC was determined by SPECT imaging in athymic nude mice bearing AR42J xenografts. In vivo biodistribution of [212Pb]PSC-PEG2-TOC in normal organs and potential redistribution of 212Bi daughter were determined in CD-1 Elite mice. Clinical imaging was conducted under appropriate regulations to determine the biodistribution of the agent in patients bearing SSTR2+ tumors as determined by gold standard 68Ga-DOTATOC PET imaging. Results: Rapid incorporation of 203Pb and 212Pb in PSC-PEG2-TOC was observed after reactions at temperatures as low as 4°C within 15 min. Greater than 95% radiochemical stability was observed for both [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC after incubation in human serum for up to 55 hours. Stable decay product [212Bi]PSC-PEG2-TOC with minimal free 212Bi was observed. Agents demonstrated superior binding affinity to SSTR2 with Kd=0.59 nM. In in vivo studies, rapid tumor uptake and renal clearance of [203Pb]PSC-PEG2-TOC were observed in athymic nude mice bearing AR42J xenografts. In the biodistribution study, nearly identical biodistribution profiles of [212Pb]PSC-PEG2-TOC and progeny [212Bi]PSC-PEG2-TOC were found. No redistribution of 212Bi activity was identified, indicating that 212Bi remained co-localized with parent [212Pb]PSC-PEG2-TOC in vivo. Therapeutic studies in this same mouse model resulted in 100% complete responses for both a single dose 4.4 MBq administration of [212Pb]PSC-PEG2-TOC or a fractionated regimen of 4 × 1.1 MBq. First in humans clinical imaging showed PK properties that included rapid tumor accumulation and retention at over 20 h post administration, coupled with fast renal clearance of residual agent. Conclusion: [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC have promising potential for image-guide alpha-particle therapy for SSTR2 positive tumors. Agents have received a Fastrack designation by the US FDA and a safe to proceed for a Phase 1 trial. Citation Format: Michael King Schultz, Yusuf Menda, Dijie Liu, Mengshi Li, Frances Johnson, Brianna Cagle, Nicholas Baumhover, Ivy Vance. Pb-203 image guided Pb-212 receptor targeted alpha particle therapy for cancer - a powerful emerging paradigm. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5049.

Noninvasive Imaging OX40+ Activated T Cells Provides Early Warning of Rheumatoid Arthritis

PurposeThe goal of this study was to develop an imaging probe—IRDye-680RD-OX40 mAb—that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA.MethodsOX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups.ResultsNIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring.ConclusionThe results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.Graphical

Halogen Replacement on the Lysine Side Chain of Lys-Urea-Glu-Based PSMA Inhibitors Leads to Significant Changes in Targeting Properties.

Investigate the impact of various halogens on pharmacokinetics, biodistribution, and micro positron emission tomography/computed tomography (PET/CT) imaging of Glu-urea-Lys-based prostate-specific membrane antigen (PSMA) inhibitors. Based on the modification of SC691, a small molecule inhibitor of PSMA previously developed by our group, we synthesized 68Ga-labeled compounds by modifying the lysine terminal amino with different halogenated phenyl substituents. After complete characterization, in vitro and in vivo properties were studied. The [68Ga]Ga-DOTA-SC691-R possesses a high radiochemical yield (98-99%). The internalization values of [68Ga]Ga-DOTA-SC691-H, [68Ga]Ga-DOTA-SC691-Cl, and [68Ga]Ga-DOTA-SC691-Br in LNCaP cells all displayed time-dependent pattern enhanced with time. The results of in vitro competitive inhibition assay showed that the affinity of natGa-DOTA-SC691-R for PSMA had a trend of H < F < Cl < Br < I. The blocking imaging and dynamic imaging on micro-PET/CT of male non-obese diabetic/severe combined immunodeficiency mice with LNCaP tumors showed the rapid tumor targeting properties of [68Ga]Ga-DOTA-SC691-R with specificity for PSMA. Static imaging of micro-PEC/CT of these compounds could rapidly localize LNCaP tumors with decent image quality (except for [68Ga]Ga-DOTA-SC691-H). Biodistribution data showed that [68Ga]Ga-DOTA-SC691-R were metabolized via the kidney and tumor accumulation followed the order of H ≈ F ≈ Cl < I < Br uptake values at 1 h. [68Ga]Ga-DOTA-SC691-Br showed the highest tumor accumulation and retention (15.21 ± 5.57%ID/g at 30 min, 20.39 ± 4.38%ID/g at 60 min, and 13.30 ± 4.39%ID/g at 120 min), which is consistent with the results of the competitive inhibition assay and cell binding assay. It was demonstrated that the halogen substituent on the lysine terminal amino group on the Glu-urea-Lys backbone did positively affect the binding of [68Ga]Ga-DOTA-SC691-R to PSMA. The bulkier and less electronegative Br (or I) elements are preferred for structural modifications here.

LOTUS suppresses amyloid β-induced dendritic spine elimination through the blockade of amyloid β binding to PirB

BackgroundAlzheimer’s disease (AD) is the most common neurodegenerative disease worldwide but has no effective treatment. Amyloid beta (Aβ) protein, a primary risk factor for AD, accumulates and aggregates in the brain of patients with AD. Paired immunoglobulin-like receptor B (PirB) has been identified as a receptor of Aβ and Aβ–PirB molecular interactions that cause synapse elimination and synaptic dysfunction. PirB deletion has been shown to suppress Aβ-induced synaptic dysfunction and behavioral deficits in AD model mice, implying that PirB mediates Aβ-induced AD pathology. Therefore, inhibiting the Aβ–PirB molecular interaction could be a successful approach for combating AD pathology. We previously showed that lateral olfactory tract usher substance (LOTUS) is an endogenous antagonist of type1 Nogo receptor and PirB and that LOTUS overexpression promotes neuronal regeneration following damage to the central nervous system, including spinal cord injury and ischemic stroke. Therefore, in this study, we investigated whether LOTUS inhibits Aβ–PirB interaction and Aβ-induced dendritic spine elimination.MethodsThe inhibitory role of LOTUS against Aβ-PirB (or leukocyte immunoglobulin-like receptor subfamily B member 2: LilrB2) binding was assessed using a ligand-receptor binding assay in Cos7 cells overexpressing PirB and/or LOTUS. We assessed whether LOTUS inhibits Aβ-induced intracellular alterations and synaptotoxicity using immunoblots and spine imaging in a primary cultured hippocampal neuron.ResultsWe found that LOTUS inhibits the binding of Aβ to PirB overexpressed in Cos7 cells. In addition, we found that Aβ-induced dephosphorylation of cofilin and Aβ-induced decrease in post-synaptic density-95 expression were suppressed in cultured hippocampal neurons from LOTUS-overexpressing transgenic (LOTUS-tg) mice compared with that in wild-type mice. Moreover, primary cultured hippocampal neurons from LOTUS-tg mice improved the Aβ-induced decrease in dendritic spine density. Finally, we studied whether human LOTUS protein inhibits Aβ binding to LilrB2, a human homolog of PirB, and found that human LOTUS inhibited the binding of Aβ to LilrB2 in a similar manner.ConclusionsThis study implied that LOTUS improved Aβ-induced synapse elimination by suppressing Aβ-PirB interaction in rodents and inhibited Aβ–LilrB2 interaction in humans. Our findings revealed that LOTUS may be a promising therapeutic agent in counteracting Aβ-induced AD pathologies.