Picomolar Concentrations Research Articles

Phage-Templated Synthesis of Targeted Photoactive 1D-Thiophene Nanoparticles.

Thiophene-based nanoparticles (TNPs) are promising therapeutic and imaging agents. Here, using an innovative phage-templated synthesis, a strategy able to bypass the current limitations of TNPs in nanomedicine applications is proposed. The phage capsid is decorated with oligothiophene derivatives, transforming the virus in a 1D-thiophene nanoparticle (1D-TNP). A precise control of the shape/size of the nanoparticles is obtained exploiting the well-defined morphology of a refactored filamentous M13 phage, engineered by phage display to selectively recognize the Epidermal Growth Factor Receptor (EGFR). The tropism of the phage is maintained also after the bioconjugation of the thiophene molecules on its capsid. Moreover, the 1D-TNP proved highly fluorescent and photoactive, generating reactive oxygen species through both type I and type II mechanisms. The phototheranostic properties of this platform are investigated on biosystems presenting increasing complexity levels, from in vitro cancer cells in 2D and 3D architectures, to the in vivo tissue-like model organism Hydra vulgaris. The phage-templated 1D-TNP showed photocytotoxicity at picomolar concentrations, and the ability to deeply penetrate 3D spheroids and Hydra tissues. Collectively the results indicate that phage-templated synthesis of organic nanoparticles represents a general strategy, exploitable in many diagnostic and therapeutic fields based on targeted imaging and light mediated cell ablation.

8530 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells

Abstract Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None. Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses. Presentation: 6/3/2024

9273 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells

Abstract Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None. Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses. Presentation: 6/3/2024

14K prolactin derived 14-mer antiangiogenic peptide targets bradykinin-/nitric oxide-cGMP-dependent angiogenesis.

Over the past few decades, VEGF-targeted antiangiogenic therapy for cancers has gained increasing attention. Nevertheless, there are still several limitations such as the potential resistance mechanisms arising in cancer cells against these therapies and their potential adverse effects. These limitations highlight the need for novel anti-angiogenesis molecules and better understanding of the mechanisms of tumor angiogenesis. In the present study, we investigated the antiangiogenic properties of a novel 14-mer antiangiogenic peptide (14-MAP) derived from N-terminal 14 kDa buffalo prolactin and characterized its mode of action. 14-MAP at the picomolar concentration inhibited VEGF- and bradykinin (an autacoid peptide expressed in vascular tissues in pathophysiology, BK)-stimulated endothelial nitric oxide (eNO) production, cell migration, and proliferation in endothelial cells and vessel development in the chick embryo. Although this peptide inhibited both VEGF- and BK-dependent angiogenic processes, its action was more pronounced in the latter. Moreover, the interference of 14-MAP with the eNO synthase (eNOS)-cyclic GMP pathway was also identified. A combination of a low dose of Avastin, a widely used drug targeting VEGF-dependent angiogenesis, and 14-MAP significantly reduced tumor size in an in vivo model of human colon cancer. Taken together, our results suggest that 14-MAP, a BK- and eNOS-dependent antiangiogenic peptide, might be useful for overcoming the limitation of VEGF-targeted antiangiogenic therapy in cancer patients. However, further studies will be required to further characterize its mode of action and therapeutic potential.

Targeting T cells with tetravalent bispecific antibodies for the treatment of graft-versus-host disease

AbstractAllogeneic hematopoietic stem cell transplantation is an established treatment for hematological malignancies and some genetic diseases. Acute graft-versus-host disease (GVHD) is the most common and debilitating side effect with poor survival rates of 5% to 30% for severe cases. In this manuscript, we describe a tetravalent T-cell–engaging bispecific antibody (BsAb) based on the immunoglobulin G-[L]-single-chain variable fragment (IgG-[L]-scFv) platform, with all 4 binding domains specific for CD3. In vitro, picomolar concentrations of the CD3×CD3 BsAb induced potent lysis of activated CD4 and CD8 T cells. In immunodeficient mice, in which human T cells induced xenogeneic GVHD, administration of 0.1 μg BsAb per dose depleted the majority of T cells from the peripheral blood, and 10 μg per dose completely reversed established GVHD and achieved a 100% survival rate. In mice bearing NALM6-luc xenografts, treatment with CD3×CD19 BsAb and activated human T cells induced complete remission of the leukemia, and all treated mice developed GVHD by 50 days after treatment. CD3×CD3 BsAb (3-30 μg doses) reversed clinical signs of GVHD, allowing long term follow-up beyond 250 days. T cells were undetectable by polymerase chain reaction in 4 of 5 mice in the 30 μg CD3×CD3 BsAb group 180 days after leukemia injection, and complete necropsies on day 259 revealed no evidence of human T cells or leukemia cells. Curing GVHD allows for long-term follow-up of tumor response heretofore impossible in humanized mouse models. Further studies are warranted to determine whether the CD3×CD3 BsAb has potential for treating clinical GVHD and other autoimmune diseases in humans.

Modular Semisynthetic Approach to Generate T Cell-Dependent Bispecific Constructs from Recombinant IgG1 Antibodies.

Redirecting T cells to tumor cells by bispecific antibodies is an effective approach to treat cancer, and T cell-dependent bispecific antibodies (TDBAs) are an emerging class of potent immunotherapeutic agents. By simultaneously targeting antigens on tumor cells and T cells, T cells are activated to kill tumor cells. Herein, we report a platform to generate a novel class of 2:1 structure of T cell-dependent bispecific antibody with bivalency for HER2 receptors on tumor cells and monovalency for CD3 receptors on T cells. For this, we use a biogenic inverse electron-demand Diels-Alder (IEDDA) click reaction on genetically encoded tyrosine residues to install one TCO handle on therapeutically approved antibody trastuzumab. Subsequent TCO-tetrazine click with a tetrazine-functionalized CD3-binding Fab yields a 2:1 HER2 × CD3 TDBA that exhibits a tumor-killing capability at picomolar concentrations. Monovalency toward the CD3 receptor on T cells can lower the chances of cytokine release syndrome, which is a common side effect of such agents. Our semisynthetic approach can generate highly potent TDBA constructs in a few chemoenzymatic and synthetic steps.

IL-1β Induces LDL Transcytosis by a Novel Pathway Involving LDLR and Rab27a.

In early atherosclerosis, circulating LDLs (low-density lipoproteins) traverse individual endothelial cells by an active process termed transcytosis. The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) treated advanced atherosclerosis using a blocking antibody for IL-1β (interleukin-1β); this significantly reduced cardiovascular events. However, whether IL-1β regulates early disease, particularly LDL transcytosis, remains unknown. We used total internal reflection fluorescence microscopy to quantify transcytosis by human coronary artery endothelial cells exposed to IL-1β. To investigate transcytosis in vivo, we injected wild-type and knockout mice with IL-1β and LDL to visualize acute LDL deposition in the aortic arch. Exposure to picomolar concentrations of IL-1β induced transcytosis of LDL but not of albumin by human coronary artery endothelial cells. Surprisingly, expression of the 2 known receptors for LDL transcytosis, ALK-1 (activin receptor-like kinase-1) and SR-BI (scavenger receptor BI), was unchanged or decreased. Instead, IL-1β increased the expression of the LDLR (LDL receptor); this was unexpected because LDLR is not required for LDL transcytosis. Overexpression of LDLR had no effect on basal LDL transcytosis. However, knockdown of LDLR abrogated the effect of IL-1β on transcytosis rates while the depletion of Cav-1 (caveolin-1) did not. Since LDLR was necessary but overexpression had no effect, we reasoned that another player must be involved. Using public RNA sequencing data to curate a list of Rab (Ras-associated binding) GTPases affected by IL-1β, we identified Rab27a. Overexpression of Rab27a alone had no effect on basal transcytosis, but its knockdown prevented induction by IL-1β. This was phenocopied by depletion of the Rab27a effector JFC1 (synaptotagmin-like protein 1). In vivo, IL-1β increased LDL transcytosis in the aortic arch of wild-type but not Ldlr-/- or Rab27a-deficient mice. The JFC1 inhibitor nexinhib20 also blocked IL-1β-induced LDL accumulation in the aorta. IL-1β induces LDL transcytosis by a distinct pathway requiring LDLR and Rab27a; this route differs from basal transcytosis. We speculate that induction of transcytosis by IL-1β may contribute to the acceleration of early disease.

New silica-based adsorbents for water purification: Removal of short- and long-chain perfluoroalkyl sulfonic acids (PFSA) at sub-nanomolar concentrations

Perfluoroalkyl acids (PFAAs) are widespread in the aquatic environment and also measurable in ground and drinking water. Because of the insufficient PFAA elimination in conventional water treatment processes, e. g. active carbon based methods, consumers in areas with contaminated water supplies are exposed to an elevated health hazard. For this purpose, the applicability of five differentially fluorinated silica-based adsorbents (HSU00107954-958) to remove the potentially human toxic perfluoroalkyl sulfonic acids(PFSAs) perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), and perfluorobutane sulfonic acid (PFBS) from water was investigated with regard to removal efficiencies (REs) and equilibrium loadings. During the adsorbent screening at nanomolar concentrations (20.0–33.4 nmol L−1) maximum REs of 46.9% for PFBS (HSU00107954), 79.4% for PFHxS (HSU00107954), and between 86.5 to 96.7% for PFOS (HSU00107956, and HSU00107954, respectively) have been achieved. Even at picomolar concentrations (< 400 pmol L−1) HSU00107954 was still able to eliminate PFBS and PFHxS with an efficiency of 46.3–51.2% and 79.1–88.2%, respectively. Analyses of the equilibrium loadings of the functionalized adsorbents in the concentration range 40.1 pmol to 3.34 nmol L−1 resulted in appropriate linearized Freundlich isotherms for all investigated PFSA. Compared to literature-based Freundlich adsorption coefficients (KF) for granular activated carbon (GAC), the determined KF values (nmol(1−n) Ln m−2) of the most efficient adsorbents HSU00107956 and HSU00107954 for each PFSA were significantly 8–10 and 50–60 times higher, respectively. These proven increased adsorption capacities relative to activated carbon possibly indicate specific PFSA selectivities of the functionalized macroporous silica adsorbents.

Designing Nanocomposite-Based Electrochemical Biosensors for Diabetes Mellitus Detection: A Review.

This review will unveil the development of a new generation of electrochemical sensors utilizing a transition-metal-oxide-based nanocomposite with varying morphology. There has been considerable discussion on the role of transition metal oxide-based nanocomposite, including iron, nickel, copper, cobalt, zinc, platinum, manganese, conducting polymers, and their composites, in electrochemical and biosensing applications. Utilizing these materials to detect glucose and hydrogen peroxide selectively and sensitively with the correct chemical functionalization is possible. These transition metals and their oxide nanoparticles offer a potential method for electrode modification in sensors. Nanotechnology has made it feasible to develop nanostructured materials for glucose and H2O2 biosensor applications. Highly sensitive and selective biosensors with a low detection limit can detect biomolecules at nanomolar to picomolar (10-9 to 10-12 molar) concentrations to assess physiological and metabolic parameters. By mixing carbon-based materials (graphene oxide) with inorganic nanoparticles, nanocomposite biosensor devices with increased sensitivity can be made using semiconducting nanoparticles, quantum dots, organic polymers, and biomolecules.

Programmable DNA Nanomachine Integrated with Electrochemically Controlled Atom Transfer Radical Polymerization for Antibody Detection at Picomolar Level.

The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background signal noise and restricted signal gain. In this work, we have developed a highly efficient electrochemical biosensor by constructing a programmable DNA nanomachine integrated with electrochemically controlled atom transfer radical polymerization (eATRP). The sensor works by binding the target antidigoxin antibody (anti-Dig) to the epitope of the recognization probe, which then initiates the cascaded strand displacement reaction on a magnetic bead, leading to the capture of cupric oxide (CuO) nanoparticles through magnetic separation. After CuO was dissolved, the eATRP initiators were attached to the electrode based on the CuΙ-catalyzed azide-alkyne cycloaddition. The subsequent eATRP reaction results in the formation of long electroactive polymers (poly-FcMMA), producing an amplified current response for sensitive detection of anti-Dig. This method achieved a detection limit at clinically relevant picomolar concentration in human serum, offering a sensitive, convenient, and cost-effective tool for detecting various biomarkers in a wide range of applications.

Mechanistic aspect of copper selenide as an electrocatalyst for the detection of hydrogen peroxide in gaseous phase

The electrocatalytic properties of copper selenide for hydrogen peroxide detection in a gas phase were investigated using cyclic and square-wave voltammetry. Copper selenide demonstrated significant electrocatalytic activity towards hydrogen peroxide oxidation in a basic medium containing polyacrylic acid as a gas-absorbing polyelectrolyte. Experimental data were corroborated by a brief theoretical analysis and simulations based on a two-electron irreversible electrode mechanism of a dissolved redox couple. The voltammetric response of a conventional glassy carbon electrode modified with copper selenide showed a linear correlation with hydrogen peroxide concentration over a range of 3.0 × 10−5 to 1.0 × 10−3 mol L−1 in solution.Practical application of this method for gaseous hydrogen peroxide detection was explored using screen-printed electrodes (SPE) coated with a thin film of polyacrylic acid containing medium, with copper selenide serving as a modifier of the working electrode. The detectable concentration range of gaseous hydrogen peroxide using the modified SPE spanned picomolar concentrations. The statistical parameters of the calibration line in terms of LOQ, LOD and R2 are 4.1 × 10−6, 1.2 × 10−6 mol L−1, and 0.996, which correspond very closely to the gas concentrations of 6.1 × 10−12 to 6.1 × 10−11 mol L−1.

Aurora: a fluorescent deoxyribozyme for high-throughput screening.

Fluorescence facilitates the detection, visualization, and tracking of molecules with high sensitivity and specificity. A functional DNA molecule that generates a robust fluorescent signal would offer significant advantages for many applications compared to intrinsically fluorescent proteins, which are expensive and labor intensive to synthesize, and fluorescent RNA aptamers, which are unstable under most conditions. Here, we describe a novel deoxyriboyzme that rapidly and efficiently generates a stable fluorescent product using a readily available coumarin substrate. An engineered version can detect picomolar concentrations of ribonucleases in a simple homogenous assay, and was used to rapidly identify novel inhibitors of the SARS-CoV-2 ribonuclease Nsp15 in a high-throughput screen. Our work adds an important new component to the toolkit of functional DNA parts, and also demonstrates how catalytic DNA motifs can be used to solve real-world problems.

Ultrafast Au(III)-Mediated Arylation of Cysteine.

Through mechanistic work and rational design, we have developed the fastest organometallic abiotic Cys bioconjugation. As a result, the developed organometallic Au(III) bioconjugation reagents enable selective labeling of Cys moieties down to picomolar concentrations and allow for the rapid construction of complex heterostructures from peptides, proteins, and oligonucleotides. This work showcases how organometallic chemistry can be interfaced with biomolecules and lead to a range of reactivities that are largely unmatched by classical organic chemistry tools.

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

AbstractIn the realm of high‐throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post‐screening. Our innovation introduces a tag‐free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity‐enhancing derivatization method, utilized in tandem with nano LC‐MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

A Tag-Free Platform for Synthesis and Screening of Cyclic Peptide Libraries.

In the realm of high-throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post-screening. Our innovation introduces a tag-free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity-enhancing derivatization method, utilized in tandem with nano LC-MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

The Detection of Trace Metal Contaminants in Organic Products Using Ion Current Rectifying Quartz Nanopipettes.

While ion current rectification (ICR) in aprotic solvent has been fundamentally studied, its application in sensing devices lacks exploration. The development of sensors operable in these solvents is highly beneficial to the chemical industry, where polar aprotic solvents, such as acetonitrile, are widely used. Currently, this industry relies on the use of inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (OES) for the detection of metal contamination in organic products. Herein, we present the detection of trace amounts of Pd2+ and Co2+ using ion current rectification, in cyclam-functionalized quartz nanopipettes, with tetraethylammonium tetrafluoroborate (TEATFB) in MeCN as supporting electrolyte. This methodology is employed to determine the concentration of Pd in organic products, before and after purification by Celite filtration and column chromatography, obtaining comparable results to ICP-MS within minutes and without complex sample preparation. Finite element simulations are used to support our experimental findings, which reveal that the formation of double-junction diodes in the nanopore enables trace detection of these metals, with a significant response from baseline even at picomolar concentrations.

A model for cooperativity of kinesin-4 motors by communicating through the microtubule track

Kinesin-4 motor proteins at nanomolar concentrations can accumulate at microtubule plus ends, regulating the microtubule length. Recent experimental studies showed that at picomolar motor concentrations, under which nearly no motor crowding can occur, the run length of the kinesin-4 motor increases whereas the velocity decreases with the motor concentration, indicating the presence of a long-range communication between the motors through the microtubule track. However, the mechanism of the long-range communication is illusive. Here, based on the proposal that large conformational changes of the tubulin induced by the strong interaction with a kinesin head can propagate along the microtubule such that influence the tubulin-binding energy of other motors distant away, the dynamics of the kinesin-4 motor at the picomolar motor concentrations is studied theoretically, explaining quantitatively the experimental data. Moreover, the lifetime of the motor when multiple motors are jammed near the plus ends at high motor concentrations is studied theoretically.

Abstract 2715: Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for solid tumors

Abstract Background: Innate immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), dominate the immune landscape within "cold" solid tumors, which typically exhibit poor responses to immune checkpoint inhibitors (ICIs). The dearth of tumor-infiltrating T cells and the prevailing immunosuppressive microenvironment contribute to this ineffectiveness. To bolster immunotherapy against such tumors, we developed SM2248, a bispecific VHH antibody. SM2248 simultaneously obstructs the CD47-SIRPα phagocytosis checkpoint pathway and conditionally activates CD40 on tumor-infiltrating innate immune cells. CD47/SIRPa blockade enhances macrophage and dendritic cell (DC) phagocytosis and cross-presentation of antigens to cytotoxic T cells. CD40 activation fosters DC licensing, reprograms macrophages, and guides MDSC differentiation towards DCs and macrophages. The combined action of CD47 blockade and CD40 stimulation remodels the tumor microenvironment, fostering T cells infiltration and activation. Methods: SM2248 was developed by fusing a highly selective CD47-blocking VHH module - minimizing binding to human erythrocytes and platelets, with a conditional CD40 agonistic VHH module - selectively activating immune cells upon CD47 engagement, incorporated into an inactivated IgG1 Fc backbone. The specificity and affinity of SM2248 binding to human CD47 and CD40 were assessed via ELISA and flow cytometry assays. CD47 and SIRPα interaction blockade was confirmed through a competitive ligand binding assay. Target-dependent CD40 activation was evaluated via a CD40-NFκB-Luc reporter assay. In vivo anti-tumor efficacy was determined using a syngeneic transplantation model of MC38-hCD47 cancer cells in B-hCD40 humanized mice. Binding to erythrocytes and platelets, compared with magrolimab, was assessed using flow cytometry. Hemolysis toxicity was evaluated through an in vitro erythrocyte lysis assay. Results: SM2248 effectively inhibits cell surface CD47-SIRPα interaction at nanomolar concentrations and activates CD40-NF-κB-luciferase reporters at picomolar concentrations in a target-dependent manner. In contrast to the CD47 monoclonal antibody magrolimab, SM2248 does not bind to human erythrocytes or platelets and does not induce hemolysis. In murine syngeneic tumor models, SM2248 completely halts the growth of MC38-hCD47 tumors in humanized B-hCD40 mice and does not induce dose-dependent hepatotoxicity at doses up to 10 mg/kg. Conclusions: SM2248, a humanized bispecific tetravalent VHH antibody targeting CD47 and CD40, exhibits promising in vivo efficacy and a favorable safety profile in preclinical studies, offering the potential for enhancing immunotherapy in solid tumors. Citation Format: Xiaodan Liu, Hong Zhou, Shihao Lu, Huiqin Geng, Wenjing Song, Siyao Xie, Yi Wei, Xing Zhang, Simin Yang, Sheila Zhou, Yanbin Liang. Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2715.

Abstract 5316: SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response

Abstract Background: Monoclonal antibodies targeting the co-stimulatory receptors CD137 (4-1BB) and CD40 have displayed encouraging activity against a wide range of solid tumors in preclinical animal models. However, their clinical development as monotherapy has been hindered by dose-related liver toxicity, leading to limited efficacy at safe doses. To address this challenge and bolster tumor-specific immunity by orchestrating the interaction between dendritic cells (DCs) and cytotoxic T cells, we introduce SM2256 - a bispecific conditional CD40 and 4-1BB dual agonist. SM2256 physically bridges DCs and T cells, enhancing T cell priming and reactivation. In preclinical animal studies, SM2256 exhibited both excellent tolerability and potent anti-tumor activity. Methods: SM2256 was engineered by fusing high-affinity, non-ligand-blocking VHH domains targeting 4-1BB and CD40 to a silenced IgG1 Fc backbone. This fusion resulted in a tetravalent bispecific antibody capable of conditionally activating CD40 and 4-1BB upon mutual receptor binding. We assessed the binding specificity and affinity of SM2256 to human CD40 and 4-1BB using ELISA and flow cytometry. The target-dependent activation of CD40 and 4-1BB was confirmed via reporter assays and co-culture systems using primary DCs and T cells. In vivo safety and anti-tumor efficacy were evaluated in a syngeneic transplantation model using the MC38 cancer cell line in B-h41BB/B-hCD40 double humanized mice. Results: SM2256 exhibited sub-nanomolar binding affinity to recombinant human and cynomolgus monkey CD40 and 4-1BB, activating 4-1BB-NFκB-luc and CD40-NFκB-luc reporter cell lines at picomolar concentrations in a strictly target-dependent manner. Furthermore, SM2256 physically facilitated the interaction between DCs and T cells, leading to increased expression of DC co-stimulatory ligands and elevated secretion of cytokines by T cells in mixed lymphocyte cultures. In a syngeneic tumor transplantation model, SM2256 significantly restrained the growth of MC38 tumors in B-h41BB/B-hCD40 double humanized mice. Conclusions: SM2256 is a novel conditional CD40 and 4-1BB dual agonist that demonstrates promising preclinical efficacy against solid tumors while boasting an improved safety profile when compared to existing monoclonal antibodies. Citation Format: Wenjing Song, Simin Yang, Yi Wei, Shihao Lu, Huiqin Geng, Xiaodan Liu, Hong Zhou, Siyao Xie, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5316.

Abstract 5821: DXC008, a novel STEAP1 antibody-tubulysin analog conjugate with a function linker, demonstrates a potential to broaden therapeutic opportunities for prostate tumors

Abstract Six-transmembrane epithelial antigen of the prostate 1 (STEAP1), is a cell surface protein frequently expressed in prostate cancer, with limited expression in non-prostate tissues. A Steap1 antibody called Vandortuzumab conjugated with MMAE (DSTP3086S) was in the clinical phase I trial for treating STEAP1-expressing metastatic castration-resistant prostate cancer and showed acceptable safety at 2.4 mg/kg once every 3 weeks. However, many patients are nonresponsive to DSTP3086S due to low target expression levels and common treatment-related AEs which were caused by MMAE payloads. DXC008 was generated through screening the therapeutical index in vitro of conjugates of an anti-Steap1 antibody with varieties of payloads of tubulysin B analogs through function peptide spacer linkers. The generated DXC008 exhibited not only good affinity for Steap1 but also moderate affinity for Prostate-specific membrane antigen (PSMA) which is as well specifically overexpressed in most prostate cancer with limited expression in normal tissue. DXC008 demonstrated couple tens to a hundred of picomolar concentration (pM) of potency against several prostate tumor cells and over 60% internalization rate within 90 min in vitro. And in vivo it showed very good durable antitumor response as low dose as 1 mg/kg one injection in both high and moderate of both Steap1 and PSMA expression xenograft models. Pharmacokinetic profiles of DXC008 were favorable and the safety of Maximal Tolerable Dose (MTD) was over 120 mg/kg in single injection in mice. DXC008 has been forward to NHP toxicity study and it has potential to be a good STEAP1/PSMA-targeting ADC with a wide therapeutic window for prostate cancers. Citation Format: Qingliang Yang, Yuanyuan Huang, Junxiang Jia, Huihui Guo, Lingli Zhang, You Zhou, Zhicang Ye, Hangbo Ye, Yifang Xu, WenJun Li, Zhiyu Zhao, Lingyao Zhao, Lu Bai, Jun Zheng, Robert Y. Zhao. DXC008, a novel STEAP1 antibody-tubulysin analog conjugate with a function linker, demonstrates a potential to broaden therapeutic opportunities for prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5821.