Target Cell Surface Receptors Research Articles

Abstract B040: Trispecific killer engagers against IGF1R and fetal form of insulin receptor induce human natural killer cell mediated killing of hormone receptor positive breast cancer in vitro and in vivo

Abstract Patients with hormone receptor-positive (HR+) breast cancers frequently recur with metastatic disease 5-20 years after the initial diagnosis and completion of chemotherapy and prolonged treatment with hormonal therapy. Thus, patients with metastatic HR+ breast cancer need new therapeutic options. A potential mechanism of therapeutic resistance in HR+ patients is the presence of slow-growing, well-differentiated cancer cells that result in clinically detectable metastases after a prolonged latency. One way to eliminate these cancer cells is with immunotherapy (IT). Unfortunately HR+ breast cancers have not responded to current T-cell based IT. The objective of this study is to target HR+ breast cancers using a natural killer (NK) cell based approach that targets cell surface receptors in HR+ breast cancer. The type I IGF receptor (IGF1R) is expressed on HR+ breast cancers and its expression is tightly related to ER function. Endocrine therapy resistance is associated with increased expression of the A-isoform or fetal form of IR (IR-A) and depend on IR signaling. We hypothesized that a drug that would target NK cells to IGF1R or IR-A expressing HR+ cells will be effective in killing them. We generated IGF1R or IR-A specific Tri specific Killer Engagers (TriKE) that leverage expression of IGF1R and IR-A on HR+ cells to cause NK directed death. The IGF1R TriKE consists of humanized single domain antibody (VHH) sequence of a CD16 nanobody to engage and activate NK cells, a humanized VHH of a nanobody against IGF1R to target HR+ breast cancer cells, and an IL-15 molecule between them to drive NK cell expansion and survival. The IGF1R TriKE did not bind embryonal fibroblasts from IGF1R knock out mice (R- cells) but bound R-/IGF1R cells (R- cells engineered to express human IGF1R) and to IGF1R+ MCF-7 and T47D breast cancer cells indicating specific binding to IGF1R on cells. It did not cause downregulation of IGF1R levels in multiple HR+ breast cancer cells and did not inhibit signaling via IGF1R. It specifically induced NK cell degranulation, measured by surface CD107a expression and intracellular IFNγ, against IGF1R+ target breast cancer cells (MCF-7, T-47D, ZR-75-1) in a functional assay compared to the anti-IGF1R nanobody. It did not induce degranulation of NK cells against IGF1R negative cells suggesting that the functionality was specific for IGF1R expressing targets. The IGF1R TriKE enhanced cell killing of HR+ breast cancer cells as measured by cytotoxicity assays. IGF1R TriKE combined with human NK cells inhibited tumor growth of MCF-7 tumors in NSG mice compared to control groups. Human NK cells persisted in peripheral blood three weeks after final NK infusion based on flow cytometry assessment. The IR-A TriKE incorporated a novel engineered non-immunoglobulin IR-A specific peptide binder. Our preliminary data show that the IR-A TriKE degranulated NK cells against breast cancer cells overexpressing IR-A. These data indicate that this strategy could be an effective treatment for HR+ metastatic breast cancer and make them responsive to IT. Citation Format: Courtney Baar, Emily Chiu, Yvette Soignier, Behiye Kodal, Jeffrey S Miller, Martin Felices, Deepali Sachdev. Trispecific killer engagers against IGF1R and fetal form of insulin receptor induce human natural killer cell mediated killing of hormone receptor positive breast cancer in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B040.

Nanotechnology: A promising tool for targeted drug delivery

Nanotechnology has eventually and strongly engaged in the field of drug delivery. It makes use of the specific properties of the substance at the Nano scale. Their primary goal is to increase therapeutic effects while reducing adverse effects. Due to their improved goods, nanotechnology has become more popular across a variety of industries. The term “Nano medicine” is used to denote the application of nanotechnology in medicine. This Nano medicine is essential for drug delivery, antibacterial, vaccine development, wearable technology, diagnostic and imaging tools, implants, high throughput screening platforms, etc. It makes use of biological, biomimetic, no biological, or hybrid materials. To attain logical drug delivery, it is important to understand the interlink age between nanoparticles and the biological environment, drug release, and targeting cell-surface receptors. We can control disease progression by using nanomaterial including peptide-based nanotubes to prey the vascular endothelial growth factor (VEGF) receptor. Also, the use of herbal medicine has been used since ancient times. The supply of active compounds is shown by the effectiveness of various species of herbal medicine. The essential requirements for extending novel nanotechnology-based medication delivery systems are highlighted in this review.

Nanotechnology-based drug delivery systems for treatment of knee injuries and Alzheimer’s disease – a review

The effective treatment of human illnesses relies on the ability to deliver therapeutic compounds to diseased sites in a highly efficient manner. However, conventional therapeutic strategies often require high systemic administration due to non-specific biodistribution and rapid metabolism of free drug molecules before they can reach their intended targets which leads to undesirable side effects. Nanotechnology offers a solution to mitigate these issues by allowing the development of drug delivery systems (DDS) within the nanometer size range, which can alter the pharmacological and therapeutic effects of drug molecules. These novel DDS have several advantages over traditional large-scale systems, including altered pharmacokinetic behavior and improved payload, owing to their small size. Moreover, their surface chemistry can be easily modified to attach targeting and therapeutic molecules for specific curative and therapeutic applications. In this comprehensive review, we explore and present the potential of nanoparticles as a highly effective drug delivery system for the treatment of knee injuries and Alzheimer’s disease. With the aim of addressing the challenges that are associated with drug delivery, nanotechnology has emerged as an increasingly important area of research. Scientists have conducted extensive investigations into nanosystems with varying compositions and biological properties for use in drug delivery applications. In order to ensure efficient drug delivery, it is critical to understand how nanomaterials interact with the biological environment, target cell-surface receptors, release drugs, administer multiple drugs, and maintain the stability of therapeutic agents. The recognition and appreciation of the molecular mechanisms that are involved in cell signaling in relation to the disease under investigation is of fundamental importance.

The CD38/CD3xCD28 Trispecific Antibody (SAR442257) Potentially Represents a Novel Therapeutic Strategy for Peripheral T-Cell Lymphomas

Introduction Peripheral T-cell lymphomas (PTCL) remain an unmet medical need and novel therapeutic options inducing longstanding responses are needed. Immunotherapies with humanized monoclonal antibodies targeting cell surface receptors have dramatically improved the prognosis of many cancers. However, so far, only two antibodies targeting CCR4 and CD30 have been approved for the treatment of subsets of PTCL patients. Currently, several bi- and tri-specific antibodies are under pre-clinical or clinical evaluation to treat lymphomas. TSAR442257, a trispecific antibody (TriAb) which targets and binds CD38, CD3 and CD28 to engage T-cells against CD38-expressing tumor cells and enhance their CD3-triggered activation via CD28 co-stimulation. Here, we performed a preclinical investigation to evaluate the potential therapeutic value of the SAR442257 to treat PTCL, knowing that both CD28 and CD38 might represent targets in tumor T cells. Material and methodsCD28 and CD38 expression was studied in tumor cells of 268 PTCL patients. In situ semi-quantitative evaluation was done using immunohistochemistry on FFPE samples (n=250), and quantitative evaluation was performed using flow cytometry (FCM) on PBMCs (n=18) and PTCL lines (n=6). Multiplex immunofluorescence stainings with tumor cell marker were also performed on biopsies sections of selected cases. In vitro cytotoxic assays were performed by using 24h co-cultures of PTCL cell lines stained with a viability dye with allogeneic blood cells from healthy donors (HD) and SAR442257 or control TriAbs with null mutations in CD28 (D28), CD38 (D38) or both recognition domains at various concentrations (0-25000 pM) at 10:1 effector-to-target ratio. For primary PTCL cells, similar experiments were done with appropriate panels of surface markers aiming to identify tumor cells. Activation assays, measuring expression of CD25 and CD69, were done on PTCL lines and PBMCs from HD using the afore mentioned TriAbs at similar concentrations. ResultsWe found a recurrent but variable expression of CD28 and CD38 among the different PTCL entities (Figure 1). Overall, CD38 expression by tumor cells was found in 42% of PTCL samples in situ and 67% of PBMCs (ranging from 2.5% to 65%; data not shown). CD28 was found in neoplastic T cells in 56% of PTCL samples by IHC and 100% of PBMCs with a strong heterogeneity ranging from 30% to 99.4% (data not shown). CD38 and CD28 showed a range of expression among PTCL categories, with CD38 being more frequently expressed (Avg. IHC score: 250-300)) in PTCL diseases with a cytotoxic profile postulated to derive from innate immune/cytotoxic cells whereas CD28 was more frequently expressed (Avg. IHC score: 150-200) in PTCLs known to derive from helper T-cells (p<0.001, Figure 2). Co-expression of both targets was rare. Multiplex stainings confirmed co-expression of CD38 and/or CD28 targets in tumor cells but consistent with the FCM analysis, CD28 and CD38 were variably expressed in tumor T-cells of TFH PTCLs. The SAR442257 induced efficient killing of all CD28+/CD38+ and CD28-/CD38+ PTCL lines (with maximum killing reaching about 35%), while the D28 and D38 control TriAbs showed less efficient cytotoxic activities in six different cell lines. SAR442257 also induced CD25 and CD69 expression on normal T-cells suggesting efficient T-cell activation, (data not shown). Conclusion Altogether, this study shows that 1) most PTCL cells express at least CD28 or CD38, and 2) SAR442257 can efficiently kill malignant PTCL cells, while ensuring effective T-cell activation; In view of these results, clinical investigation of SAR442257 in PTCL is warranted.

Targeting glioblastoma multiforme using a novel fusion protein comprising interleukin-13 and staphylococcal enterotoxin B in vitro

Targeting cell surface receptors with immunotoxins provides a novel, unique and highly potent treatment against cancers. A high expression of interleukin-13 (IL13) receptor α2 (IL13Rα2) has been reported in different types of cancers including glioblastoma multiforme (GBM). In this paper, to target IL13Rα2 on GBM cells, a fusion protein was generated comprising human IL13 and staphylococcal enterotoxin B (SEB), termed IL13-linker-SEB. The fusion protein was cloned into pET28a(+) and expressed in Escherichia coli strain BL21 (DE3); U251 (IL13Rα2-positive) and T98G (IL13Rα2-negative) GBM cell lines were employed and the functional activity of IL13-linker-SEB was evaluated by cell ELISA, cytotoxicity (MTT and LDH), apoptosis (flow cytometry and caspase-3 activity), adhesion, scratch and RT-PCR tests. SEB and chemotherapeutic drugs were employed to be compared to IL13-linker-SEB function. The IL13-linker-SEB exhibited higher binding affinity and cytotoxicity compared to SEB on U251 cells, although both recombinant proteins had shown similar behavior regarding T98G cells. Furthermore, the highest induction of apoptosis was observed in U251 cells treated with IL13-linker-SEB which was confirmed by Bax/Bcl-2 ratio. The expression of MMP2, MMP9 and VEGFR2 in U251 cells experienced a significant reduction after treatment with IL13-linker-SEB compared to SEB and T98G treated cells. The data showed that IL13-linker-SEB can be considered as a novel potential agent for GBM treatment; however, further research is needed to investigate the efficacy.

Fate and Efficacy of Engineered Allogeneic Stem Cells Targeting Cell Death and Proliferation Pathways in Primary and Brain Metastatic Lung Cancer.

Primary and metastatic lung cancer is a leading cause of cancer-related death and novel therapies are urgently needed. Epidermal growth factor receptor (EGFR) and death receptor (DR) 4/5 are both highly expressed in primary and metastatic non-small cell lung cancer (NSCLC); however, targeting these receptors individually has demonstrated limited therapeutic benefit in patients. In this study, we created and characterized diagnostic and therapeutic stem cells (SC), expressing EGFR-targeted nanobody (EV) fused to the extracellular domain of death DR4/5 ligand (DRL) (EVDRL) that simultaneously targets EGFR and DR4/5, in primary and metastatic NSCLC tumor models. We show that EVDRL targets both cell surface receptors, and induces caspase-mediated apoptosis in a broad spectrum of NSCLC cell lines. Utilizing real-time dual imaging and correlative immunohistochemistry, we show that allogeneic SCs home to tumors and when engineered to express EVDRL, alleviate tumor burden and significantly increase survival in primary and brain metastatic NSCLC. This study reports mechanistic insights into simultaneous targeting of EGFR- and DR4/5 in lung tumors and presents a promising approach for translation into the clinical setting.

Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox.

In recent years, targeted protein degradation (TPD) of plasma membrane proteins by hijacking the ubiquitin proteasome system (UPS) or the lysosomal pathway has emerged as a novel therapeutic avenue in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful in targeting cell surface receptors, these approaches are limited by the availability of suitable binders to generate heterobifunctional molecules. Here, we present the development of a nanobody (VHH)-based degradation toolbox termed REULR (Receptor Elimination by E3 Ubiquitin Ligase Recruitment). We generated human and mouse cross-reactive nanobodies against five transmembrane PA-TM-RING-type E3 ubiquitin ligases (RNF128, RNF130, RNF167, RNF43, and ZNRF3), covering a broad range and selectivity of tissue expression, with which we characterized the expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interactions with a variety of disease-relevant target receptors (EGFR, EPOR, and PD-1) by induced proximity, resulting in effective membrane clearance of the target receptor at varying levels. In addition, we designed E3 ligase self-degrading molecules, "fratricide" REULRs (RNF128, RNF130, RENF167, RNF43, and ZNRF3), that allow downregulation of one or several E3 ligases from the cell surface and consequently modulate receptor signaling strength. REULR molecules represent a VHH-based modular and versatile "mix and match" targeting strategy for the facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

Global asymptotic stability of a delay differential equation model for SARS-CoV-2 virus infection mediated by ACE2 receptor protein

The COVID-19 pandemic has brought a serious threat to human life safety worldwide. SARS-CoV-2 virus mainly binds to the target cell surface receptor ACE2 (Angiotensin-converting enzyme 2 ) through the S protein expressed on the surface of the virus, resulting in infection of target cells. During this infection process, the target cell ACE2 receptor plays a very important mediating role. In this paper, a delay differential equation model containing the mediated effect of target cell receptor is established according to the mechanism of SARS-CoV-2 virus invasion of target cells, and the global stability of the infection-free equilibrium and the infected equilibrium of the model is obtained by using the basic reproduction number ℛ0 and constructing the appropriate Lyapunov functional. The expression of the basic reproduction number ℛ0 intuitively gives the dependence on the expression ratio of the target cell surface ACE2 receptor, which is helpful for the understanding of the mechanism of SARS-CoV-2 virus infection.

A Conjugate between Lqh-8/6, a Natural Peptide Analogue of Chlorotoxin, and Doxorubicin Efficiently Induces Glioma Cell Death.

Natural peptides isolated from animal venoms generally target cell surface receptors with high affinity and selectivity. On many occasions, some of these receptors are over-expressed in cancer cells. Herein, we identified Lqh-8/6 as a natural peptide analog of chlorotoxin, a proven and useful compound for the diagnosis and treatment of glioma. Lqh-8/6 and two other natural analogues were chemically synthesized for the first time and evaluated for their ability to label, detect and prevent glioma growth in vitro. We demonstrate that a biotinylated version of Lqh-8/6 allows both the labeling of glioma cell lines and the detection of glioma in brain sections of glioma allograft Fisher rats. Lqh-8/6 has intrinsic anti-invasive properties but is non-toxic to glioma cells. To confer anti-tumor properties to Lqh-8/6, we chemically coupled doxorubicin to the glioma-targeting peptide using click chemistry. To this end, we successfully chemically synthesized Lqh-8/6-azide and doxorubicin-alkyne without impairing the toxic nature of doxorubicin. The toxin-drug conjugate efficiently promotes the apoptosis of glioma cells in vitro. This example contributes to the concept that animal venom peptides constitute exquisite warheads for delivering toxic chemical conjugates, a parallel to the popular concept of antibody-drug conjugates for the treatment of cancer.

G-Quadruplex-Proximized Aptamers (G4PA) Efficiently Targeting Cell-Surface Transferrin Receptors for Targeted Cargo Delivery.

DNA-assembled multiaptamer systems have been demonstrated to significantly promote the aptamer capacity of binding cell-surface-expressed proteins. However, how to conveniently harness them for efficient transmembrane delivery of targets remains a challenge. Toward this goal, here we engineer a G-quadruplex-proximized aptamer (G4PA) system in which a DNA aptamer specific for transferrin receptor (TfR) is guided by a bimolecular G4 and assembles into a dimerized proximity form that well matches homodimeric TfR highly expressed on the cancer cell surface. This system displays a higher capacity for targeting cell-surface TfR than the monomeric aptamer and super transmembrane transportation of nucleic acid cargoes, which is comparable to that of conventional liposome transfection but overcomes the lack of targeting ability of the latter. The G4PA system is then applied to the targeted delivery of siRNA for PLK1 gene silencing in positive cells rather than negative controls, showing great promise for use in precise anticancer therapy.

Antibody-Recruitment as a Therapeutic Strategy: A Brief History and Recent Advances.

Antibodies are a significant and growing sector within the global pharmaceutical industry. The popularity of antibodies as therapeutics derives from - at least in part - evolvable affinity for virtually any disease-relevant cell surface receptor, as well as unique immunotherapeutic mechanisms of action, including neutralization, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC). While advances in the large-scale expression and purification of therapeutic antibodies have been made, these remain costly and laborious tasks. Agents that redirect endogenous antibodies to target a pathogen or malignant cell obviate the need for new antibody discovery and production. Chimeric antibody-recruiting technologies consist of a target cell surface receptor binding domain, and an endogenous antibody-binding domain. By design, these agents bring endogenous antibodies to the surface of a target pathogen or diseased cell, which can result in targeted cytotoxicity by antibody-dependent mechanisms. This review highlights seminal contributions and recent advances in this growing and important therapeutic field.

Preparation and characterization of amine-functionalized mupirocin-loaded zinc oxide nanoparticles: A potent drug delivery agent in targeting human epidermoid carcinoma (A431) cells

Skin cancer is one of the conventional malignities hastened in humans associated with sunburns, exhibiting considerable morbidity rate disparity. Besides the current treatments, nanotechnology alongside combination therapy holds enormous potential in treating metastatic tumors by selectively targeting cell surface receptors with a suitable carrier for drug delivery. The primary purpose of this research involves investigating and scrutinizing the physicochemical characteristics and antiproliferative effects of amine-functionalized and mupirocin-loaded zinc oxide nanoparticles (AZNP-MUP). Physicochemical characterization of the AZNP-MUP revealed spherical NPs (18.6 nm) on the drug-loaded matrix developed by EDC/NHS mediation, with an average crystallite grain size of 24.75 nm. The 50% hydroxyl radical scavenging activity for the conjugate (AZNP-MUP) was achieved at 91.89 μg/ml, the best concentration evinced when juxtaposed with the other antioxidant assays in the study. Moreover, AZNP-MUP inhibited the growth of V. cholera, E. faecalis and L. monocytogenes more significantly, with a greater zone of inhibition (25 mm) than AZNP. The conjugate AZNP-MUP manifested a strong antiproliferative effect with a higher percentage of necrotic cells (63%) and more DNA fragmentation than AZNP. Our results evince that AZNP-MUP could be adeptly proposed as a skin cancer therapy strategy to improve current patients' treatments.

Transferrin receptor-mediated internalization and intracellular fate of conjugates of a DNA aptamer

Aptamers have excellent specificity and affinity in targeting cell surface receptors, showing great potential in targeted delivery of drugs, siRNA, mRNA, and various nanomaterials with therapeutic function. A better insight of the receptor-mediated internalization process of aptameric conjugates could facilitate the design of new targeted drugs. In this paper, human transferrin receptor-targeted DNA aptamer (termed HG1-9)-fluorophore conjugates were synthesized to visualize the internalization, intracellular transport, and nano-environmental pH of aptameric conjugates. Unlike transferrin that showed high recycling rate and short duration time in cells, the synthetic aptameric conjugates continuously accumulated within cells at a relatively slower rate, besides recycling back to cell surface. After long incubation (≥2 h), only very small amounts of HG1-9 conjugates (approximately 5%) entered late endosomes or lysosomes, and more than 90% of internalized HG1-9 was retained in cellular vesicles (pH 6.0–6.8), escaping from degradation. And among the internalized HG1-9 conjugates, approximately 20% was dissociated from transferrin receptor. The lower recycling ratios of HG1-9 conjugates and their dissociation from receptors promote the accurate and efficient release of their loaded drugs. These results suggest that aptamer HG1-9 could be provided as a versatile tool for specific and effective delivery of diverse therapeutic payloads.

Specificity of CD8-Targeted Fusosomes in Human PBMCs Using Single Cell RNA and T Cell Receptor Sequencing

Introduction: The ability to deliver genes to specific cell types in vivo would have a profound therapeutic impact for a diverse set of diseases. For example, targeting T cells for in vivo delivery of a chimeric antigen receptor (CAR) to treat B cell malignancies would improve access to CAR T therapies by overcoming the limitations of ex vivo manufacturing such as high costs, wait times and manufacturing failures. We have developed a novel paramyxovirus-based integrating vector (fusosomes) that specifically targets cell surface receptors for targeted gene delivery. Fusosomes, engineered to target CD8α, a cell surface protein expressed on CD8+ T cells, can bind and specifically deliver a genetic payload through membrane fusion. To evaluate the specificity of fusosome-mediated delivery to cells expressing CD8α in vitro, single cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (scTCR-seq) were performed on human PBMCs treated with CD8α-targeted fusosomes with a GFP payload. scRNA-seq is a tool that can be used to detect the transgene delivered by our fusosomes in specific cell populations by measuring mRNA expression of the receptor targeted by the fusosome (e.g., CD8α) and the genetic payload delivered by the fusosome in the same cell. Transcriptome information to understand potential pathway changes induced by delivery of the transgene is also captured.Methods: scRNA-seq was performed using the 10X Genomics system on human PBMCs in vitro. Activated and resting PBMCs from a single donor were transduced with CD8α-targeted fusosomes. Cells were then harvested 3 days post-transduction for scRNA-seq and scTCR-seq. Following library preparation and Illumina sequencing, read processing and bioinformatics analyses were performed using 10X Genomics Cell Ranger and the Seurat R package.Results: In the PBMCs transduced with fusosomes, > 9,000 cells were barcoded with > 1,900 median genes detected per cell. scRNA-seq identified multiple cell types in PBMCs with approximately 25% of cells expressing CD8α transcripts. Fusosome-associated transcripts were seen in about 54% of the cells expressing CD8α and in particular, T cells classified as CD8+ using known markers and classification algorithms based on reference data sets. Subsequently, scTCR-seq data were used to confirm the identity of T cells. Comparison of the results showed an overlap of > 87% of cells classified as T cells by the two independent methods. Visualization by UMAP and inference based on a reference dataset showed that naïve (Tn), central memory (Tcm), effector memory (Tem) and mucosal-associated invariant T cells were transduced by the CD8α-targeted fusosomes. In addition, fusosome-associated transcripts were detected in about 19% of NK cells where approximately 62% of these NK cells also expressed CD8α. Overall, our CD8α-targeted fusosomes have a specificity of > 93% in resting PBMCs based on CD8α expression. A subset of cells may have detectable GFP transcripts at the time of analysis, but not CD8α transcripts due to limited sequencing depth per cell.Summary: Our in vitro scRNA-seq and scTCR-seq data demonstrate that our CD8α-targeted fusosomes are highly specific for cells expressing CD8α transcripts in resting PBMCs. These data highlight the potential for utilizing single cell sequencing technologies to comprehensively characterize the specificity of our fusosomes, and to identify key biological pathways that may play a role in specificity, transduction efficiency and clinical efficacy. As next steps, we will use similar approaches to characterize in vivo transduction in animal models. DisclosuresIftikhar: Sana Biotechnology: Current Employment. Balanis: Sana Biotechnology: Ended employment in the past 24 months. Fonseka: Sana Biotechnology: Current Employment. Bandoro: Sana Biotechnology: Ended employment in the past 24 months. Cruite: Sana Biotechnology: Current Employment. Davis: Sana Biotechnology: Current Employment. Amatya: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Laska: Sana Biotechnology: Current Employment. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Shah: Sana Biotechnology: Current Employment. Paliwal: Sana Biotechnology: Current Employment. Chaivorapol: Sana Biotechnology: Current Employment.

A comparative analysis of cell surface targeting aptamers

Aptamers represent a potentially important class of ligands for the development of diagnostics and therapeutics. However, it is often difficult to compare the function and specificity of many of these molecules as assay formats and conditions vary greatly. Here, with an interest in developing aptamer targeted therapeutics that could effectively deliver cargoes to cells, we chemically synthesize 15 aptamers that have been reported to target cell surface receptors or cells. Using standardized assay conditions, we assess each aptamer’s binding properties on a panel of 11 different cancer cell lines, correlate aptamer binding to antibody controls and use siRNA transfection to validate each aptamer’s binding to reported target receptors. Using a subset of these molecules known to be expressed on prostate cancers, we use near-infrared in vivo imaging to assess the tumor localization following intravenous injection. Our data demonstrate some surprising differences in the reported specificity and function for many of these molecules and raise concerns regarding their cell targeting capabilities. They also identify an anti-human transferrin aptamer, Waz, as a robust candidate for targeting prostate cancers and for future development of aptamer-based therapeutics.

Elongated PEO-based nanoparticles bind the high-density lipoprotein (HDL) receptor scavenger receptor class B I (SR-BI)

Targeting cell-surface receptors with nanoparticles (NPs) is a crucial aspect of nanomedicine. Here, we show that soft, flexible, elongated NPs with poly-ethylene-oxide (PEO) exteriors and poly-butadiene (PBD) interiors – PEO-PBD filomicelles - interact directly with the major high-density lipoprotein (HDL) receptor and SARS-CoV-2 uptake factor, SR-BI. Filomicelles have a ~ 6-fold stronger interaction with reconstituted SR-BI than PEO-PBD spheres. HDL, and the lipid transport inhibitor, BLT-1, both block the uptake of filomicelles by macrophages and Idla7 cells, the latter are constitutively expressing SR-BI (Idla7-SR-BI). Co-injections of HDL and filomicelles into wild-type mice reduced filomicelle signal in the liver and increased filomicelle plasma levels. The same was true with SCARB1−/− mice. SR-BI binding is followed by phagocytosis for filomicelle macrophage entry, but only SR-BI is needed for entry into Idla7-SR-BI cells. PEO-PBD spheres did not interact strongly with SR-BI in the above experiments. The results show elongated PEO-based NPs can bind cells via cooperativity among SR-BI receptors on cell surfaces.

Determinants of Ligand-Functionalized DNA Nanostructure-Cell Interactions.

Synthesis of ligand-functionalized nanomaterials with control over size, shape, and ligand orientation facilitates the design of targeted nanomedicines for therapeutic purposes. DNA nanotechnology has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential. In this study, we systematically investigate tunable design parameters of antibody-functionalized DNA nanostructures binding to therapeutically relevant receptors, including the programmed cell death protein 1, the epidermal growth factor receptor, and the human epidermal growth factor receptor 2. We show that, although the native affinity of antibody-functionalized DNA nanostructures remains unaltered, the absolute number of bound surface receptors is lower compared to soluble antibodies due to receptor accessibility by the nanostructure. We explore structural determinants of this phenomenon to improve efficiency, revealing that receptor binding is mainly governed by nanostructure size and DNA handle location. The obtained results provide key insights in the ability of ligand-functionalized DNA nanostructures to bind surface receptors and yields design rules for optimal cellular targeting.

Carbon dots-embedded N-acetylneuraminic acid and glucuronic acid-imprinted polymers for targeting and imaging of cancer cells.

Diagnosis, treatment, and prediction of cancer progression require new targeting agents to specifically target cell surface receptors. Herein, we demonstrated fluorescent carbon quantum dots-molecularly imprinted polymer (CQD-MIP) for selective targeting and imaging of cancer cells. Carbon quantum dots (CQDs) were synthesized and characterized. The synthesized CQDs had average size of 1.5nm and show intense fluorescence emission at wavelength of 450nm with excitation at 370nm. CQD-MIP nanoparticles imprinted with N-acetylneuraminic acid and glucuronic acid were prepared and characterized. CQD-MIPs were successfully applied for selective targeting and imaging of MCF-7, HepG-2, and NIH-3T3 cell lines. Non-imprinted polymer (NIP) showed no binding properties toward a target molecule. Non-imprinted polymer (NIP) and non-cancerous human cell lines were used for controlling the imprinting and targeting effects, respectively. Acceptable results were obtained with imprinted polymers on cancer cells.

Self-assembled chitosan polymer intercalating peptide functionalized gold nanoparticles as nanoprobe for efficient imaging of urokinase plasminogen activator receptor in cancer diagnostics

Targeting cell surface receptors for specific drug delivery in cancer has garnered lot of attention. Urokinase plasminogen activator receptor (uPAR), a surface biomarker, is overexpressed on many tumours including breast, colorectal, prostate, and ovarian cancers. Binding of growth factor domain (GFD) of urokinase plasminogen activator (uPA) with uPAR lead to its close conformation, and allow somatomedin B domain (SMB) of vitronectin binding by allosteric modulation. In-silico docking of uPAR with GFD and SMB peptides was performed to identify potential binding affinity. Herein, we report fluorescently labeled peptide functionalized AuNPs with a mixed self-assembled monolayer of intercalating chitosan polymer for efficient targeting and imaging of uPAR-positive cells. The biophysical characterization of nanoconjugates and uPAR-specific targeting was assessed by FACS, cell adhesion, and fluorescence imaging. AuNPs/chitosan/GFD+SMB peptides showed higher uptake as compared to AuNPs/chitosan/GFD, and AuNPs/chitosan/SMB that can be utilized as a tool for molecular targeting and imaging in metastasis.

SARS-CoV-2 Entry Inhibitors Targeting Virus-ACE2 or Virus-TMPRSS2 Interactions.

COVID-19 is an infectious disease caused by SARS-CoV-2. The life cycle of SARS-CoV-2 includes the entry into the target cells, replicase translation, replicating and transcribing genomes, translating structural proteins, assembling and releasing new virions. Entering host cells is a crucial stage in the early life cycle of the virus, and blocking this stage can effectively prevent virus infection. SARS enters the target cells mediated by the interaction between the viral S protein and the target cell surface receptor angiotensin- converting enzyme 2 (ACE2), as well as the cleavage effect of a type-II transmembrane serine protease (TMPRSS2) on the S protein. Therefore, the ACE2 receptor and TMPRSS2 are important targets for SARS-CoV-2 entry inhibitors. Herein, we provide a concise report/information on drugs with potential therapeutic value targeting virus-ACE2 or virus-TMPRSS2 interactions to provide a reference for the design and discovery of potential entry inhibitors against SARS-CoV-2.